SignalTransBook FINAL

SIGNAL TRANSDUCTION
A short overview of its role in health and disease
Chandra Mohan, Ph.D. EMD Chemicals, San Diego, California 2009 Edition
Signal Transduction
A Short Overview of Its Role in Health and Disease
Cells respond to extracellular and
intracellular signals by using a complex network of communication pathways,
collectively known as Signal Transduction. Any abnormality in even a single pathway can lead to serious complications and debilitating diseases.
This concise overview of the role of signal transduction in health and disease is our modest attempt to introduce newcomers to this fascinating field of biology. Each chapter is a self-contained summary with diagrams that show the relationship between
various signaling components in an easy to understand manner. We hope that you will find this information useful.
A Word to Our Colleagues in Life Science Research

Research over the past thirty years, evolved from genome to proteome to kinome, has contributed immensely to our understanding of various biological processes including cell division, differentiation, and death. Such processes are governed by complex cell signaling pathways and interactions that involve a vast number of ligands, receptors, enzymes, and messengers. Internal programming and a variety of external factors delicately regulate complex interactions in various signaling pathways. It is impossible to embellish the significance and contribution of signal transduction research to our understanding of the variety of biological processes in health and disease. Investigating and understanding cellular signaling pathways is important in developing pharmaceutical agents and reducing human suffering. Although activation of enzymes has been exploited therapeutically, most drugs are based on enzyme inhibition that normalizes an overactive pathway. Furthermore, years of research has shown that inhibitors are useful for mechanistic studies aimed at understanding how enzymes interact with their substrates, the roles inhibitors play in enzyme regulation, and structure-activity relationships that are the basis for developing drugs that inhibit aberrant biochemical reactions. Rapid advances in basic and clinical sciences and the vast number of publications in these areas may create a delusion that we understand everything about cells and their interactions, but the reality is that we know only a fraction of what we want to know. We are pleased to present to you this short collection of overviews on signal transduction. This book is intended to provide a concise overview of some of the major pathways involved in cell signaling. We hope that newcomers to the area of signaling biology will find it useful and it will also serve as a quick reference resource for our more experienced colleagues. This practical resource is a part of our continuing commitment to provide useful information and exceptional service to you, our customers. If you have used Merck/EMD products (Calbiochem, Novagen, and Novabiochem) in the past, we thank you for your support and confidence in our products, and if you are just beginning your research career, please give us an opportunity to demonstrate our exceptional customer and technical service.
Hans J. Ahl Senior Sales Director, International Sales Operations EMD Chemicals, Inc. 10394 Pacific Center Court San Diego, CA 92121
Merck
A name synonymous with commitment to high quality and exceptional service
Chandra Mohan
received his
Ph.D. in 1976 from Bangalore University and did his post-doctoral work from 1977 to 1983 at the University of Southern California, School of Medicine. From 1983 to 1993 he was an Assistant Professor of Pharmacology and Nutrition at the University of Southern California, Medical School in Los Angeles. During this time he also served as an Associate Editor of the journal Biochemical Medicine and Metabolic Biology. He joined Calbiochem (EMD) in 1993 and is currently the Senior Director of Technical Services and Senior Technical Writer. His research interests include diabetes, biochemical basis of stress, inflammation, and the biology of aging.
Acknowledgments
I am grateful for the generous input of my colleagues at EMD Chemicals whose wisdom and experience contributed to this work. My special thanks go to Michelle Minix for her painstaking work in organizing and enhancing its presentation.
-Chandra Mohan
Table of Contents
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Signal Transduction: A Brief Introduction ............................................................... 1 Cell Cycle: A Brief Overview ...................................................................................... 5 Phosphoinositide 3-Kinase (PI 3-K): A Multifunctional Signaling Molecule ....... 9 Akt Signaling in Cell Death and Cell Survival ....................................................... 13 Receptor and Non-receptor Protein Tyrosine Kinases ........................................... 17 MAP Kinase Signaling: Synergistic Response to Upstream Signals .....................21 Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential .... 25 Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease . 29 Organizational and Functional Diversity of Protein Phosphatases ..................... 33 Acetylation and Methylation: Epigenetic Modulators .......................................... 39 Nuclear Import - Export: A Nuclear Revolving Door ............................................ 45 Poly(ADP-ribosylation): PARP in DNA Repair and Apoptosis ............................ 49 Heterotrimeric G-Proteins: An Introduction ......................................................... 53 Tubulin Polymerization and Depolymerization: Exploiting the Dynamic Instability of Microtubules ....................................................................................... 57 APOPTOSIS: Receptor and Mitochondrial Gateways to Cell Death .....................61 p53: Choice of Response: Repair or Death .............................................................. 67 Ubiquitnation and Proteasomal Degradation of Proteins ......................................71 Cancer Stem Cells: The Real Perpetrators in Cancer Growth and Progression ... 75 Sonic Hedgehog Signaling ....................................................................................... 79 TGF- Signaling: Dual Role in Tumor Suppression and Oncogenesis ................ 83 Angiogenesis: Its Role in Tumor Growth and Metastasis ..................................... 87 NF-B Activation: A Link Between Chronic Inflammation and Cancer ..............91 Tumor Metastasis: Role of Cell Adhesion and Matrix Metalloproteinases.......... 95 Nitric Oxide and Nitric Oxide Synthases ................................................................ 99 From Physiology to Pathology: Maintenance of the Critical Balance by Antioxidants ............................................................................................................ 105 Lipid Signaling.......................................................................................................... 111
Table of Contents
Calcium Signaling ....................................................................................................115
28 29 30 31 32
Cytokine and Chemokine Signaling ...................................................................... 121 Collagens and Other Extracellular Matrix Proteins ............................................. 127 Integrins: Molecular Adhesives in Cell Survival and Cell Death ........................ 133 Diabetes, Obesity, Dyslipidemia, and Hypertension ............................................ 137 Alzheimers Disease: The Role of -Amyloid Peptides and Oxidative Stress.... 143
Signal Transduction: A Short Overview of its Role in Health and Disease | Merck KGaA
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Chapter One
Signal Transduction: A Brief Introduction
Mechanisms involved in intercellular and intracellular communication and their effects on metabolic regulation have largely formed the basis of modern cell biology. This field of biological sciences extends into several other scientific domains including biochemistry, immunology, molecular biology, pharmacology, physiology and others.
Interaction between cells, tissues, and organs is controlled by internal and external signals that either stimulate or inhibit biological processes. The mechanism by which cells recognize and respond to these signals is one of the central issues in current research in cell biology. The biological effects of hormones, neurotransmitters, growth factors, cytokines, and other regulatory molecules are transferred from the cell membrane to intracellular
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targets such as the mitochondria, the endoplasmic reticulum, and the nucleus by a series of reactions that are collectively known as Signal Transduction. This transduction of information from membrane to internal targets leads to a cascade of molecular events that translate into the ultimate biological response to the effector molecule. During the past two decades we have seen a colossal development in biochemical and molecular biological techniques that have led not only to the identification of key enzymes involved in the signal transduction process, but also to the development of several natural and synthetic modulators of various biological processes. These tools have helped to elucidate molecular events under normal and pathological conditions. With the advent of vast information on molecular interactions that regulate cellular responses, the
| Signal Transduction: A Brief Introduction
potential for the design and development of new drugs to treat cancer, hypertension, cardiovascular complications, and other debilitating diseases has become most intriguing. In order to respond to a variety of signaling molecules, multi-cellular organisms have developed an extensive intracellular communication network, which when activated leads to growth, differentiation, and changed metabolism in a variety of cells. Cells respond to signals received from outside the cells by a variety of cell membrane and internal receptors. Receptors play a very significant role in signal transduction and without them large molecules such as hormones and growth factors would not be able to elicit any biological response. Receptors can be classified under three general classes:
(a) Plasma membrane receptors with intrinsic enzymatic activity (e.g. tyrosine kinase linked receptors: PDGF-R, insulin-receptor, EGF-R, etc.). (b) Receptors coupled to GTP-binding and hydrolyzing proteins (G-proteins) (e.g., glucagon, follicle stimulating hormone). (c) Intracellular receptors that migrate to the
nucleus upon ligand binding and affect gene transcription (e.g., corticosteroids).
Once activated, these receptors initiate a series of events, such as activation or inhibition of protein kinases, release of intracellular modulators of enzyme activities, movement of activated transcription factors into the nucleus, and transcription of proteins that ultimately change the metabolic activities of cells. Signal transduction involves thousands of different molecules and hundreds of different pathways in the cell. Signaling processes often involve signal amplification through the action of second messengers, and one signaling molecule can affect several different processes in the cell. For example, insulin can increase protein synthesis, fat synthesis, glucose transport, DNA synthesis, and cell division and reduce gluconeogenesis and glycogenolysis. A typical example of a signaling process is presented in the figure below and some of the major molecules and their roles in cell signaling is described in the next few pages. We have tried to highlight the biological role of many of these molecules in different pathways and their effects on metabolic activity in health and disease conditions.
A typical signaling cascade
G-Proteins (GTP Binding Proteins):

G-proteins are membrane associated heterotrimeric (, , and - subunits) proteins. They couple hormones, neurotransmitters, and other bioactive molecules to receptors within the cell. Depending upon their stimulatory or inhibitory action they are categorized as Gs and Gi proteins, respectively.
protein kinases and other proteins. They act on proteins involved in muscle contraction, protein synthesis, cholesterol metabolism, and several other biological processes. They can be inhibited by okadaic acid, calyculin, tautomycin, certain vanadium compounds, and other phosphatase inhibitors.
Nitric Oxide:
Nitric oxide is a very short-lived molecule that is synthesized from L-arginine by the action of nitric oxide synthase. It acts as a second messenger molecule that can readily diffuse through cell membranes. Three types of nitric oxide synthase are found in cells and their expression is cell-type dependent. Inducible nitric oxide synthase plays a major role in killing infectious microbes. Nitric oxide is also important in modulating vascular tone. Several actions of nitric oxide are attributed to its stimulation of guanylate cyclase, the enzyme that synthesizes cGMP from GTP.
Growth Factors:
Growth factors participate in the regulation of cell growth, replication, and differentiation. They act through binding to cell surface receptors and elicit their responses by activation of signal transduction pathways resulting in phosphorylation of tyrosine residues on latent cytoplasmic transcription factors.
Cytokines:
Cytokines mediate communication among cells involved in immune function by binding to specific receptors on target cells. Their biological actions vary widely depending upon the type of target tissue involved. Some of them have anticancer properties and regulate the synthesis of acute phase proteins following injury, trauma, and sepsis.
Second Messengers:
The interaction of hormone with plasma membrane receptor leads to the generation of a second messenger. Several second messengers have been postulated. Most notable second messengers are cAMP, cGMP, calcium, inositol triphosphate (IP3), PIP2, and PIP3. Most of these second messengers activate different protein kinases that phosphorylate a variety of proteins.
Cell Adhesion Molecules:

Cell adhesion molecules (CAM) are multifunctional proteins involved in cell growth, cell differentiation, cell motility, cell proliferation, and apoptosis. Two types of cell adhesion processes common to cells are: cell-cell adhesion and cellmatrix adhesion. The mechanism of cell adhesion is of great significance in studying cancer metastasis. Increased expression of CAM leads to the increased adhesive activity of cancer cells.
Ion Channels
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Protein Kinases:
Protein kinases are enzymes that phosphorylate proteins on specific serine, threonine, or tyrosine residues. Stimulation of protein kinases is one of the most common mechanisms of signal transduction in cellular growth and differentiation. Protein kinases can be activated by calcium/ calmodulin, phospholipids, cAMP and cGMP, growth factors, cytokines, and even toxins.
Cell membranes, made up of phospholipid bilayers, create a hydrophobic, low dielectric barrier to hydrophilic molecules and ions. Hence, cells have a specialized need for systems to transport ions across their membranes. Ion channels are membrane protein complexes that facilitate the passage of ions across the cell membrane. They may be in an open or closed mode at any given time. Once an ion enters or exits the cell it changes electrical membrane potential, which forms the basis for nerve conduction. Ions such as calcium (Ca2+) can act as messengers within the cell to affect cell metabolism. Ion channels can be classified based on the nature of the chemical or physical modulator that controls their opening and closing (gating). Hence, the channel can be ligand-gated, voltagegated, or second messenger-gated.
| Signal Transduction: A Brief Introduction
Protein Phosphatases:
Protein phosphatases modulate physiological responses by causing dephosphorylation of
Chapter Two
Cell Cycle: A Brief Overview
In all multi-cellular organisms proliferating cells must make a decision to either enter the cell cycle or reach a quiescent state. Quiescent cells must also decide whether to stay in a non-proliferative state or re-enter the cell cycle.
During early embryonic development most cells divide rapidly, while in adults most cells, with the exception of cells of hematopoietic origin and those lining the digestive tract, are in a quiescent state. Interphase, which is considered the resting stage between cell division, is actually a period of diverse activities that are important in making the next mitosis possible. In mammalian tissue, interphase normally lasts from 12 to 24 hours. During this period, cells synthesize RNA, produce protein and grow in size. Interphase precedes mitosis and can be divided into 4 steps: Gap 0 (G0) phase: In this phase cells leave the cycle and stop dividing. Gap 1 (G1) phase: In this phase cells increase in size, produce RNA, and synthesize protein. This is the only part of the cell cycle regulated primarily by extracellular stimuli. S phase: This is a DNA replication phase. Gap 2 (G2) phase: This is a gap between DNA synthesis and mitosis. During this phase cells continue to grow and synthesize new proteins. Mitosis or M phase: In this stage cells stop growing and protein synthesis ceases. Cells divide into two identical daughter cells. Mitosis is much shorter than interphase and lasts only one to two hours. The lengths of S and M phases are usually similar in most mammalian cells; however, the duration of the G1 phase is highly variable and depends on the cell type. Cells that are
2 | Cell Cycle: A Brief Overview
arrested in G1 phase for a very long period of time are often said to be in G0 state. For example, nerve cells that are destined never to divide again are in a permanent G0 state. Cells can also be arrested in the G2 phase, lasting about 4 to 6 hours. In multi-cellular organisms, generation times vary depending upon the type of cells and their role in the whole organism. Cells that are continuously destroyed and face much wear and tear have a shorter generation time. For example, skin cells and epithelial cells lining the intestine have a shorter generation time. On the other hand, cells in slow growing tissues, such as liver cells may have a generation time of several days. To ensure accurate progression through the cell cycle, cells have a series of checkpoints that prevent them from entering into a new phase until they have successfully completed the previous one. For example, the G1 checkpoint in the G1 phase guarantees that everything is ready for DNA synthesis. Towards the end of the G2 phase is the G2 checkpoint that checks the entry of cell into the M (mitosis) phase and during mitosis the metaphase checkpoint ensures that the cell is ready to complete the division. Multi-cellular organisms also control the number of cells in each organ. This coupled with cellular size determines the size of the whole organism. In the early G1 phase, cells respond to external mitogenic stimuli and nutrient availability, preparing themselves for passing through the various phases of the cell cycle. Cell cycle progression is regulated by the sequential events that include activation and subsequent inactivation of cyclin dependent kinases (Cdk) and cyclins. Cdks are a group of serine/threonine kinases that form active heterodimeric complexes following binding to their regulatory subunits, cyclins. Several Cdks, mainly Cdk2, Cdk4, and Cdk6, work cooperatively to drive cells from G1 into S phase. Cdk4 and Cdk6 are involved in early the G1 phase, whereas Cdk2 is required to complete the G1 phase and initiate the S phase. Both Cdk4 and Cdk6 form active complexes with D-type cyclins (cyclins D1, D2, and D3). Cdk2 is sequentially activated by E-type cyclins, cyclins E1 and E2, during the G1/S transition stage. A-type cyclins, cyclin A1 and A2 play a role during the S phase. Cdk2-cyclin A complexes appear during the late S phase and play a role in the progression of DNA replication. Cyclins that are involved in
regulating the passage of cell from G2 checkpoint into M phase are known as mitotic cyclins and they associate with mitotic Cdks. Similarly, cyclins that are involved in the passage of cells from the G1 checkpoint into S phase are called G1 cyclins. Cyclins contain the nuclear localization sequence (NLS) and help move Cdks into the nucleus. They also contain PEST (Pro, Glu, Ser, and Thr) sequences that target them for degradation by the ubiquitin-proteasomal pathway. Once the Cdks have completed their role, they undergo a rapid programmed proteolysis via ubiquitin-mediated delivery to the proteasome complex.
The enzymatic activity of a Cdk is regulated at three levels: cyclin association, subunit phosphorylation, and association with Cdk inhibitors (CKIs). When cyclins initially bind to Cdks, the resulting complex is inactive. For example, phosphorylation of Thr172 in Cdk4 or Thr160 in Cdk2 T loop ensures their proper catalytic activity. Such phosphorylations are brought about by Cdk activating kinases. However, before phosphorylation of Cdks occurs, an inhibiting kinase phosphorylates them at two other locations that block the active site. The inhibitory phosphorylation of adjacent threonine and tyrosine residues, for example Thr14/Tyr15 in Cdk1, is mediated by dual-specificity kinases. This inhibition can be relieved by the action of Cdc25 phosphatase, which then triggers the entry of cells into the mitosis phase. As negative cell cycle regulators, the CKIs may be suitable targets for inactivation in oncogenesis and tumor progression. They are induced in response to different cellular processes. For example, p21/ WAF1 is one of the effectors of p53, which is important in the DNA-damage checkpoint.
Two main categories of CKIs have been reported in cells. They are the INK and WAF/Kip families. The members of the INK family, INK4A (p16), INK4B (p15), INK4C (p18), and INK4D (p19), bind to Cdk4 and Cdk6 and block their interaction with D-type cyclins, thereby inhibiting Cdk activities. The members of the WAF/Kip family, WAF1 (p21), Kip1 (p27), and Kip2 (p57), form heterotrimeric complexes with the G1/S Cdks. Their major action is reported to be inhibition of the kinase activity of the Cdk/cyclin-E complex. p21 proteins bind to cyclins and prevent Cdks from phosphorylating retinoblastoma (Rb) proteins. p21 can also bind and inhibit PCNA, a subunit of DNA polymerase synthesized during the S phase. Members of the retinoblastoma protein family, Rb, p107, and p130 serve as the primary substrates of Cdk2, Cdk4, and Cdk6 in G1 progression. Rb proteins control the expression of genes that code for molecules required for passage of the cell through the G1 checkpoint into the S phase. They act as docking sites for a number of proteins involved in the cell cycle. For example, Rb proteins bind to E2F transcription factors and maintain them in their inactive state. This prevents the cell from entering into the S phase. The RbE2F complex also participates in the active repression of selected promoters of the cell cycle. The activity of Rb proteins is modulated by the sequential phosphorylation by Cdk4/6-cyclin D and Cdk2/cyclin E complexes. Rb proteins are inactivated by phosphorylation that allows the release of E2F to initiate the cell cycle. Rb proteins are also regulated by histone acetylase mediated acetylation, which prevents phosphorylation of Rb protein by Cdk2/cyclin E. In cells that are treated with growth hormones, the activation of Cdks catalyzes the phosphorylation of Rb proteins and these phosphorylated Rb proteins lose their ability to bind to E2F. This allows E2F to activate the transcription of genes that produce essential components for entry of cell into the S phase. Studies of human tumors have revealed that cell-cycle regulators are frequently mutated in human cancers. These mutations can lead to over expression of cyclins and Cdks, as well as loss of Rb and CKI activities, mainly of p16, p15, and Kip1. Other genes that are mutated in cancer include those that inactivate apoptotic pathways. Cancer cells often show alterations in the signal-
transduction pathways that lead to proliferation in response to external signals. Indeed, many growth factors and their receptors, as well as their membrane, cytoplasmic, and nuclear downstream effectors have been identified either as oncogenes or as tumor-suppressor genes. Genetically altered mice have provided much insight into the role of cell cycle regulators in normal as well as in pathological processes. For example, Rb deficiency is shown to be lethal in mouse embryos and mice with the Rb+/- gene develop tumors of endocrine origin. The combined deficiency of Rb+/- and p107 is reported to cause retinoblastoma in mice. Although the activation of proto-oncogenes to oncogenes can be accomplished by a number of different molecular mechanisms, it is noteworthy that cells have mechanisms to block this activation and prevent the onset of cancer. Tumor suppressor genes provide a safety mechanism to accomplish this. The p53 gene, located on chromosome 17 in humans, is one of the most prominent tumor suppressor genes. p53 protein, sometimes called the guardian of the genome, contains 393 amino acids, and even a single amino acid substitution can lead to loss of its function. About 50% of human cancers have been shown to be associated with p53 mutations and these cancers are more aggressive and difficult to treat. p53 prevents the cell from completing the cell cycle if its DNA is not properly replicated in the S phase. It does this by binding to the E2F transcription factor. This binding prevents E2F from interacting with promoters of such proto-oncogenes as c-myc and c-fos. p53 protein senses DNA damage and can stop the cell cycle in both G1 and G2 phases and can trigger apoptosis if the damage is severe enough.
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DNA damaging agents, inhibitors of microtubule assembly, and topoisomerase inhibitors that disrupt the normal cell cycle are important chemotherapeutic agents. DNA damaging agents can activate checkpoint controls and arrest the cell cycle in G1 and G2 phases. Arrest before the G1 phase allows repairs prior to DNA replication and the arrest before the G2 phase permits repairs before the separation of chromosomes and mitosis. G1 arrest is largely dependent on p53 that induces the transcription of p21, which in turn blocks the activity of Cdks. From a therapeutic standpoint Cdks are considered as promising targets in cancer chemotherapy. The most promising strategies
| Cell Cycle: A Brief Overview
involve designing inhibitors that either block Cdk activity or prevent its interaction with cyclins. Most of the currently available molecules target the ATP-binding site of the enzyme. Such an approach may create serious problems, as catalytic residues are well conserved across eukaryotic protein kinases. However, compounds such as flavopiridol, olomucine, and butyrolactone-1 that exhibit greater specificity for Cdks have shown promise.
References: Myatt, S.S., and Lam, E.W-F. 2007. Cell Division 2, 6. Golias, C.H., et al. 2004. Int. J. Clin. Pract. 58, 1134. Scafoglio, C., and Weisz, A. 2004. Cancer Res. 64, 4122. Vousden, K.H., and Lu, X. 2002. Nat. Rev. Cancer 2, 594. Adams, P.D. 2001. Biochim. Biophys. Acta 1471, 123. Chan, H.M. et al. 2001. Nat. Cell Biol. 3, 667. Ezhevsky, S.A., et al. 2001. Mol. Cell. Biol. 21, 4773. Morris, E.J., and Dyson, N.J. 2001. Adv. Cancer Res. 82, 1. Alt, J.R., et al. 2000. Genes Dev. 14, 3102. Ekholm, S.V., and Reed, S.I. 2000. Curr. Opin. Cell Biol. 12, 676.
Chapter Three
Phosphoinositide 3-Kinase (PI 3-K): A Multifunctional Signaling Molecule
The PI 3-kinases are ubiquitous, heterodimeric enzymes that play a crucial role in the regulation of many cellular processes, including cell growth, proliferation, motility, and survival. The PI 3-kinase family constitutes a large family of lipid and serine/ threonine kinases, including a number of phosphatidylinositol kinases, as well as the ATM and ATR kinases.
PI 3-kinases are divided into three classes based on their structure and substrate specificity. Class I PI 3-kinases, the best studied class, were the first to be characterized and include receptor-regulated heterodimeric enzymes, which contain a 110 kDa catalytic subunit and an 85 kDa regulatory subunit (p85/p110; p85/p110; p101/ p110). The catalytic subunit of class IA PI 3-kinases (p110, p110 and p110 isozymes) associates with an 85 kDa adaptor/regulatory subunit that is essential for interaction of these PI 3-kinases with receptor tyrosine kinases and intracellular proteins, such as PKC, SHP1, Rac, and Rho. The class IB PI 3-kinases (p110) are activated by heterotrimeric G protein -subunits and associate with a p101 adaptor/regulatory subunit that is important for full responsiveness to G heterodimers. PI 3-kinases of both class IA and IB are also activated by Ras. They can use PI, PI(4)P and PI(4,5) P2 as substrates in vitro. Their major in vivo substrate appears to be PI(4,5)P2 (PIP2). The members of this class are sensitive to wortmannin. Class II PI 3- kinases are larger monomeric enzymes (~170 kDa) and show variable responses to wortmannin. Three isoforms of class II PI 3-kinases have been described in mammals: the ubiquitously expressed PI 3-kinase C2 and PI 3-kinase C2, and a liver-specific PI 3-kinase C2. This class of enzymes
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| Phosphoinsositide 3- Kinase (PI 3-K): A Multifunctional Signaling Molecule
contains a C-2 domain at the C-terminal region that binds phospholipids in a Ca2+-dependent manner. They lack the adapter/regulatory subunit and use PI and PI(4)P as substrates. However, some studies have suggested that clathrin functions as an adaptor subunit in PI 3-kinase C2. These PI 3-kinases participate in integrin signaling in platelets and are involved in cell migration and
Catalytic Subunit P110 P110 P110 IB II P110 C2 C2 C2 III Vps34p Adaptor Subunit p85, p85 p85, p85 p85, p85 p101 Clathrin Vps15p; p150
cytosolic rearrangement. PI 3-kinases of class III are heterodimeric enzymes consisting of a p150 adaptor unit, and catalytic subunit Vps34 of 100 kDa size, which can phosphorylate PI(3) P. The human homolog of Vps34 is reported to be sensitive to wortmannin and participates in the regulation of endocytic membrane trafficking and autophagy.
Regulation/ Activation by RTK RTK; GPCR RTK GPCR RTK; GPCR RTK, GPCR Constitutive; TLR PI PIP2 PI In vivo Substrates PIP2
Class IA
Following ligand binding to receptor and activation of receptor tyrosine kinase, the p85/ p110 complex is recruited to the receptor by interaction of the SH2 domain of p85 with consensus phosphotyrosine residues on receptor tyrosine kinase. This allows the p110 catalytic subunit to come in close proximity to its lipid substrates in the cell membrane. The interaction of a receptor tyrosine kinase with the p85
subunit relieves the inhibitory effect of p85 on the p110 catalytic subunit. Activated PI 3-kinase phosphorylates phosphoinositol (PI) substrates to produce PI(3)P, PI(3,4)P2, and PI(3,4,5)P3 (PIP3). These molecules act as second messengers and recruit PI 3-kinase-dependent serine/threonine kinases (PDK1) and Akt from the cytoplasm to the plasma membrane. Lipid binding and membrane translocation of Akt leads to conformational
RTK
PI(4,5)P2
PLC
DAG IP3
GPCR
IRS
PTEN
p110
p85
PI 3-K
PIP3
SH IP
PDK1
p10 p10 1 1
PIP2
PI(3,4)P2
PI 3-K
Akt
p70S6K
mTOR
4E-BP1
Cell Growth
Translation
elf-4E
PI 3-Kinase Signaling - An Overview
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changes, which allow it to become phosphorylated on Thr308 in the activation loop and Ser473 in the hydrophobic phosphorylation motif by PDK1 and PDK2, respectively. This dual phosphorylation causes full activation of Akt. The PI 3-kinase pathway diverges at many points in the metabolic scheme, resulting in a variety of physiological effects. PI 3-kinase activation of Akt and subsequent activation of mTOR stimulates cell proliferation and the translation process in response to nutrients and growth factors. It phosphorylates p70S6 kinase and 4E-binding protein (4E-BP). Phosphorylation of 4E-BP allows the release of translation initiation factor, elF4E. The PI 3-kinase/Akt pathway is also involved in the LKB1-mediated activation of AMP-activated protein kinase (AMPK) that inhibits mTOR activity, thereby allowing energy conservation in the cell. Akt also phosphorylates p21/WAF1 and p27KIP1, allowing them to be exported to the cytoplasm for sequestration and degradation. This allows cell proliferation to proceed unimpeded. Several other proteins have also been identified as intracellular targets of PI 3-kinase/Akt. Class IA PI 3-kinase signaling is considered to be important in mediating insulin responses in cells. Any loss in the activity of these kinases can result in metabolic defects linked to type 2 diabetes. Inhibition of PI 3-kinase and overexpression of dominant negative PI 3-kinase mutants are shown to block many of the physiological responses to insulin. Pharmacological inhibition of PI 3-kinase by wortmannin or LY294002 is shown to diminish insulin-stimulated translocation of GLUT4 to the cell surface and reduce glucose uptake into cells. Overexpression of constitutively active forms of PI 3-kinase p110 catalytic subunit stimulates insulin-mediated metabolic effects and dominant-negative p85 regulatory subunit constructs block insulin-mediated metabolic effects. Akt2, a downstream target of PI 3-kinase, is highly expressed in insulin-responsive tissues, such as muscle and adipose tissue, and is essential for the maintenance of glucose homeostasis. Mice deficient in Akt2 display classical features of type 2 diabetes. Under resting conditions, PIP3 levels are practically undetectable in mammalian cells. Levels of PIP3 are controlled tightly by the action of several PIP3
phosphatases, such as PTEN (Phosphatase and Tensin homolog), SHIP (SH2-domain containing Insositol 5-Phosphatase) 1 and 2. PTEN acts on the 3-position to convert PIP3 back to PIP2, which can also function as a second messenger to recruit PH-domain-containing proteins, such as Akt. Over-expression of PTEN is sufficient to lower basal levels of 3-phosphorylated phosphoinositide in cells. PTEN-/- mice exhibit higher levels of 3-phosphorylated phospholipids and die during embryogenesis due to the failure of developmental apoptosis. PTEN-/- mouse embryo fibroblasts are shown to be resistant to apoptotic stimuli. PTEN is the most important negative regulator of PI 3-kinase. It has multiple conserved domains, including a C2 phospholipid binding domain, two PEST sequences, a PZD binding domain, and a PIP2 binding motif. SHIP removes phosphate from the 5-position to produce PI(3,4)P2. SHIP1, a 145 kDa protein, is expressed predominantly in cells of the hematopoietic lineage that respond to cytokine and growth factor stimulation and participates in regulating apoptosis, cell differentiation, and migration. On the other hand, SHIP2, a 150 kDa protein, is ubiquitously expressed and responds predominantly to insulin stimuli. A lack of SHIP2 is shown to cause a dramatic increase in insulin sensitivity in tissues. Deletion of SHIP also leads to an increase in PIP3 levels and a decrease in PIP2 levels. SHIP-/- mice are also shown to have defective apoptosis and excessive cell survival and proliferation in the myeloid lineages. This is associated with elevated levels of PIP3 and increased Akt phosphorylation. PI 3-kinase signaling is crucial to many aspects of cell growth and survival and this pathway is stimulated by many growth factors. Hence, PI 3-kinase activity is tightly regulated in normal cells. Overactivation of this pathway can perturb the control mechanisms for cell growth and survival and contribute to metastatic competence and resistance to chemotherapy. Abnormal activation of PI 3-kinases is seen in several forms of cancer. About 30% of solid tumors contain mutations in the catalytic unit of their PI 3-kinase, which increase its enzymatic activity and produce excessive Akt signaling. Somatic missense mutations in the p110 gene are reported in HER2amplified and hormone-receptor-positive breast
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| Phosphoinsositide 3- Kinase (PI 3-K): A Multifunctional Signaling Molecule
cancers. Also, p110 is frequently overexpressed and mutated in gliomas, colon, prostate, and gynecological tumors. Several tumors also exhibit either defective or diminished activity in the tumor suppressor, PTEN. Deletion of PTEN in T cells or B cells is shown to enhance their proliferation. Mice heterozygous for PTEN show a predisposition for developing leukemia and lymphoma. Additionally, PI 3-kinase inhibitors, wortmannin and LY294002 exhibit antitumor activities and sensitize tumor cells to chemotherapeutic agents. Given the importance of the PI 3-kinase signaling pathway, several new drug candidates are being sought. A few isoform-selective inhibitors have also been identified. Stable, water-soluble conjugates of wortmannin are being developed to improve its pharmacological characteristics.
References: Ito, K., et al. 2007. J. Pharmacol. Exp. Therap. 321, 1 Medina-Tato, D.A., et al. 2007. Immunology 121, 448. Leslie, N.R. 2006. Antioxid. Redox Signal. 8, 1765. Workman, P., et al. 2006. Nat. Biotech. 24, 794. Samuels, Y., and Ericson, K. 2006. Curr. Opin. Oncol. 18, 77 Bader, A.G., et al. 2005. Nat. Rev. Cancer 5, 921. Hennessy, B.T., et al. 2005. Nat. Drug Disc. 4, 988 Fruman D.A. 2004. Biochem. Soc. Trans. 32, 315. Scheid, M.P., and Woodgett, J.R. 2003. FEBS Lett. 546, 108. Cantley, L.C. 2002. Science 296, 1655. Comer, F.I., and Parent, C.A. 2002. Cell 109, 541 Gaidarov, I., et al. 2001. Mol. Cell 7, 443. Lawlor M.A., and Alessi, D.R. 2001. J. Cell Sci. 114, 2903. Toker, A., and Newton, A.C. 2000. J. Biol. Chem. 275, 8271. Stein, R.C, and Waterfield, M.D. 2000. Mol. Med. Today 6, 347. Datta, S.R., et al. 1999. Genes Dev. 13, 2905. Dong, Z., et al. 1999. Anticancer Res. 19, 3743. Prior, I.A., and Clague, M.J. 1999. Mol. Cell Biol. Res. Commun. 1, 162. Shepherd, P.R., et al. 1996. J. Mol. Endocrinol. 17, 175.
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Chapter Four
Akt Signaling in Cell Death and Cell Survival

Akt (protein kinase B), a serine/threonine kinase, has emerged as a critical enzyme in several signal transduction pathways involved in cell proliferation, apoptosis, angiogenesis, and diabetes. In mammals three isoforms of Akt have been reported (, , or Akt 1, 2, 3) that exhibit a high degree of homology, but differ slightly in the localization of their regulatory phosphorylation sites.
Akt is the predominant isoform in most tissues, whereas the highest expression of Akt is observed in the insulin-responsive tissues, and Akt is abundant in brain tissue. Each Akt isoform is composed of three functionally distinct regions: an N-terminal pleckstrin homology (PH) domain that provides a lipid-binding
4 | Akt Signaling in Cell Death and Cell Survival
module to direct Akt to PIP2 and PIP3, a central catalytic domain, and a C-terminal hydrophobic motif. Akt is constitutively phosphorylated on Ser124, in the region between PH and catalytic domains, and on Thr450, in the C-terminal region (in Akt, the most widely studied isoform) in unstimulated cells. Activation of Akt involves growth factor binding to a receptor tyrosine kinase and activation of PI 3-K, which phosphorylates the membrane bound PI(4,5)P2 (PIP2) to generate PI(3,4,5)P3 (PIP3). The binding of PIP3 to the PH domain anchors Akt to the plasma membrane and allows its phosphorylation and activation by 3-phosphoinositide-dependent kinase-1 (PDK1). Akt is fully activated following its phosphorylation at two regulatory residues, a threonine residue on the kinase domain and a serine residue on the hydrophobic motif, which are structurally and functionally conserved within the AGC kinase family. Phosphorylation
13
at Thr308 and Ser473 is required for the activation of Akt, while phosphorylation at Thr309 and Ser474 activates Akt1 and 2. Phosphorylation at Thr305 activates Akt. Phosphorylation of a threonine residue on the kinase domain, catalyzed by PDK1, is essential for Akt activation. It causes a charge-induced conformational change, allowing substrate binding and increased rate of catalysis. Akt activity is augmented by about 10-fold by phosphorylation at the serine residue by PDK2. DNA-PK and PKCII are reported to phosphorylate the serine residue on the regulatory subunit. Without threonine phosphorylation, the hydrophobic motif of Akt is more susceptible to the action of phosphatases; however, the dually phosphorylated and fully active enzyme is stable, allowing its localization to the nucleus and other sites. The activity of Akt is negatively regulated by PTEN (phosphatase and tensin homolog deleted on chromosome 10, a PIP3-specific phosphatase) and SHIP (SH2-domain containing inositol 5-phosphatase). The principal role of Akt is to facilitate growth factor-mediated cell survival and to block apoptotic cell death. This is achieved by phosphorylating and deactivating pro-apoptotic factors such as Bad, caspase-9, and Forkhead transcription factors (AFX, Daf-16, FKHR). The phosphorylation of Bad at Ser136 promotes its association with 14-3-3 proteins in the cytosol, which prevents Bad from localizing at the mitochondria to induce apoptosis. Akt is also known to promote cell survival by inactivating caspase-9 by phosphorylating it at Ser196. Likewise, activated Akt phosphorylates Forkhead family members, resulting in their sequestration in the cytoplasm. In the absence of survival factors and Akt activity, Forkhead family members translocate to the nucleus, where they initiate a program of gene expression (e.g., FasL) that promotes cell death. Akt is also reported to phosphorylate IKK at Thr23 and activate it. The activated IKK, in turn, phosphorylates IB, targeting it for ubiquitination and proteasomal degradation. This leads to the activation and nuclear translocation of NF-B, and transcription of NF-B-dependent pro-survival genes, including Bcl-xL and caspase inhibitors. Akt is also known to phosphorylate and thereby inactivate GSK-3, allowing the activation of glycogen synthase to proceed. An important point to note here is that phosphorylation of cyclin D by GSK-3 targets it for proteolysis; hence the inactivation
of GSK-3 by Akt may promote the up-regulation of cyclin D and enhance cell cycling. Recently it has been shown that if Chk1, a DNA damage effector kinase, is phosphorylated by Akt at Ser280 it can no longer be phosphorylated by ATM/ATR at Ser345 to undergo activation. This may be of therapeutic significance as Chk1 inhibition is shown to enhance sensitization of tumors to chemotherapeutic agents. Akt also phosphorylates Cdc25B on Ser353, resulting in its cytoplasmic accumulation. Cdc25B undergoes activation during S phase and plays a role in activating the mitotic kinase Cdk1/cyclin B in the cytoplasm. In relocating Cdc25B to the cytoplasm, Akt regulates its function and participates in controlling the entry of cells into mitosis. The identification of Akt as a key regulator of cellular survival also has significant implications for oncogenesis. A number of oncogenes and tumor suppressor genes that function upstream of Akt influence cancer progression by regulating Akt. Akt is expressed to various degrees in breast cancer cell lines and has been shown to be important in estrogen-stimulated growth. Treatment of multiple myeloma cell lines with the Akt inhibitor, 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (Calbiochem Cat. No. 124005), results in reduced survival of both drug resistant and drug sensitive cells. Akt plays a critical role in tumorigenesis, becoming activated when tumor suppressors such as p27Kip1 and PTEN lose their functions. Phosphorylation of p27 at Thr157 by Akt impairs its nuclear import and leads to its cytoplasmic accumulation. In human breast cancers, cytoplasmic mislocalization of p27 has been strongly linked to loss of differentiation and poor patient outcome. Akt is also reported to physically associate with endogenous p21, a cell cycle inhibitor, and phosphorylate it at Thr145, causing its localization to the cytoplasm and subsequent degradation. Akt and p53 play opposing roles in signaling pathways that determine cell survival and the interaction between these two molecules is becoming an important area of study. Under conditions where the apoptotic effect of p53 is dominant, destruction of Akt plays a role in accelerating the apoptotic process. In apoptosisprone cells, p53-dependent signaling enables downregulation of Akt, which predisposes cells
14
p27 p27 Mdm2 Mdm2 RTK PI-3 Kinase IKK IkB p50 p65 p Nuclear import Nuclear import p53 3 Mdm2 P Proteasomal degradation Cytoplasmic retention
Growth factor
Survival
PIP2
IKK PTE PTEN PTEN TEN T Activated Akt C Caspase-9
IkB p50 p65 p p50 p65 N NF-kB
Survival
PIP3 Akt
Apoptosis Caspase-9 Survival
PDK1 Other h kinases
Fo Forkhead o Forkhead
FasL transcription
Caspase a activation
Apoptosis Survival
mTOR Rictor
Bad Apoptosis Bad

14 -3 -3
Survival
GSK3
Cyclin D
Proteolytic d degradation Glycogen synthase activation
C Cell cycle arrest
GSK3
p21 p21 C Cytoplasmic retention
Cell cycle arrest
Apoptosis Survival
degradation
Multiple roles of activated Akt in cell survival and death
4 | Akt Signaling in Cell Death and Cell Survival
to rapid apoptosis in response to stress signals. Under certain circumstances Akt activation may overcome the death promoting effects of p53 and may rescue cells from apoptosis. It has been reported that Akt can phosphorylate the murine double minute-2 oncoprotein (Mdm2) on Ser166 and Ser188 and promote its translocation to the nucleus where it destabilizes p53 and enhances its degradation via the proteasomal pathway.
References: Feng, J., et al. 2004. J. Biol. Chem. 279, 41189. Kawakami, Y., et al. 2004. J. Biol. Chem. 279, 47720. King F.W., et al. 2004. Cell Cycle 3, 634. Franke, T.F., et al. 2003. Oncogene 22, 8983. Zhou, B-B. S., and Sausville, E.A. 2003. Prog. Cell Cycle Res. 5, 413. Cantley, L.C. 2002. Science 296, 1655. Graff, J.R. 2002. Expert Opin. Ther. Targets 6, 103. Hill, M.M., and Hemmings B.A. 2002. Pharmacol. Therap. 93, 243.
Liang, J., et al. 2002. Nat. Med. 8, 1153. Mitsiades, C.S., et al. 2002. Oncogene 21, 5673. Shiojima, I., and Walsh, K. 2002. Circ. Res. 90, 1243. Whiteman, E.L., et al. 2002. Trends Endocrinol. Metab. 13, 444. Yang, J., et al. 2002. Nat. Struct. Biol. 9, 940. Zhou, B.P., and Hung, M.C. 2002. Semin. Oncol. 29, 62. El-Deiry, W.S. 2001. Nat. Cell Biol. 3, E71. Martin, D., et al. 2001. J. Neurochem. 78, 1000. Mayo, L.D., and Donner, D.B. 2001. Proc. Natl. Acad. Sci. USA 98, 11598. Somervaille, T.C.P., et al. 2001. Blood 98, 1374. Dimmeler, S., and Zeiher, A.M. 2000. Circ. Res. 86, 4. Obata, T., et al. 2000. J. Biol. Chem. 275, 36108. Toker A., and Newton, A.C. 2000. J. Biol. Chem. 275, 8271. Datta, S.R., et al. 1999. Genes Dev. 13, 2905. Kandel E.S., and Hays, N. 1999. Exp. Cell Res. 253, 210. Ozes, O.N., et al. 1999. Nature 401, 82. Peterson, R.T., and Schreiber, S.L. 1999. Curr. Biol. 9, R521. Sabbatini P., and McCormick, F. 1999. J. Biol. Chem. 274, 24263. Alessi, D.R. et al. 1996. EMBO J. 15, 6541.
15
Chapter Five
Receptor and Non-Receptor Protein Tyrosine Kinases
Although tyrosine phosphorylation by tyrosine kinases accounts for only about 0.1% of all protein phosphorylation in mammalian cells, tyrosine kinases have been recognized as key players in cellular regulation. Protein tyrosine kinases (PTKs) catalyze the transfer of the -phosphoryl group from ATP to tyrosine hydroxyls of proteins.
Phosphorylation of tyrosine residues modulates enzymatic activity and creates binding sites for the recruitment of downstream signaling proteins. PTKs are involved in regulating signaling processes in a wide variety of cellular functions, including cell
5 | Receptor and Non-Receptor Protein Tyrosine Kinases
growth, proliferation, migration, and differentiation. Unregulated activation or over expression of PTKs has been linked to various forms of cancers and benign proliferative conditions. Abnormal PTK signaling has also been reported in diabetes, inflammation, and other diseases. PTKs have been classified as receptor PTKs and non-receptor PTKs. Thus far 59 receptor PTKs and 32 non-receptor PTKs have been identified. Receptor PTKs contain a single polypeptide chain with a transmembrane segment. The extracellular end of this segment contains a high affinity ligand-binding domain, while the cytoplasmic end comprises the catalytic core and the regulatory sequences. The cytosolic end also contains tyrosine residues that become the substrates or targets for the tyrosine kinase portion of the receptor. The transmembrane domain anchors the receptor in the plasma membrane, while the extracellular domain binds the growth factor. The extracellular domain also has one or more identifiable structural motifs, which include cysteine-rich regions, immunoglobulin-like domains, EGF-like domains, glycine-rich
17
regions, leucine-rich regions and other motifs. The receptor PTK remains inactive until a ligand binds to the receptor, which leads to the dimerization of two ligand-bound receptors (exception- insulin receptor is a tetramer). Once activated, receptors are able to autophosphorylate tyrosine residues outside the catalytic domain. This autophosphorylation stabilizes the active receptor conformation and creates phosphotyrosinedocking sites for proteins that transduce signals within the cell. The unphosphorylated receptor lacks the accurate conformation for recognition. The cytosolic portion of phosphorylated receptor is capable of recruiting a number of cytosolic adapter proteins via interactions between phosphorylated tyrosine residues on the receptor and the SH2 (Src homology 2) domain on the adapter molecule. SH2 domains contain a stretch of amino acids, which can recognize phosphotyrosines on the receptor. Several signaling proteins, such as Ras-GAP, PI 3-kinase, and phospholipase C, can bind to the intracellular domain of receptor PTKs in a phosphotyrosinedependent manner. It is worth noting here that different proteins have different SH2 domains that recognize specific phosphotyrosine residues. Thus, it is possible that the same SH2-containing protein is activated by different growth factors. An SH2-containing protein, Grb2, acts as a common adapter protein in a majority of growth factor related signaling events.
Sos, a GDP-GTP exchange protein. This binding displaces an inhibitory domain in Sos. Activated Sos translocates to the plasma membrane where it activates Ras by inducing it to exchange GDP for GTP. A wide variety of effectors of Ras activation have been reported, however, activation of Raf, a cytoplasmic protein kinase, is one of the beststudied example. Ras binds to the N-terminus of Raf and recruits it to the inner surface of the plasma membrane, where it is phosphorylated by protein kinase C. Translocation of Raf to the membrane positions it in direct proximity of MAP kinase kinase (MEK; MAPKK). Raf phosphorylates MEK, which in turn phosphorylates MAP kinase (MAPK). In a resting cell MAPK remains inactive because its phosphorylation lip excludes ATP access to the binding pocket. However, MEK binding destabilizes the lip thereby exposing the buried tyrosine residues. Phosphorylation of the exposed tyrosine and nearby threonine residue causes the lip to alter its conformation allowing ATP binding. The only exception to the above activation scheme is the activation of insulin receptor. The insulin receptor is already dimerized and consists of two extracellular subunits that bind insulin and two transmembrane subunits, which possess tyrosine kinase activity. When subunits bind insulin, a conformational change occurs that activates the tyrosine kinase. Each subunit then phosphorylates tyrosine residues on the other subunit and cytoplasmic proteins known as insulin receptor substrates. Our discussion so far was limited to receptors with intrinsic tyrosine kinase activities. However, in some cases the receptor and the tyrosine kinase are two separate proteins. Such tyrosine kinases are referred to as the non-receptor or cellular PTKs. They include members of the Src, Tec, JAK, Fes, Abl, FAK, Csk, and Syk families. They are located in the cytoplasm as well as in the nucleus and play important roles in cellular signaling. They are also activated by a large number of stimuli, including hormones, neurotransmitters, growth factors, and cytokines. While there is a striking homology in the catalytic sequences of these enzymes, they diverge greatly in their
Grb2 binding to phosphotyrosine residues changes its conformation and allows it to bind to prolinerich sequences in the carboxy terminal tail of
18
regulatory and non-enzymatic sequences. They exhibit distinct kinase regulation, substrate phosphorylation, and function. For example, Src is involved in cell differentiation, whereas Abl is involved in growth inhibition, and FAK activity is linked with cell adhesion. Deregulation of these kinases has also been linked to several human diseases. For example, oncogenic forms of Abl, JAK, and Src kinases have been reported in several human cancers and are shown to be involved in carcinoma development. Unlike the receptor PTK, not much is known about the mechanism of activation of non-receptor PTKs. In most cases, their activation also begins with the phosphorylation of a tyrosine residue present in an activation loop. The best studied enzymes in this group include Src kinases. Src is believed to be negatively regulated by phosphorylation at Tyr527 present at the C-terminus by Csk and other cellular kinases. The enzyme assumes an inactive conformation when this phosphotyrosine is bound by the Src SH2 domain in an intramolecular fashion. In this structure, the Src SH3 domain interacts with a single proline, Pro250, in the linker region between the SH2 and catalytic domain. In contrast to Src activation, c-Abl kinase activity is stimulated by phosphorylation of a catalytic domain tyrosine residue, Tyr412, either via autophosphorylation or via transphosphorylation by c-Src. Recent studies have indicated that dimerization or oligomerization of c-Abl might also be sufficient to activate Abl kinase activity in vivo. Syk, an essential enzyme for immune system development and function, is reportedly activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs). More recently, it is also shown to be activated by binding to the cytoplasmic tail of the integrin 3 receptor through its SH2 domain. Due to their involvement in various forms of cancers, PTKs have become prominent targets for therapeutic intervention. The clinical application of selected tyrosine kinase inhibitors for cancer treatment was a major therapeutic breakthrough. The rationale for using these inhibitors rests on the observation that tyrosine kinases are critical components of cellular signaling and
are frequently mutated or otherwise deregulated in malignancies. Quinazoline compounds, such as PD 168393 and PD156273 (Calbiochem Cat. No. 513033 and Cat. No. 513032, respectively) are highly potent and selective inhibitors of EGF receptor tyrosine kinase activity and are shown to block tumor cell growth both in vitro and in vivo. Selected indolinone compounds, such as SU6656 (Calbiochem Cat. No. 572635), have been shown to be potent and selective inhibitor of Src family of non-receptor tyrosine kinases. The design of specific inhibitors of tyrosine kinases is important both for fundamental research and for developing therapeutic strategies for the treatment of disorders such as cancer, atherosclerosis, psoriasis, and septic shock in which increased tyrosine kinase activity has been reported. Two classes of protein tyrosine kinase inhibitors have been developed. One class acts by binding to the ATP binding site and the other by binding to the substrate-binding site of the enzyme. Among the inhibitors that act at the ATP binding site, genistein is the most commonly used. One drawback of this class of inhibitors is that they exhibit greater cytotoxicity and cause nonspecific inhibition of serine/threonine kinases. Gazit and others have developed a series of synthetic compounds, tyrphostins (also known as AG compounds) that inhibit protein tyrosine kinases by binding to the substrate binding site. Structurally they resemble tyrosine and erbstatin moieties and have hydrophobic characteristics, which allow them to readily cross the cell membrane. Many of the tyrphostins have selective and distinct inhibitory activities in various tyrosine kinase assay systems. It is important to note that signals from various pathways may converge to bring about a desired effect in the cell in a synergistic manner. For example, the stimulation of cell proliferation may occur by a combination of receptor PTK activation, activation of non-receptor PTKs and G-proteincoupled receptors. Sub-threshold combinations of various ligands can act synergistically to stimulate the action of downstream effectors to fully promote a biological response.
19
5 | Receptor and Non-Receptor Protein Tyrosine Kinases
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References: Reiter, A., et al. 2007. Curr. Drug Targets 8, 205. Schmidt-Arras, D.E., et al. 2005. Mol. Cell. Biol. 25, 3690 Krause, D.S., and Van Etten, R.A. 2005. N. Engl. J. Med. 353, 172. Medinger, M., and Drevs, J. 2005. Curr. Pharm. Des. 11, 1139 Tibes, R., et al. 2005. Annu. Rev. Pharmacol. Toxicol. 45, 357. Mazitschek, R., and Giannis, A. 2004. Curr. Opin. Chem. Biol. 8, 432. Rowinsky E.K. 2003. Oncologist 8 (suppl. 3), 5. Martin, G.S. 2001. Nat. Rev. Mol. Cell Biol. 2, 467. Smith, K.M., and Van Etten, R.A. 2001. J. Biol. Chem. 276, 24372. Woodside, D.G., et al. 2001. Curr. Biol. 11, 1799. Sada, K. et al. 2001. J. Biochem. (Tokyo) 130, 177. Brasher, B.B., and Van Etten, R.A. 2000. J. Biol. Chem. 275, 35631 Plattner, R., et al. 1999. Genes Dev. 13, 2400. Manes, G., et al. 1999. Gene Ther. Mol. Biol. 4, 417. Stratowa, C., et al. 1999. Anticancer Drug Des. 14, 393. Kyriakis, J.M. 1999. J. Biol. Chem. 274, 5259. Pawson T., and Scott, J.D. 1997. Science 278, 2075. Lemmon M.A., and Schlessinger J. 1994. Trends Biochem. Sci. 19, 459. Taniguchi, T., et al. 1991. J. Biol. Chem. 266, 15790. Gazit, A., et al. 1989. J. Med. Chem. 32, 2344.
Chapter Six
MAP Kinase Signaling: Synergistic Response to Upstream Signals
The mitogen-activated protein (MAP) kinases are a group of evolutionarily conserved proline-directed protein serine/ threonine kinases that are activated in response to a variety of extracellular stimuli and mediate signal transduction from the cell surface to the nucleus. They regulate several physiological and pathological cellular phenomena, including inflammation, apoptotic cell death, oncogenic transformation, tumor cell invasion, and metastasis.
In combination with several other signaling pathways, they can differentially alter the phosphorylation status of transcription factors. The controlled regulation of MAP kinase cascades is involved in cell proliferation and differentiation, whereas an unregulated activation can result in oncogenesis. Although MAP kinases are expressed in all cell types, they regulate very specific biological responses that differ from cell type to cell type. Four major types of MAP kinase cascades have been reported in mammalian cells that respond synergistically to different upstream signals. MAP kinases are part of a three-tiered phospho-relay cascade consisting of MAP kinase, a MAP kinase kinase (MEK or MKK) and a MAP kinase kinase kinase (MEKK or MKKK). In this relay system MKKKs phosphorylate and activate MEKs, which in turn phosphorylate and activate MAPKs. MEKs are dual specificity enzymes that phosphorylate hydroxyl side chains of serine/threonine and tyrosine residues in their MAP kinase substrates. MEKs are activated by phosphorylation of serine or threonine residues in their activation loop. There are at least 11 MAPKs, but only 7 MEKs, and at least 20 MKKKs. It is this diversity of MKKKs that allow
6 | MAP Kinase Signaling: Synergistic Response to Upstream Signals
21
the integration of specific MAPK pathways in the cellular response to diverse stimuli, including cytokines, growth factors, drugs, and stress signals. Controlled regulation of these cascades is involved in cell proliferation and differentiation, whereas unregulated activation of these MAP kinases can result in oncogenesis. The most widely studied cascade is that of ERK1/ERK2 (p44/p42) MAP kinases. A general activation scheme involves the activation of receptor tyrosine kinases by growth factors, such as EGF, which provides the binding site for the adapter protein Grb2 which in turn localizes Sos to the plasma membrane. Sos activates Ras by exchange of GTP for GDP. The Ras- GTP binds directly to a serine/threonine kinase, Raf, forming a transient membrane anchoring signal. Raf family is composed of A-Raf, B-Raf, and Raf-1 (C-Raf). Of these Raf-1 is most widely studied and is stabilized in association with 14-3-3 protein in low or high activity conformation. Raf is present in cells as a
multi protein complex of Raf, Hsp90, and 14-3-3. Active Raf kinase phosphorylates a dual specificity kinase, MEK, on Ser218 and Ser222 and activates it. MEK can also be phosphorylated by Mos, a protein kinase expressed during meiotic maturation of oocytes and by MEKK1. A generally held belief is that MEK1 binds ERK and phosphorylates either a threonine or a tyrosine residue and then dissociates. The monophosphorylated ERK then rebinds to an active MEK1 for dual phosphorylation and complete activation. The activated MEK phosphorylates ERK1/ERK2 on Thr183 and Tyr185 (at the TEY motif). The major targets of activated ERKs are p90 ribosomal S6 kinase (Rsk) and the cytoplasmic phospholipase A2. ERK also translocates to the nucleus to phosphorylate the transcription factor Elk-1 (on Ser383 and Ser389). Recently another related kinase, ERK3, a nuclear protein kinase, has been cloned. It exhibits about 50% homology with ERK1/ERK2 within its catalytic domain. However, it does not phosphorylate any typical ERK substrates.
Map Kinase signaling cascade
22
Usually only one highly active form of ERK1 or ERK2 (dual phosphorylated) exists in the cell, exhibiting over 1000-fold greater activity than the unphosphorylated form. Within the cell, at any time, one may find three less active forms of ERKs: one unphosphorylated enzyme, and two singly phosphorylated forms that contain phosphate either at the tyrosine or threonine residue. The JNK/SAPK (c-Jun kinase/stress activated protein kinase) cascade is activated following exposure to UV radiation, heat shock, or inflammatory cytokines. The activation of these MAP kinases is mediated by Rac and Cdc42, two small G-proteins. The activated Cdc42 binds to PAK65 protein kinase and activates it. The activated PAK65 can activate MEKK which in turn phosphorylates and activates SEK/JNKK at Ser219 and Ser223. The active SEK/JNKK phosphorylates JNK/SAPK (at the TPY motif) that in turn binds to the N terminal region of c-Jun and phosphorylates it at Ser63 and Ser73. The sites of activating phosphorylation are conserved between ERK and JNK, however, these sites are located within distinct dual specificity phosphorylation motifs (TPY for JNK and TEY for ERK). Molecular cloning studies show about 40 - 45% sequence homology between JNK/SAPK and the classical MAP kinases. The p38 kinase, another member of the MAP kinase family, bears similarity to the yeast MAPK, Hog-1. Mammalian cells contain four different isoforms of the p38 MAPK. They are known as p38 SAPK2a), p38 SAPK2b), p38 SAPK3), and p38 SAPK4). p38 is the most physiologically relevant and best characterized isoform - it is ubiquitous in its distribution. p38 is found mainly in the testes, pancreas, small intestine, as well as in CD4+ T cells and p38 is expressed in muscle. p38 MAPKs are activated in response to inflammatory cytokines, endotoxins, and osmotic stress. They share about 50% homology with ERKs. The upstream steps in their activation are not well defined. However, downstream activation of p38 occurs following its phosphorylation (at the TGY motif) by MKK3, a dual specificity kinase. Following its activation, p38 translocates to the nucleus and phosphoryates ATF-2. Other known targets of p38 are MAPKAPK2 that is involved in the phosphorylation and activation of heat-
shock proteins and MNK1 and MNK2. p38 also phosphorylates a number of downstream MAPKactivated protein kinases, such as RSK and MSK. The fourth and least studied mammalian MAP kinase pathway is big MAP kinase 1 (BMK1), also known as extracellular signal regulated kinase 5 (ERK5). BMK1 is activated in response to growth factors and stress. Activation of the BMK1 signaling pathway has not only been implicated in normal cell survival, cell proliferation, cell differentiation, but also in pathological states such as carcinogenesis, cardiac hypertrophy, and atherosclerosis. BMK1 can be activated following exposure to EGF, BDNF, NGF, VEGF, FGF-2, phorbol esters, and oxidative stress. The signaling molecules in the ERK5 cascade include MEKK2/3, MEK5, and ERK5. In spite of its similarities to MEK1 and MEK2, MEK5 is not phophorylated or activated by Raf-1. Since BMK1 is the only known substrate of MEK5, all effects of MEK5 have been attributed to its ability to activate BMK1. Thus far, myocyte enhancer factor 2 (MEF2), Ets-domain transcription factor (Sap1a), Bad, and serum- and glucocorticoid-inducible kinase (SGK) have been identified as substrates for BMK1. Although different MAP kinase cascades show a high degree of specificity and functional separation, some degree of cross-talk is observed between these pathways. For example, JNKK, an activator of JNK/SAPK, is reported to activate p38, whereas MKK3 activates only p38 and not JNK/SAPK. MEKK1, which stimulates SEK/JNKK1 in the JNK/SAPK cascade, has only a trivial effect on p38 activation. In the upstream signaling, Sos stimulates only the ERK pathways without affecting either JNK or p38 cascade. Another important observation is that in mammalian cells treated with mitogenic agents, ERKs are significantly activated whereas JNK/ SAPK are not affected. Conversely, cells exposed to stress activate only the JNK/SAPK pathway without altering the activity of ERKs. At the transcription level, ATF-2 is phosphorylated and activated by all three MAP kinases, whereas c-Jun and Elk-1 are phosphorylated only by ERKs and JNK/SAPK, yet all these pathways result in transcriptional activity that is unique for a particular external stress.
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6 | MAP Kinase Signaling: Synergistic Response to Upstream Signals
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References: Hitti, E., and Kotlyarov, A. 2007. Anti-Inflammatory Anti-Allergy Agents in Med. Chem. 6, 85-97 Johnson, G.L. et al. 2005. Curr. Opin. Chem. Biol. 9, 325. Wong, C. H., et al. 2005. Dev. Biol. 286, 1. Hayashi, M., and Lee, J.D. 2004. J. Mol. Med. 82, 800. Murry, J. A., 2003. Curr. Opin. Drug. Discov. Develop. 6, 945. Pearson, G., et al. 2001. Endocrine Rev. 22, 153. Burack, W.R., and Sturgill, T.W. 1997. Biochemistry 36, 5929. Sagata, N. 1997. BioEssays 19, 13. Sivaraman, V.S., et al. 1997. J. Clin. Invest. 99, 1478. Tong, L., et al. 1997. Nat. Struct. Biol. 4, 311. Su, B., and Karin, M. 1996. Curr. Opin. Immunol. 8, 402. Cheng, M., et al. 1996. J. Biol. Chem. 271, 8951. Cobb, M.H., et al. 1996. Adv. Pharmacol. 36, 49. Das, R., and Vonderhaar, B.K. 1996. Breast Cancer Res. Treat. 40, 141. Moriguchi, T., et al. 1996. Adv. Pharmacol. 36, 121. Winston, L.A., and Hunter, T. 1996. Curr. Biol. 6, 668. Larochelle, S., and Suter, B. 1995. Gene 163, 209. Lin, A., et al. 1995. Science 268, 286. Martin, G.A., et al. 1995. EMBO J. 14, 1970. Raingeand, J., et al. 1995. J. Biol. Chem. 270, 7420. Waskiewicz, A.J., and Cooper, J.A. 1995. Curr. Opin. Cell Biol. 7, 798. Avruch, J., et al. 1994. Trends Biochem. Sci. 19, 279. Davis, R.J., 1994. Trends Biochem. Sci. 19, 470. Han, J., et al. 1994. Science 265, 808. Kyriakis, J.M., et al. 1994. Nature 369, 156. Hibi, M., et al. 1993. Genes Dev. 7, 2135.
Chapter Seven
Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential
Protein phosphorylation and dephosphorylation serve as molecular switches in signal transduction pathways. A variety of cellular processes, including gene expression, cell growth and differentiation are controlled by the complex interplay of
7 | Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential
protein kinases and phosphatases. Any malfunctioning in these switches may lead to cancer, apoptosis, or neurodegenerative disease. Hence, clinical manipulation of these reactions has become a major therapeutic target in the management of disease.
Protein kinase C (PKC), a ubiquitous, phospholipid dependent serine/threonine kinase, is involved in signal transduction associated with cell proliferation, differentiation, and apoptosis. At least eleven closely related PKC isozymes have been reported that differ in their structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity. They are classified as conventional (, 1, 2, ), novel (, , , , ), and atypical (, ) PKC isozymes. Conventional PKC isozymes are Ca2+-dependent, while novel and atypical isozymes do not require Ca2+ for their activation. All PKC isozymes, with the exception of and , are activated by diacylglycerol (DAG), a product of receptor-mediated hydrolysis of inositol phospholipids. Although specific functions of individual PKC isozymes and of the molecular pathways through which they exert their divergent effects are not well understood, their roles in cell growth and proliferation remain undisputed. PKC isozymes negatively or positively regulate critical cell cycle transitions, including cell cycle entry and exit and the G1 and G2 checkpoints.
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The catalytic domain of PKC contains sequences, including an ATP-binding site, which resemble other protein kinases. The regulatory domain of PKC contains a Ca2+ binding site that is found only on , , and -isozymes. The amino terminal half of the regulatory domain of the , , and -isozymes contains two conserved regions, C1 and C2 that play a vital role in the regulation of
enzyme activity. The catalytic domain is composed of highly conserved C3 and C4 regions. The C3 region contains the ATP-binding consensus sequence, whereas the C4 region is responsible for protein substrate binding. Other PKC isozymes (, , , , , and ) lack the C2 region and do not require Ca2+ for activation.
Tissue and Cell Specific Distribution of Protein Kinase C Isozymes

Tissue/Cells Brain Fibroblasts Heart Intestine Kidney + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Liver Ling B lymphocytes T lymphocytes Neutrophils Ovary Pancreas Placenta Platelets Retina Smooth muscle Spleen Testis Thymus Uterus PKC + + + PKC1 + + + PKCII + + + PKC + PKC + + + + + + + + + + + + + + + + + + + + + + PKC + + + + PKC + PKC + + + + + + + + + PKC + PKC +
+ = present; - = absent; blank = unknown
In its unstimulated state most of the PKC resides in the cytosol. In this state, the pseudosubstrate sequence of the regulatory domain of PKC interacts with the catalytic domain and prevents access of the substrate to the catalytic site. Binding of a hormone or other effector molecule to the membrane receptor results in activation of phospholipase C (PLC) or phospholipase A2 (PLA2) via a G-protein-dependent phenomenon. The activated PLC hydrolyzes phosphatidylinositol4,5-bisphosphate (PIP2) to produce DAG and inositol-1,4,5-trisphosphate (IP3). The IP3 causes the release of endogenous Ca2+ that binds to the cytosolic PKC and exposes the phospholipidbinding site. The binding of Ca2+ translocates PKC
to the membrane, where it interacts with DAG and is transformed into a fully active enzyme. Arachidonic acid released by PLA2 action also activates cytosolic PKC. Sustained activation or inhibition of PKC plays a critical role in cell proliferation, differentiation, and tumorigenesis. Altered PKC activity has been linked with various types of malignancies. Higher levels of PKC and differential activation of various PKC isozymes have been reported in breast tumors, adenomatous pituitaries, thyroid cancer tissue, leukemic cells, and lung cancer cells. Specifically, over expression of PKC and 2 has been linked to uncontrolled cell growth and transformation.
26
7 | Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential
Protein Kinase C signaling
On the other hand, overexpression of PKC , 1, and in certain carcinoma cell lines and in nontransformed intestinal epithelial cells, is reported to retard cell growth and inhibit tumorigenicity. Downregulation of PKC is also reported in a majority of colon adenocarcinomas and in the early stages of intestinal carcinogenesis. Hence, any mutations involving PKC isozymes may lead to perturbations in cellular functions and susceptibility to tumor development. PKC inhibitors, such as tamoxifen and safingol, are currently being used in the treatment of breast cancer. More recently, the chemotherapeutic
potential of antioxidants, which react with the cysteine residues in the catalytic domain of PKC and inhibit its activity, has also been considered. The involvement of PKC in the regulation of apoptosis adds yet another dimension to the effort in developing drugs that will specifically target PKC. PKC inhibitors are often used to induce apoptosis in a variety of cell lines. Studies in which cells are treated only with PKC inhibitors have shown that the active form of PKC shields cells from apoptosis. However, the question as to whether PKC is a positive or a negative regulator of apoptosis is still controversial and requires further research.
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References: Gould, C.M., and Newton, A.C. 2008. Curr. Drug Targets 9, 614. Vignot, S., et al. 2008. Bull. Cancer 95, 683. Breitkreutz, D., et al. 2007. J. Cancer Res. Clin. Oncol. 133, 793. Yamaguchi, T., et al. 2006 Allergol. Int. 55, 245. Pickett, C.A., et al. 2002. Mol. Endocrinol. 16, 2840. Black, J.D. 2000. Front. Biosci. 5, 406. Musashi, M., et al. 2000. Int. J. Hematol. 72, 12. Gopalakrishna, R., and Jaken, S. 2000. Free Radic. Biol. Med. 28, 1349. Cooper, D.R., et al. 1999. Arch. Biochem. Biophys. 372, 69. Yamamoto, M., et al. 1998. Exp. Cell Res. 240, 349. Rasmussen, H., et al. 1995. Endocr. Rev. 16, 649. Taylor, S.S., et al. 1995. FASEB J. 9, 1255. Nishizuka, Y. 1995. FASEB. J. 9, 484. Newton, A.C. 1995. J. Biol. Chem. 270, 28495. Hanks, S., and Hunter, T. 1995. FASEB J. 9, 576. Blobe, G.C., et al. 1994. Cancer Metastasis Rev. 13, 411. Basu, A. 1993. Pharmacol. Ther. 59, 257. Gopalakrishna, R., et al. 1992. Biochem. Biophys. Res. Commun. 189, 950. Walaas, S.I., and Greengard, P. 1991. Pharmacol. Rev. 43, 299.
Chapter Eight
Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease
Glycogen synthase kinase-3 (GSK-3), a multifunctional serine/ threonine kinase, is a key regulator of numerous signaling pathways during embryogenesis and in metabolic control. Two isoforms of GSK-3 are reported in mammals: a 51 kDa GSK3 and a 47 kDa GSK-3. The GSK-3 contains a glycine-rich extension at its N-terminal. These two isoforms exhibit about 98% homology in their kinase domains, but share only about 36% identity in the last 76 C-terminal amino acid residues.
A minor (~15% of total) splice variant of GSK-3, GSK-32, has also been identified, which contains a 13-residue insert within the kinase domain. It exhibits reduced kinase activity towards the tau protein compared with unspliced GSK-3. GSK-32 is localized primarily to neuronal cell bodies, unlike unspliced GSK-3 that is also found in neuronal processes. GSK-3 is constitutively active in cells and is regulated through inhibition of its activity. GSK-3 shows a preference for target proteins that are pre-phosphorylated at a `priming residue located C-terminal to the site of GSK-3 phosphorylation. Priming phosphorylation, although not absolutely required, enhances the efficiency of phosphorylation of most GSK-3 substrates. Phosphorylation of a threonine (Thr) residue in the activation loop (T-loop) is considered to be essential for several protein kinases, such as Cdk2, p38, and ERK2 that are closely related to GSK-3. This phosphorylation at Thr is also required by p38 and ERK2 to open up the catalytic site for substrate access. The T-loop of GSK-3 is phosphorylated at Tyr279 and GSK-3 at Tyr216, which play a role in forcing open the substrate-binding site of the
8 |
29
Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease
enzyme. Uniquely, the T-loop of GSK-3 does not undergo any Thr phosphorylation. The function of missing pThr in the T-loop of GSK-3 is carried out by the phosphorylated residue of a primed substrate that binds to a positively charged pocket consisting of Arg96, Arg180, and Lys205 (for GSK3). This arrangement optimizes the orientation of the kinase domain and places the substrate at the proper position within the catalytic groove for phosphorylation to take place. Arg96, Arg180, and Lys205 are conserved in all GSK-3 homologs, indicating that the priming phosphate-binding site is well conserved in all organisms. GSK-3 is constitutively active in resting cells and treatment of cells with an agent, such as insulin, causes GSK-3 inactivation through a PI 3-kinase (PI 3-K)-dependent mechanism. PI 3-K-induced activation of PKB/Akt results in phosphorylation of Ser21 on GSK-3 and Ser9 on GSK-3, which inhibit GSK-3 activity. The phosphorylated N-terminus becomes a primed pseudosubstrate that occupies the positive binding pocket and the active site of the enzyme, and acts as a competitive inhibitor for true substrates. This prevents phosphorylation of substrates. An Arg96 is shown to be a crucial component of the positive pocket that binds primed substrates. Arg96 to Ala96 mutation disrupts the pocket in a way that primed substrates can no longer bind and, hence, the enzyme remains active. Also, Ser9-phosphorylated pseudosubstrate is no longer capable of inactivating the enzyme. Small molecule inhibitors that fit in the positively charged pocket of the kinase domain of GSK-3 are useful for selectively inhibiting primed substrates. Several known GSK-3 substrates participate in a wide spectrum of cellular processes, including glycogen metabolism, transcription, translation, cytoskeletal regulation, intracellular vesicular transport, cell cycle progression, and apoptosis. Phosphorylation of these substrates by GSK-3 usually has an inhibitory effect. GSK-3 plays a key inhibitory role in the Wnt signaling pathway. Wnt genes encode a large family of secreted, cysteine-rich proteins that are important in development and in maintenance of adult tissues. Abnormalities in Wnt signaling are reported to promote both human degenerative diseases and cancer. Several groups have shown that -catenin is a primed substrate for GSK3, with casein kinase I (CKI) acting as the
priming kinase. In this capacity CKI functions as a negative regulator of Wnt signaling since it promotes GSK-3 function. In unstimulated cells, CKI phosphorylates -catenin on Ser45, priming it for further phosphorylation on Ser41, 35, and 33 by GSK-3 in a sequential manner, thereby allowing -catenin to be ubiquitinated for proteasomal degradation. It has been suggested that ankyrin repeat protein, Diversin, may help recruit CKI to the destruction complex. Wnt stimulation activates the receptor Frizzled, which then signals through Dishevelled (Dvl) to inactivate -catenin phosphorylation. Unphosphorylated -catenin translocates to the nucleus where it transactivates genes regulated by TCF/LEF transcription factors. Another key player in the regulation of the Wnt signaling pathway is GBP/FRAT, a GSK-3binding protein. Binding of GBP/FRAT to GSK-3 prevents GSK-3 from binding to axin and thus it interferes with -catenin phosphorylation. GBP/ FRAT also plays a significant role in the nuclear export of GSK-3. This suggests that GBP/FRAT may be involved in regulating the access of GSK-3 to substrates that are partitioned between the nucleus and the cytoplasm. Any mutation that prevents the binding of GSK-3 to GBP/ FRAT causes nuclear localization of GSK-3. A small peptide derived from FRAT, FRATtide, is reported to prevent axin-GSK-3 interaction and thereby block phosphorylation of both axin and -catenin. In the Wnt signaling pathway GSK-3 appears to be insulated from regulators of GSK3 that lie outside of the Wnt pathway. Insulin signaling that leads to inhibition of GSK-3 via phosphorylation at either Ser9 or Ser21 does not cause accumulation of -catenin. Mutations in -catenin that prevent its phosphorylation by GSK-3 are common in skin, colon, prostate, liver, endometrial, and ovarian cancers. GSK-3 also plays a significant role in Hedgehog signaling. Sonic Hedgehog (Shh) has been implicated in several embryonic developmental processes and it displays inductive, proliferative, neurotrophic, and neuroprotective properties. Response to Shh signaling is controlled by two transmembrane proteins, the Patched (Ptc), a twelve-span transmembrane protein, and Smoothened (Smo), a seven transmembrane receptor protein. Ptc acts an inhibitor of Smo activation. Binding of Shh to Ptc lifts the inhibitory effect on Smo, leading to the activation of the Shh signaling cascade. Wnt and Shh often
30
work in concert to set the embryonic development pattern. The Wnt pathway uses -catenin to transduce its signals to the nucleus, whereas the Shh pathway employs a 155 amino acid protein, Cubitus interruptus (Ci155) in Drosophila or Gli in mammals. In vertebrates three Gli proteins (Gli1, Gli2, and Gli3) have been reported, Gli1 and Gli2 function primarily as activators and Gli3 has a
repressor role. In the absence of any Shh signal, Ci is targeted for proteolysis, which generates a truncated 75-residue amino acid form (Ci75), which functions as a transcriptional repressor. GSK-3, in combination with CKI and the priming PKA, phosphorylates Ci155 (and probably Gli) and targets it for proteolytic processing in the absence of a Shh signal. Activation of Shh signaling results
Shh Wnt Fzl LRP G Ptc Smo Ptc
CKI CKI Diversin Dvl FRAT Diversin Axin CKI APC PKA GSK-3 Ci155/Gli
GSK-3
| Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease
-catenin Ub Axin APC CKI Dvl Ub -catenin Ub Ub sufu FRAT GSK-3 (inactive) Gli
Proteasome
-catenin
Lef/Tcf responsive Gli responsive gene transcription gene transcription RNA pol -catenin Lef/Tcf Dyrk1 CREB Gli
GSK-3,Wnt and Shh signaling
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in translocation of Ci155/Gli to the nucleus, where it activates Hh target genes. Although GSK-3 phosphorylation of the mammalian homologs of Ci has yet to be reported, they all contain multiple GSK-3 consensus sites next to PKA sites. Supressor of Fused (SuFu) interacts directly with Gli proteins, repressing Shh signaling while Dyrk1 acts by a distinct pathway to stimulate Gli1 activation of transcription. Over signaling by Shh appears to be involved in the initiation and propagation of some tumors of the muscle, skin, and nervous system. GSK-3 is also involved in the pathogenesis of Alzheimers disease. Both GSK-3 and are activated in the brain of parental APP-V717I amyloid mice. Even in very young mice, before any amyloid deposits are detected, higher GSK-3 activities can be observed. Activated GSK-3 contributes to multi-site hyperphosphorylation of tau proteins that form neurofibrillary tangles in Alzheimers disease. Abnormalities in pathways that use GSK-3 as a regulator have been linked to several disease conditions. Hence, GSK-3 has emerged as a potential therapeutic target, particularly in noninsulin-dependent diabetes mellitus, Alzheimers disease, developmental disorders, and cancer. Several new GSK-3 inhibitors have recently been developed, most of which act in an ATP competitive manner. Inhibitors belonging to aloisines, the paullones, and the maleimide families, have shown promise as therapeutic agents. Due to its involvement in multiple pathways, selectivity of GSK-3 inhibition is an important factor in developing inhibitors for therapeutic applications.
References: Terwel, D., et al. 2008. Amer. J. Pathol. 172, 786. Rankin, C.A., et al. 2007. Mol. Neurodegenetaion 2, 12. Amerongen R.V., et al. 2005. Genes Dev. 19, 425. Jiang, H., et al. 2005. Cell 120, 123. Mill, P., et al. 2005. Dev. Cell 9, 293. Beachy, P.A., et al. 2004. Nature 432, 324. Cohen, P., and Goedert, M. 2004. Nat. Rev. Drug Discovery 3, 479. Meijer, L., et al. 2004. Trends Pharmacol. Sci. 25, 471. Doble, B.W., and Woodgett, J.R. 2003. J. Cell Sci. 116, 1175. Amit, S., et al. 2002. Genes Dev. 16, 1066. Fang, X., et al. 2002. Mol. Cell. Biol. 22, 2099. Huelsken, J., and Behrens, J. 2002. J. Cell Sci. 115, 3977. Jia, J., et al. 2002. Nature 416, 548. Jiang, J. 2002. Gene Dev. , 2315. Mukai, F., et al. 2002. J. Neurochem. 81, 1073. Price, M. A., and Kalderon, D. 2002. Cell 108, 823. Tanji, C., et al. 2002. J. Biol. Chem. 277, 36955. Ali, A., et al. 2001. Chem. Rev. 101, 2527. Dajani, R., et al. 2001. Cell 105, 721. Frame, S., et al. 2001. Mol. Cell 7, 1321. Frame, S., and Cohen, P. 2001. Biochem. J. 359, 1. Ingham, P. W., and McMahon, A. P., 2001. Genes Dev. 15, 3059. Sharpe, C., et al. 2001. Bioessays 23, 311. Taipale, J., and Beachy P.A. 2001. Nature 411, 349. Hoeflich, K. P., et al. 2000. Nature 406, 86. Polakis, P. 2000. Genes Dev. 14, 1837. Brown, N. R., et al. 1999. Nat. Cell Biol. 1, 438. Thomas, G. M., et al. 1999. FEBS Lett. 458, 247. Brady, M. J., et al. 1998. J. Biol. Chem. 273, 14063. Canagarajah, B. J., et al. 1997. Cell 90, 859. Cross, D. A., et al. 1995. Nature 378, 785. Woodgett, J. R. 1990. EMBO J. 9, 2431.
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Chapter Nine
Organizational and Functional Diversity of Protein Phosphatases
Phosphorylation and dephosphorylation of structural and regulatory proteins are major intracellular control mechanisms in eukaryotes. Protein Kinases transfer a phosphate from ATP to a specific protein, typically at serine, threonine, or tyrosine residues.
The covalent attachment of a phosphoryl group changes the conformation of the protein and alters its ability to interact with a ligand. Phosphatases remove the phosphoryl group and restore the protein to its original dephosphorylated state. Hence, the phosphorylation-dephosphorylation cycle can be regarded as a molecular on-off switch. In contrast to the large number of protein kinases that have been discovered and studied in detail, relatively few protein phosphatases have received similar attention.
9 | Organizational and Functional Diversity of Protein Phosphatases
Protein phosphatases (PPs) have been classified into three distinct classes: serine/threonine (Ser/Thr)-specific, tyrosine-specific, and dual-specific phosphatases. Based on biochemical parameters, substrate specificity, and sensitivity to various inhibitors, Ser/Thr
33
protein phosphatases are divided into two major classes. Type I phosphatases, which include PP1, can be inhibited by two heat-stable proteins known as Inhibitor-1 (I-1) and Inhibitor-2 (I-2). Type II phosphatases are insensitive to heat-stable inhibitors and are subdivided into spontaneously active (PP2A), Ca2+-dependent (PP2B), and Mg2+dependent (PP2C) classes of phosphatases. PP1 and PP2A share about 43% overall sequence identity, whereas PP2C is structurally distinct and belongs to a completely different gene family. Both PP1 and PP2A have a complex holoenzyme structure and their catalytic subunits (PP1c and PP2Ac) share about 50% sequence homologies. Their catalytic subunits associate with a variety of regulatory units that assure the substrate specificity of these enzymes. In rat, cDNA cloning has shown the existence of at least four isoforms of PP1, termed , 1, 2, and . It is shown that bacterially expressed PP1c differs in some aspects from native PP1c. The native structure of PP1 is a 1:1 complex between the catalytic and a number of different regulatory subunits that are involved in targeting the catalytic subunit towards specific subcellular locations in order to increase its activity towards particular substrates. High levels of PP1 are found in the nuclei of eukaryotic cells. Over 90% of this activity is located in the chromatin fraction. Part of the nuclear PP1 is present in a latent form (PP1N) where PP1c is complexed to an inhibitory (NIPP-1) polypeptide. The latent PP1N can be activated in vitro through phosphorylation of NIPP-1 by either PKA or casein kinase II. This activation process can be reversed by the
action of PP2A. A cytosolic form of PP1, termed PP1s, consists of PP1c complexed to I-2. In vitro studies have shown that during purification PP1s gradually switches to an inactive conformation. Even after proteolysis of I-2 in this complex, PP1c remains in the inactive conformation. In vitro, phosphorylation of I-2 on Thr72 by GSK-3 initiates a reactivation process. Hence, it is suggested PP1s serves as a pool of inactive phosphatase, from which the catalytic subunit can be recruited into other subcellular compartments. PP2A consists of a 36 kDa catalytic subunit (PP2Ac), which is complexed with a regulatory subunit of 65 kDa (PR65). This dimer associates with variable regulatory subunits of 55 kDa (PR55), 72 kDa (PR72), or 130 kDa (PR130). These various regulatory subunits are believed to affect substrate specificity and the subcellular distribution of PP2A. The PR72 unit contains a potential nuclear localization signal in its primary sequence, which may help PP2A to translocate to the nucleus. The catalytic subunit of PP2A can be phosphorylated in vitro at the C-terminus Tyr307 by the tyrosine kinases p60v-src, p56lck, and by EGFRkinase, which reduces enzyme activity by over 90%. Dephosphorylation restores the catalytic activity of PP2A. Two isoforms of the catalytic subunit (, ), two isoforms of PR65 (, ), three isoforms of PR55 (, , ) and two isoforms of PR72 have been reported. Most PP2A subunits are ubiquitously expressed, however, PR55 and are mainly expressed in neuronal tissue and PR72 is found only in the muscle tissue.
Major Protein Phosphatases Involved in Signal Transduction

Protein Phosphatase PP1 PP2A PP2B Cytosol, nucleus, myofibrils, glycogen particles Cytosol, mitochondria, nucleus Cytosol, plasma membrane, synaptosome, nucleus G and M targeting subunits A and B subunits Calyculin A, Microcystins, Nodularin, Okadaic Acid, Endothall B subunit; calmodulin Cyclosporin A and FK 506/ Immunophilin complexes, Cypermethrin, Deltamethrin, Fenvalerate PTP Plasma membrane, nucleus various bpV(phen), Dephostatin, mpV(pic), DMHV, Sodium Orthovanadate Calyculin A, Nodularin, NIPP-1 Localization Regulatory Subunits Potent Inhibitors
34
PP2B (calcineurin, Ca2+/calmodulin dependent protein phosphatase) was first identified as a major calmodulin-binding protein from brain, where it may account for up to 1% of the total protein. It is a heterodimer consisting of a 60 kDa catalytic A-subunit and a 19 kDa regulatory B-subunit. The catalytic subunit of PP2B shares about 40% sequence homology with catalytic subunits of PP1 and PP2A. However, it contains an additional 170-amino acid C-terminal region, which functions as the calmodulin-binding domain. The regulatory subunit contains four Ca2+ -binding sites. The activity of PP2B is modulated by Ca2+, calmodulin, FK506-binding proteins, and cyclophilins. Although the activity of purified PP2B is shown to be dependent on Ca2+ and calmodulin, it also requires bivalent Mn2+ or Mg2+ for optimal activity. Interestingly, PP2B is activated at much lower Ca2+ concentrations (Kd = 100 pM) than CaM kinase II (Kd = 45 nM). A partial proteolysis of PP2B, which removes the autoinhibitory domain from the C-terminus region of the enzyme, can eliminate the Ca2+ requirement for its activity. In the brain, PP2B is believed to play a major role in memory by modulating longterm potentiation. It is abundantly expressed in areas of the brain that are vulnerable to stroke, epilepsy, and neurodegenerative diseases. PP2B also plays a significant role in inflammation and immunosuppression. Inhibition of PP2B by cyclosporin A and FK506 suppresses the production of IL-2 and other cytokines in activated T cells. PP2C is a monomeric Mg2+/Mn2+-dependent enzyme that does not exhibit any significant homology with other protein phosphatases. However, it displays some degree of overlapping substrate specificity with PP1 and PP2A. Multiple isoforms of PP2C have been recognized in humans, yeast, and mice. Although PP2C is widely ex-pressed is mammalian tissues, it is most abundant in heart and skeletal muscle. PP2C negatively regulates cell cycle in a manner similar to that of PP1 and PP2A via dephosphorylation and inactivation of Cdks. It has also been implicated in cystic fibrosis due to its role in the dephosphorylation and inactivation of cystic fibrosis transmembrane conductance regulator (CFTR).
In mammals several other members of Ser/Thr phosphatases have been reported. PP4 phosphatase that contains a complex of PP4C, PP4R2, and PP4R3, dephosphorylates ATR-mediated -H2AX that is generated during DNA replication. PP5, an evolutionarily conserved Ser/Thr phosphatase, is unique in that it contains a C-terminal catalytic domain and an amino-terminal TPR domain, which functions both to maintain low basal activity and to bind to target proteins. It functions as an inhibitor of glucocorticoid- and p53-induced signaling cascades leading to growth suppression. The precise regulatory mechanism of PP5 activity is not fully established. However, its aberrant expression has been linked to development of MCF-7 cells with an estrogen-independent phenotype. PP6 is shown to regulate cell cycle progression in human cells through control of cyclin D1. It exhibits 57% sequence homology with PP2A and 40% with PP1. It suppresses cyclin D1 levels in the G1 phase of cell cycle and reduces the phosphorylation of RB1 at Ser 807/811. PP7, a recently identified protein Ser/Thr phosphatase, is largely confined to the plant kingdom. It is not closely related to any protein phosphatases in animals and shows resistance to okadaic acid, calyculin, and fumonisin B1. Protein tyrosine phosphatases (PTPs), remove phosphate groups from phosphorylated tyrosine residues of proteins. PTPs can be classified as either tyrosine-specific or dual-specific phosphatases based on their phosphatase activity. They display diverse structural features and play important roles in the regulation of cell proliferation, differentiation, cell adhesion and motility, and cytoskeletal function. They are either transmembrane receptor-like PTPs or cytosolic enzymes. Each PTP contains a highly conserved catalytic domain of about 240 residues that exhibits high sequence homology throughout the family. Highly conserved arginine and cysteine residues at the catalytic domain are considered to be essential for enzyme activity. The diversity of PTPs is primarily due to the variety of noncatalytic regulatory sequences and targeting domains attached to both N- and C-terminals. PTP-1B, a 37 kDa enzyme, is one of the moststudied non-receptor PTPs. It was the first PTP to
35
be isolated in a homogeneous form. It consists of a single domain organized into 8 -helices and 12 -sheets. Situated on loop 15, which connects -12 and -4, is Cys215, which is critical to the catalytic function of PTP-1B. The base of the catalytic site is formed by the residues His214 through Arg221. A structural feature that is highly conserved in PTP1B is the catalytic or PTP loop, which consists of 11 residues: (I/V)HCXAGXXR(S/T)G. In this sequence, Cys215 and Arg221 are critical for catalysis to proceed. Another highly conserved region is the recognition pocket, which plays a role in substrate recognition. Tyr46 and Val49 assist the substrates insertion into the catalytic site. Ser216 of the PTP loop forms a hydrogen bond with the recognition loop, which stabilizes the active site cleft. When a phosphorylated tyrosine residue comes in contact with the active site cleft, Tyr46 and Val49 of the recognition loop facilitate its entry into the site. It is interesting to note that the phosphorylated end of tyrosine is polar and the phenol ring is non-polar. Normally, phosphotyrosine should be repelled from a polar catalytic site. However, Tyr46 and Val49 provide a non-polar pocket for the phenol ring of the phosphotyrosine substrate while the phosphorylated end is firmly attached to the catalytic cleft. The phosphorylated tyrosine residue gets situated in such a manner that the phosphorus atom and the sulfur atom of Cys215 become juxtaposed. This is an important feature in the catalytic scheme because the Cys215 residue of the PTP loop removes the phosphate from tyrosine and stores it briefly as an intermediate. In this reaction scheme, Asp181 adds a proton (H+) to the oxygen of tyrosine, which neutralizes the tyrosine allowing it to diffuse away from the catalytic cleft. The phosphate then binds to the sulfur on Cys215 and forms the cysteinyl-phosphate intermediate. The phosphate is removed from the cysteine via a nucleophilic attack of a water molecule. CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases that are located in the leukocyte plasma membrane. They are considered to be essential for antigenreceptor-mediated activation of T and B cells. CD45 is a long, single chain, type I transmembrane molecule consisting of 1120 to 1281 amino acids residues. Of these about 391 to 552 amino acids reside in the extracellular domain. The size variability is reported to be due to differential splicing of 3 exons near the amino-terminus of
the molecule. It contains a long cytoplasmic tail of about 707 amino acids, which includes a direct repeat of two phosphatase domains. However, only the first domain has any significant phosphatase activity. CD45 is expressed at high levels on all hematopoietic cells, however, higher density expression occurs on lymphocytes where about 10% of the surface area may be CD45. It has been proposed that spatial and temporal controls allow CD45 to promote B cell antigen receptor signal transduction by constitutively maintaining the activity of Src kinases in a partially active state. This helps B cells to effectively respond to any antigenic challenge. Another important member of the PTP family is the LAR (Leukocyte Antigen Related) PTP, which is a receptor-type PTP and is widely distributed. It is involved in neuronal development, regulation of cell adhesion, and modulation of insulin signaling. It plays a major role in dephosphorylation of phosphotyrosines in the regulatory domain of the insulin receptor. LAR PTP contains a soluble catalytic domain of about 350 amino acids. It also contains an extracellular domain, consisting of a combination of immunoglobulin and fibronectin type III domains, which is linked via a transmembrane region to two tandem PTP domains. Another class of protein phosphatases is the dual-specificity phosphatases (DSP), which play a key role in the dephosphorylation of MAP kinases. Hence, they are also termed as MAP kinase phosphatases (MKPs). Thus far about 10 genes encoding members of the classical MKP family have been isolated and characterized in mammals. On the basis of structures, predicted from genomic sequence, MKPs have been divided into three subgroups. Group I contains DSP1, DSP2, DSP4, and DSP5; group II enzymes are DSP6, DSP7, DSP9, and DSP10; and group III consists of DSP8 and DSP16. All the DSPs share strong amino-acid sequence homology in their catalytic domains. The catalytic domain contains a highly conserved consensus sequence DX26(V/L) X(V/I)HCXAG(I/V)SRSXT(I/V)XXAY(L/I)M, where X could be any amino acid. The three amino acids (bold) are reported to be essential for the catalytic activity of DSPs. The cysteine is required for the nucleophilic attack on the phosphorus of the substrate and the formation of the thiol-phosphate
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intermediate. The conserved arginine binds the phosphate group of the phospho-tyrosine or phospho-threonine, enabling transition-state stabilization; and the aspartate enhances catalysis by protonating oxygen on the departing group (tyrosine or threonine). All DSPs contain two conserved regions, known as the CH2 domains, at their amino-terminus, which are believed to be involved in substrate binding. In addition, all DSPs contain a MAP kinase-docking site at their amino-terminus, which consists of a cluster of positively charged amino acids. The corresponding docking site on the MAP kinases is shown to consist of negatively charged residues indicating that electrostatic interactions are important for the binding of MAP kinases and MKPs. The group three DSPs also have an extended carboxyl terminus containing PEST sequences [abundant in proline (P), glutamate (E), serine (S) and threonine (T) residues] that are normally found in rapidly degrading proteins. Removal of the PEST sequences from these proteins can result in their stabilization. Most DSPs display wide tissue distribution, however, some exhibit a tissue-specific expression pattern. For example, DSP2 is predominantly expressed in hematopoietic tissues; DSP8 is expressed mainly in brain, heart, and lung; DSP9 is expressed in placenta and kidney; and DSP10 is seen only in liver and skeletal muscle. The intracellular distribution of DSPs is also variable. DSP1, 2, 4, and 5 are located exclusively in the nucleus whereas DSP6, 7, and 16 are predominantly expressed in the cytoplasm. DSP 8, 9, and 10 exhibit both cytosolic and nuclear expression. Newly reported DSP18 and DSP21 are localized in the mitochondria, and while DSP18 is expressed in several mammalian tissues, DSP21 expression is limited only to the testes. Cdc25 phosphatases are another group of dual specificity phosphatases that play a significant role in the dephosphorylation and subsequent activation of cyclin-dependent kinases (Cdk). Three isoforms of Cdc25 have been described in humans (Cdc25A, B, and C). They are 300-600 residues in length and posses an N-terminus with numerous phosphorylation and ubiquitination sites that are involved in regulating phosphatase activity, protein levels, and/or association with other proteins. The N-terminal region also has the
nuclear import-export signaling sequences, which control intracellular localization of Cdc25. The C-terminal region contains the catalytic domain where the signature motif (HCX5R) containing the catalytic cysteine is found. An important feature of Cdc25 is that their catalytic domains lack of any apparent substrate recognition site. Cdc25A controls chromosome condensation, Cdc25B regulates the centrosomal activation of Cdk/cyclin complexes, and Cdc25C is involved in the amplification and maintenance of the Cdk/ cyclin activity during mitosis. Changes in Cdc25A and Cdc25B expression have been linked with high aggression and poor prognosis for a number of tumor types. Hence, Cdc25 phosphatases are considered as promising targets in cancer chemotherapy. PTEN (phosphatase and tensin homologue deleted on chromosome 10), a 55 kDa protein belongs to the family of lipid phosphatases. It contains an N-terminal phosphatase catalytic domain, a C2-domain that binds to phospholipid membranes, and a C-terminal regulatory tail. It is a dualspecific phosphatase, which dephosphorylates both lipid and protein substrates. PTEN blocks the phosphatidylinositol 3-kinase/Akt signaling pathway by removing the phosphate in the 3-position on phosphoinositide-3, 4, 5-trisphosphate (PIP3). Dephosphorylation of PIP3 is an important factor in cell growth, proliferation, apoptosis, and survival. Dephosphorylation of PIP3 blocks Akt signaling that can lead to higher activity of pro-apoptotic molecules such as Bad and Caspase-9. PTEN is considered a major tumor suppressor and its mutations are reported in several human tumors. Over seventy different types of PTEN mutation have been recorded in patients with Cowden syndrome. PTEN -/mice exhibit higher levels of 3-phosphorylated phospholipids and die during embryogenesis due to the failure of developmental apoptosis. SHIP (SH2-domain-containing inositol 5- phosphatase), another family of lipid phosphatases has two members, SHIP-1 and SHIP-2. They have a Src homology 2 domain containing inositol polyphosphate 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOHterminal proline-rich domain. SHIP-1 is an 145kDa protein that gets tyrosine-phosphorylated
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upon IL-3 stimulation. Alternatively spliced isoforms of SHIP-1 (135, 125, and 110 kDa) have also been reported. SHIP removes the phosphate group from 5-position of PIP3. In contrast to SHIP-1, which has a restricted hematopoietic expression, SHIP2 is more widely expressed and is localized in a perinuclear cytosolic region at the leading edge of the cell. It undergoes cytokine, growth factor, and insulin-stimulated phosphorylation in a number of cell lines. SHIP-2 negatively regulates insulin signaling, whereas SHIP-1 negatively regulates the activation of immune cells. The brief discussion presented above, although neither comprehensive and nor all-inclusive, does indicate that protein phosphatases play a significant role in signal transduction during cell proliferation and cell death. Hence, protein phosphatase inhibitors and activators could be of great therapeutic importance in the treatment of cancer and stimulation of cell death. Several serine/threonine phosphatases are known to alter the phosphorylation state of anti-apoptotic molecules, such as Bcl-2 and Bcl-xL, and of pro-apoptotic molecules, such as Bad and Bid. Dephosphorylation of Bad by PP1, PP2A, and PP2B allows it to interact with Bcl-xL and initiate cell death. PTP-1B is considered as a negative regulator of insulin signaling. Hence, PTP-1B inhibitors have been studied as possible therapeutic candidates in the treatment of obesity and type II diabetes. Recent genetic studies support the association between PTP-1B and insulin resistance. PTP-1B has also been shown to negatively regulate leptin signaling. Mice
deficient in PTP-1B are resistant to diabetes and diet-induced obesity. Another area of clinical importance is Alzheimers disease where abnormal deposits of highly phosphorylated tau proteins have been linked to higher levels of phosphorylation and defective PP2A and PP2B activities.
References: Chowdhury, D., et al. 2008. Mol. Cell 31, 33. Rardin, M.J., et al. 2008. J. Biol. Chem. 283, 15440. Redfern, R.E., et al. 2008. Biochemistry 47, 2162. Cazales, M., et al. 2007. Mol. Cancer Therap. 6, 318. Rudolph, J. 2007. Biochemistry 46, 3595. Stefansson, B., and Brautigan, D.L. 2007. Cell Cycle 6, 1386. Stoker A.W. 2005. J. Endocrinol. 185, 19. Farooq, A., and Zhou, M.M. 2004. Cell Signal 16, 769. Kirstjansdottir, K., and Rudolph, J. 2004. Chem. Biol. 11, 1043. Guzeloglu-Kayisli, O., et al. 2003. J. Clin. Endocrinol. Metab. 88, 5017. Bollen, M., and Beullens, M. 2002. Trends Cell Biol. 12, 138. Cheng, A., et al. 2002. Dev. Cell 2, 497. Cook W.S., and Unger, R.H. 2002. Dev. Cell 2, 385. Dombradi, V. 2002. Eur. J. Biochem. 269, 1049. Goldstein, B.J. 2002. J. Clin. Endocrinol. Metab. 87, 2474. Klumpp, S., and Krieglstein J. 2002. Curr. Opin. Pharmacol. 2, 458. Theodosiou, A., and Ashworth, A. 2002. Genome Biol. 3, 3009.1. Ukkola, O., and Santaniemi, M. 2002. J. Intern. Med. 251, 467. Dyson, M.J., et al. 2001. J. Cell Biol. 155, 1065. Justement, L.B. 2001. Int. Rev. Immunol. 20, 713. Urban, G., et al. 2001. J. Biol. Chem. 276, 27638. Berndt, N. 1999. Front. Biosci. 4, 22. Honkaniemi J, et al. 1998. Brain Res. Mol. Brain Res. 60, 1. Kutuzov., M. A. 1998. FEBS Lett. 440, 147. Oliver, C.J., and Shenolikar, S. 1998. Front. Biosci. 3, D961. Neel, B.G., and Tonks, N.K. 1997. Curr. Opin. Cell Biol. 9, 193. Jia, Z., et al. 1995. Science 268, 1754. Trojanowski, J.Q., and Lee, V. M-Y. 1995. FASEB J. 9, 1570. Wera, S., and Hemmings, B.A. 1995. Biochem. J. 311, 17. Stone, R.L., and Dixon, J.E. 1994. J. Biol. Chem. 269, 31323. Zhang, Z-Y. et al. 1992. J. Biol. Chem. 267, 23759. Charbonneau, H., et al. 1988. Proc. Natl. Acad. Sci. USA 85, 7182.
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Chapter Ten
Acetylation and Methylation: Epigenetic Modulators

Guest Author: Huda E. Shubeita, Ph.D., EMD Chemicals, San Diego, CA 92121
Mechanisms involved in intercellular and intracellular communication and their effects on metabolic regulation have largely formed the basis of modern cell biology. This field of biological sciences extends into several other scientific domains including biochemistry, immunology, molecular biology, pharmacology, physiology and others.
Although the general structure of chromatin is similar in the cells of all eukaryotes, it is in fact a dynamic structural and functional scaffold that plays major roles in many cellular processes such as transcription, DNA replication and repair, mitosis, differentiation, and apoptosis. Chromatin structure and function are in turn influenced by both genetic and epigenetic factors. DNA methylation and the post-translational modification of histones are among the major epigenetic factors. Each of the histones that make up the nucleosome core contains a flexible N-terminus of 19-39 residues. H2A and H2B also contain a flexible C-terminus. These termini, called histone tails, are subject to multiple potential post-translational modifications that include, but are not limited to acetylation, methylation, ubiquitination, phosphorylation and sumoylation. H3 and H4 have short C-terminal tails that are not modified. Posttranslational modifications also occur within the globular domain of histones that interacts with DNA. The particular combination of histone post-translational modifications constitutes a so called histone code that influences chromatin structure and function by altering nucleosomenucleosome interactions as well as interactions with various
10 | Acetylation and Methylation: Epigenetic Modulators
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protein factors that participate in or regulate processes such as gene expression, and DNA replication, repair and recombination. The many possible combinations of these post-translational modifications lead to different functional outcomes, the subject of much ongoing research.
Histone Acetylation
Histone acetylation and deacetylation are reversible and play a major role in determining chromatin structure and function. In the deacetylated form, specific basic amino acids in histones are positively charged and interact with DNAs negatively charged phosphate groups and with negatively charged patches on neighboring nucleosomes, which promotes chromatin condensation to form heterochromatin. On the other hand, acetylation neutralizes these positive charges, promoting the less condensed and more accessible chromatin conformation known as euchromatin. Thus, histone acetylation, particularly of H3 and H4, has been linked to actively-transcribed genomic regions, and HATs (histone acetylases) and HDACs (histone deacetylases) have been traditionally linked to transcriptional activation and repression, respectively. HATs and HDACs also have nonhistone target protein substrates, such as p53, NFB, STAT3 and Hsp90, as well as non-protein substrates such as polyamines or metabolic intermediates (as in bacteria). HATs catalyze the transfer of an acetyl group from acetyl-CoA to the -amino groups of specific histone lysines and are classified into several families based on sequence conservation. These include the GNAT (Gcn5-related N-acetyltransferase) superfamily, e.g., Gcn-5, Hat1, Elp3, Hpa2 and PCAF; the p300/CBP family; the TFIIIC family; the MYST family (named after its founding members, which include MOZ, YBF2/ SAS3, SAS2 and TIP60); TATA-box binding protein-associated factor TAFII-p250; and the nuclear receptor co-activators, e.g., ACTR, SRC-1, and TIF2. HATs have been reported to act as tumor suppressors or activators depending on tumor type and tumor development stage. HDACs catalyze the removal of the acetyl moiety from the -amino groups of histone lysines. Each cell is reported to have several different types of HDACs. For example, in the yeast Saccharomyces cerevisiae there are as many as 10 different HDACs that have the potential to participate in
transcriptional regulation. In mammals, eighteen different HDACs have been reported and are subdivided into four classes based on phylogenic analysis, sequence similarity to their founding yeast homologs, subcellular localization, and enzymatic activities. Class I HDACs (HDAC 1, 2, 3, and 8) are homologous to yeast Rpd3, are widely expressed in tissues, and are primarily located in the nucleus. Class II HDACs (HDAC4, 5, 6, 7, 9, and 10) are homologous to yeast Hda1, are much larger in size, display limited tissue distribution, and can shuttle between nucleus and cytoplasm. Class I and II HDACs are Zinc-dependent enzymes. Class III HDACs (human SIRT 1 to 7) consist of a large family of sirtuins (silent information regulators or SIR) that are evolutionarily distinct and related to yeast Sir2, with unique enzymatic mechanisms dependent on NAD+; they do not contain a zincbinding site. Class IV HDAC has only one member (HDAC 11); it localizes primarily in the nucleus and its catalytic core shares sequence homology with those of class I and II HDACs. In humans, HDAC 11 is expressed mainly in brain, heart, muscle, testis, and kidney, and also in several human cancer cell lines. In brain, it is reported to play a role in the development of oligodendrocytes and some neurons. Histone acetylation/deacetylation has been implicated in many biological processes, such as cell survival and differentiation, double-strand DNA repair, cell cycle progression, tumorigenesis, cardiac function and remodeling in health and disease, and plant acclimation to cold stress. Also, studies have linked the proliferative activity of many solid tumors to the histone acetylation status, associated Class I and Class II HDACs with malignant transformation, and identified the loss of acetylation at Lys16 of H4 as a common hallmark of human cancer. Consequently, HATs and HDACs are among the most promising targets in cancer therapy, and the quest for modulators of their activities has attracted much attention in the last few years. Recently, the HAT inhibitory activity of allspice extract has been correlated with reduced androgen receptor (AR) and histone acetylation, with decreased expression of the AR-regulated genes PSA and TSC22, and with the inhibition of prostate cancer cell growth. Other studies demonstrated that ITF2357, an inhibitor of class I and class II HDACs, has anti-leukemic activity in vitro and in vivo, induces apoptosis of hepatoma cells but not primary hepatocytes,
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and has anti-inflammatory activity in peripheral blood mononuclear cells.
Histone Methylation
More recently, lysine and arginine methylation in histone has attracted a lot of attention due to their involvement in many epigenetic processes, and each was found to contribute to both active and repressive chromatin functions. In the corresponding methylation reactions, the -amino group of certain lysine residues and the guanidinium group of certain arginine residues are methylated. For these methylation reactions S-adenosyl-methionine is used as the methyl group donor. Except for yeast Dot1 and mammalian Dot1L, all known histone lysine methyltransferases (HKMTs; also known as protein lysine methyltransferases (PKMTs)) contain the conserved SET enzymatic domain (originally recognized as a conserved sequence in three Drosophila genes: Su(var)3-9; En(zeste); and Trx). Dot1 and mDot1L contain novel enzymatic domains. Methylation of lysines 4, 36 and 79 of H3 is associated with active chromatin regions, while methylation of lysines 9 and 27 of H3 and lysine 20 of H4 is generally associated with silenced chromatin regions. Furthermore, lysine methylation can occur in the monomethyl, dimethyl or trimethyl state, which further expands its epigenetic information potential. Arginine methylation is catalysed by the PRMTs (protein arginine methyltransferases) and, in
mammals, is typically seen on residues 2, 8, 17, and 26 of H3 and residue 3 of H4. Methylation at H3R17, H3R26 and H4R3 has been associated with gene activation, while methylation at H3R8 has been associated with gene repression. PRMT family members are classified as type I (PRMT1, 3, 4, 6, and 8) and type II (PRMT5 and 7); the enzymatic activity PRMT2 remains to be characterized. Type I PRMTs catalyze the formation of -NG monomethylarginines (MMA) and -NG, NG-asymmetric dimethylarginines (aDMA); type II PRMTs catalyze the formation of MMA and -NG, N G-symmetric dimethylarginines (sDMA). Histone methylation is a more stable modification as compared to acetylation and phosphorylation. Hence, it was long regarded as a static modification. However, the recent discoveries of the amine oxidase (AO) LSD1 and hydroxylase enzymes that catalyze the removal of histone lysine methylation, and of peptidylarginine deiminase that converts methylarginine to citrulline (arginine demethylimination) clearly demonstrate that histone methylation, is indeed reversible. Recent studies have implicated histone methylation in the maintenance of embryonic stem (ES) cells in the undifferentiated state, arginine demethylimination in transcriptional repression, histone lysine demethylases in transcriptional regulation, cancer cell proliferation and normal neuronal function, and the loss of trimethylation at Lys20 of H4 in human cancer. Studies have suggested that histone demethylation and deacetylation are tightly coupled.
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DNA Methylation
DNA methylation in mammalian cells is catalyzed by DNA cytosine-5 methyltransferases (DNMTs), which transfer a methyl group from S-adenosylmethionine to C-5 of cytosine. Five related mammalian DNMTs have been reported: DNMT1, 2, 3a, 3b, and 3L. DNMT1 binds preferentially to hemimethylated DNA, is known as the maintenance methyltransferase, and plays a role in DNA mismatch repair. DNMT2 contains the conserved methyltransferase motif found in the other methyltransferases, but lacks the amino terminal regulatory domain, and was shown to be an RNA methyltransferase. DNMTs 3a and 3b bind both unmethylated and hemimethylated CpG sites, are known as de novo methylases, and their activities may be dependent on other proteins. DNMT3L (DNMT 3-like) lacks a catalytic domain and is devoid of DNA methyltransferase activity; it is believed to function as partner for the de novo DNMTs 3a and 3b, enhancing their catalytic activities. In addition to the post-translational modification status of histones in chromatin, the DNA methylation pattern is also an important epigenetic regulator of gene expression. In the mammalian genome, about 70% of the CpG dinucleotides are methylated. Many of the remaining nonmethylated CpGs are in CpG islands typically found in functional promoter regions. DNA methylation has long been viewed as an epigenetic marker of gene repression and plays important roles in heterochromatin formation, long-term silencing of repetitive elements, X-chromosome inactivation, and in the establishment and maintenance of imprinted genes. However, more recent studies show that transcriptional activation is associated with cycles of DNA methylation, that DNMTs are involved in both addition and removal of methyl groups, and that differentiation of ES cells is associated with changes in DNA methylation that involve both demethylation and de novo methylation. While it
is clear that DNA methylation plays an important role during development, and that the particular DNA methylation patterns at CpG islands are among the factors that underlie the production of various cell types in the body, there is still no consensus as to why development fails when DNA methylation is deficient. Many research efforts have linked the DNA methylation status to neoplasia. Although early studies reported global genome hypomethylation in cancer, tumorigenesis is frequently associated with hypermethylation of CpG islands in promoters of tumor suppressor genes. DNA methylation status can affect gene expression by various mechanisms. For example, DNA methylation could sterically hinder the binding of activating transcription factors to gene promoters; DNA methylation could recruit repressor-type protein factors that specifically bind methylated DNA via their methyl-CpGbinding domains (MBDs); Methyl-CpG-binding proteins or DNMTs may bind/recruit HDACs or histone methyltransferases and thus influence histone modifications. However, cumulative data from studies using the HDAC inhibitor Trichostatin A or Dnmt [3a-/-, 3b-/-] ES cells suggest that the recruitment of the majority of histone deacetylase activity to the chromatin is not directly dependent on DNA methylation. Clearly, the diverse combinations of the different histone post-translational modifications and the DNA methylation states lead to different functional consequences that influence gene expression and affect both normal development and disease progression. Consequently, the development of specific modulators of chromatin modifying enzymes is crucial for studying the contribution of each of these epigenetic modifications in different biological processes, as well as for providing leads in the development of therapies for cancer, cardiac hypertrophy and other disease states.
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References: Escargueil, A., et al. 2008. Mutat. Res. 658, 259. Kangaspeska, S., et al. 2008. Nature 452, 112. Latham, T., et al. 2008. Cell Tissue Res. 331, 31. Liu, H., et al. 2008. J. Neurosci. Res. 86, 537. Mtivier, R., et al. 2008. Nature 452, 45. Suzuki, M., and Bird, A. 2008. Nat. Rev. Gen. 9, 465. Zheng, Y., et al. 2008. Med. Res. Rev. 28, 645. Zhu, J., et al. 2008. Proc. Natl. Acad. Sci. USA 105, 4945. Miranda, T.B., and Jones, P. 2007. J. Cell. Physiol. 213, 384. Gilbert, N., et al. 2007. J. Cell Biol. 177, 401. Golay, J., et al. 2007. Leukemia 21, 1892. Guo, H-B., and Guo, H. 2007. Proc. Natl. Acad. Sci. USA 104, 8797. Klose, R.J., and Zhang, Y. 2007. Nat. Rev. Mol. Cell Biol. 8, 307. Lee, Y-H., et al. 2007. Biosci. Biotechnol. Biochem. 71, 2712. Montgomery, R., et al. 2007. Genes Dev. 21, 1790. Ruthenburg, A.J., et al. 2007. Nat. Rev. Mol. Cell Biol. 8, 983. Yeo, S., et al. 2007. Biochem. Biophys. Res. Commun. 359, 536. Backs, J., and Olson, E. 2006. Circ. Res. 98, 15. Bolden, J.E., et al. 2006. Nat. Rev. Drug Dis. 5, 769. Minucci, S., and Pelicci, P. 2006. Nat. Rev. Cancer 6, 38. Cheng, X., et al. 2005. Annu. Rev. Biophys. Biomol. Struct. 34, 267. Cheung, P., and Lau, P. 2005. Mol. Endocrinol. 19, 563. Fraga, M.F., et al. 2005. Nat. Genetics 37, 391. Miao, F., and Natarajan, R. 2005. Mol. Cell. Biol. 25, 4650. Tamburini, B., and Tyler, J., 2005. Mol. Cell. Biol. 25, 4903. Villar-Garea, A., and Esteller, M. 2004. Int. J. Cancer 112, 171. Shiota, K., et al. 2002. Genes Cells 7, 961. Zhang, Y., and Reinberg, D. 2001. Genes Dev. 15, 2343.
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Chapter Eleven
Nuclear Import - Export: A Nuclear Revolving Door
The field of nuclear protein transport has gained prominence during the past two decades. Although most of the protein synthesis occurs in the cytoplasm, proteins required for nuclear functions are specifically transported from the cytoplasm into the nucleus. In general, proteins larger than 25 nm (~40 kDa) are excluded from entry into the nucleus and can only enter the nucleus in an energy-dependent process, which calls for the participation of a variety of soluble import factors.
Trafficking between the nucleus and the cytoplasm occurs via the nuclear pore complexes (NPCs), which are large supramolecular assemblies of ~125 mDa and contain about 100 polypeptides embedded in the double-membrane nuclear envelope. It serves as a gateway of macromolecular trafficking between the nucleus and the cytoplasm. The NPC consists of a central core with a ringspoke structure exhibiting 8-fold radial symmetry. From this central ring, 50 to 100 nm fibrils extend into the nucleoplasm and the cytoplasm. The NPC is attached in the nuclear envelop by the nuclear lamina. The NPCs allow passive diffusion of ions and small molecules, whereas nuclear proteins, RNAs, and ribonucleoproteins larger than ~9 nm are selectively and actively transported by a signal-mediated and energy-dependent mechanism. It is estimated that the average mammalian nucleus contains approximately 5600 pores. Most of these pores function in protein transport in ribosome biosynthesis. The signal for import is provided by a polypeptide sequence in the encoded protein known as the nuclear localization signal (NLS). Several unique NLSs have been described that modulate nuclear
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| Nuclear Import - Export: A Nuclear Revolving Door
import of proteins through a specific nuclear import pathway. The presence of several different NLSs and import factors suggests the existence of multiple pathways for such an import. Conversely, there are protein sequences, which can signal the export of a protein from the nucleus to the
cytoplasm. These proteins contain a nuclear export sequence (NES) that keeps these proteins exclusively in the cytoplasmic compartment. Some proteins possess a nuclear retention signal (NRS), which guarantees their retention in the nuclear compartment.
An Overview of nuclear protein import and export
A number of nuclear transport receptors known as importins (karyopherins), transportins, and Ran-binding proteins recognize NLS and mediate docking at the nuclear pore. These receptors are generally large acidic proteins that share the ability to bind components of the NPC and contain both an N-terminal RanGTP-binding domain and a C-terminal protein or RNA-binding domain. Protein and RNA can bind their cognate nuclear transport receptor directly through NLS and NES signals or via adaptor proteins. Import receptors bind their cargos in a RanGDP-dependent manner and release this cargo in the presence of RanGTP, which accumulates in the nucleus through the action of RCC1 (RanGEF). In a reciprocal approach, export receptors bind their cargo in a stable manner in the presence of RanGTP, but the release of this cargo requires GTP hydrolysis by Ran and the accumulation of RanGDP. Thus, GTP hydrolysis by Ran is required to accumulate cargo against a concentration gradient between the nucleus and cytoplasm. Ran is maintained as RanGTP in the nucleus by its association with the Ran GTP-GDP exchange factor (RanGEF or RCC1). In the cytoplasm, the RanGTPase activating protein (RanGAP) simulates the GTPase activity of
Ran, leading to the accumulation of RanGDP in the cytoplasm. In a classic protein import cycle anywhere from 100 to 1000 cargos are carried by each NPC per minute. Cargo proteins are imported by the carrier importin-, which binds them through the adaptor protein importin-. Importin- (karyopherin-), the cytoplasmic receptor for NLS-bearing proteins, binds importin- via its importin- binding domain. Importin- interacts with the importin- bound to the NLS and acts as a carrier of the NLS/importin/ trimer. This trimeric complex docks to the cytoplasmic filaments of the NPC via importin-. Subsequent passage of import substrate from the NPC into the nuclear interior requires the small GTPase Ran, which plays a crucial role in both import/export pathways and determines the directionality of the nuclear transport. The translocation of the import complex through the NPC requires energy and this process is facilitated by nuclear transport factor 2 (NTF2/p10). Once in the nucleus, importin- binds RanGTP and the import complex is disassembled and the substrate is retained in the nucleus. Ran is reported to shuttle between the nucleus and the cytoplasm
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and its recycling is essential for the nuclear transport. In the cytoplasm, Ran is kept primary in its GDP bound form to facilitate import. On the other hand, in the nucleus, Ran is bound to GTP through RCC1, which facilitates exports of proteins. Importin-, probably bound to RanGTP, is re-exported immediately by an unknown mechanism. In contrast to importin-, importin- is exported into the cytoplasm bound to a specific export factor. Malfunctioning of the nucleo-cytoplasmic transport is profoundly involved in a number of diseases. Defects in NPC components have been implicated in several autoimmune disorders and cancer. At least 11 chromosomal rearrangements in acute leukemia involve nuclear pore protein (nucleoporin) genes; 9 of those involve the nucleoporin Nup98. Leukemias associated with nucleoporin gene rearrangements have been reported to be more refractory to therapeutic intervention. Leptomycin B, an unsaturated, branched chain fatty acid with a terminal lactone ring is shown to be a potent inhibitor of nuclear export. It targets CRM-1, an evolutionarily conserved
receptor for the nuclear export signal of proteins. Leptomycin B covalently binds to a single cysteine residue (Cys529) to inactivate CRM1. Leptomycin B is shown to protect p53 from Mdm-2- mediated degradation. It is an important tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins and may be a potential therpeutic agent for disorders associated with mislocalization of regulatory proteins.
References: Xu, X.M., and Meier, I. 2008. Trends Plant Sci. 13, 20. Sorokin, A.V., et al. 2007. Biochem. (Mosc). 72, 1439. Stewart, M. 2007. Nat. Rev. Mol. Cell Biol. 8, 195. Menendez, S., et al. 2003. Br. J. Cancer 88, 636. Weis, K. 2003. Cell 112, 441. Yashiroda,Y., and Yoshida, M. 2003. Curr. Med. Chem. 10, 741. Lyman, S.K., and Gerace L. 2001. J. Cell Biol. 154, 17. Kroon, E., et al. 2001. EMBO J. 20, 350. Arai, Y., et al. 2000. Leukemia 14, 1621. Barry, D.M., and Wente, S.R. 2000. Essays Biochem. 36, 89. Fontoura, B. M. A., 2000. J. Biol. Chem. 275, 31289. Talcott, B., and Moore, M.S. 2000. J. Biol. Chem. 275, 10099. Kudo, N., et al. 1999. Proc. Natl. Acad. Sci. USA 96, 9112. Nakielny, S., and Dreyfuss, G. 1999. Cell 99, 677. Stoffler, D., et al. 1999. Curr. Opin. Cell Biol. 11, 391. Truant, R., et al. 1998. Eur. J. Cell Biol. 77, 269. Pennisi, E. 1998. Science 279, 1129. Kutay, U., et al. 1997. Cell 90,1061. Grlich, D., et al. 1995. Nature 377, 246.
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11 | Nuclear Import - Export: A Nuclear Revolving Door
Chapter Twelve
Poly (ADP-ribosylation): PARP in DNA Repair and Apoptosis
Poly(ADP-ribosyl)ation (pADPr), a covalent post-translational modification process that occurs during DNA repair, replication and transcription, is brought about by poly(ADPribose)polymerase (PARP), which is activated by breaks in DNA strands. PARPs are a group of Zn2+ -binding enzymes that catalyze the transfer of ADP-ribose (ADPr) units from NAD+ on to protein acceptors to produce linear and/or branched polymers of ADPr.
PARP activity has been reported in all eukaryotes except yeast. The classical 113 kDa type I PARP is an abundant protein and is the major contributor of the poly(ADP-ribosyl)ating activity in higher eukaryotes. It has become a major target for drug design for the treatment of cancer, diabetes, inflammation, retroviral infection, and other diseases. Type II PARP is smaller (62 kDa) than the classical zinc-finger-containing PARP and is believed to participate in DNA repair during apoptosis. Type III PARP is large protein containing ankyrin repeats and a PARP catalytic domain and the only known members of this subgroup are tankyrase 1 and 2. Human tankyrase is localized at telomeres in living cells, and is reported to be involved in the regulation of telomeric function. Most of the physiological substrates of poly(ADP-ribosyl)ation reactions are nuclear proteins, including those that are involved in the metabolism of nucleic acids and in the maintenance of chromatin architecture. PARP is a multifunctional enzyme consisting of three domains: a DNA-binding domain (DBD), an automodification domain, and a catalytic domain. The DBD, a 42 kDa N-terminal
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PARP in DNA Repair and Apoptosis
Oxidative stress (O2, NO, OH)
Endotoxins
NAD+
ATP
PARP activation
Pro-inflammatory signaling DNA damage NF-kB activation Poly(ADP-ribosyl)ation of proteins Nicotinamide DNA repair
Signal Transduction: A Short Overview of its Role in Health and Disease | Merck KGaA | Merck KGaA
Oxidative stress (O2, NO, OH)
Carcinogenesis
NAD+
ATP
PARP over-activation
Ionizing radiation Massive DNA damage
Apoptosis
Nicotinamide
Energy Depletion
AIF
Necrosis
Oxidative stress, DNA damage, and PARP activation
region, extends from the initiator Met to Thr373 in human PARP. It contains two zinc fingers and two helixturnhelix motifs and is rich in basic residues, which are involved in the interaction of the enzyme with DNA. Zinc finger 1 (F1) occupies the region between Cys21 and Cys56, while zinc finger 2 (F2) is found between Cys125 and Cys162. The automodification domain is located in the central region of the enzyme and resides between Ala374 and Leu525 in human PARP. This domain contains the majority of the glutamic acid residues that are involved in PARP automodification. In addition, the automodification domain contains a BRCT (BRCA1 C-terminus) domain that lies between Ala384 and Ser479 and consists of about 95 amino acids found in several proteins that regulate cell-cycle checkpoints and DNA repair. BRCT domains are protein-protein interaction modules that allow BRCT-motif-containing proteins to establish strong and specific associations. The catalytic domain, a 55 kDa segment, is located in the C-terminal region of the enzyme and spans from residues Thr526 to Trp1014 in human PARP. The catalytic activity of this fragment is not stimulated by DNA strand breaks, and it corresponds only to the basal activity of the native enzyme. The ADPr transferase activity has been confined to a 40 kDa region at the extreme C-terminus of the enzyme, which is referred to as the minimal catalytic domain. This region can catalyze the initiation, elongation, and branching of ADPr polymers
independently of the presence of DNA. The loss of the last 45 amino acids at the C-terminal end of this domain completely abolishes enzyme activity. Residues spanning positions Leu859 to Tyr908 in human PARP are well conserved and comprise the PARP signature sequence. The extent of poly(ADP-ribosyl)ation is an important determinant of NAD+ levels in cells. Upon binding to DNA breaks, activated PARP cleaves NAD+ into nicotinamide and ADP-ribose. The ADP-ribose then polymerizes onto nuclear acceptor proteins including histones, transcription factors, and PARP itself. In normal, undamaged cells, NAD+ levels range from 400 to 500 M. However, PARP activation following DNA damage by radiation or cytotoxic agents reduces NAD+ levels to about 100 M with in about 15 minutes. It is believed that during its automodification PARP becomes more charged, since each residue of ADPr adds two negative charges on to the molecule. This establishes an electro-repulsive gradient between the polymers of ADPr covalently linked to the enzyme and DNA. When the charge becomes too negative, the reaction reaches a point of repulsion and the interaction between PARP and DNA is lost. The poly(ADP-ribosyl) ated PARP molecule is consequently freed from the DNA strand break and its catalytic activity is abolished. Subsequently, poly(ADP-ribose) glycohydrolase (PARG) hydrolyses the polymers
50
present on PARP, thereby allowing it to resume a new cycle of automodification in response to DNA damage. The presence of PARG during PARP automodification restores both its affinity for DNA and its catalytic activity. DNA damage, the single most important factor in the regulation of poly(ADP-ribosyl)ation reactions, can stimulate the catalytic activity of PARP by about 500 folds. PARP is required for repair of single strand breaks and base excision repair. Inhibition of PARP is shown to reduce DNA repair, increase the cytotoxicity of DNAdamaging agents, cause telomere shortening, and enhance apoptosis. The cytotoxicity of PARP inhibitors is due to an increase in the half-life of DNA strand break, which increases genomic instability. PARP cleavage by caspase-3 is considered as an early event in apoptotic cell death. PARP degradation has also been reported during necrosis, although believed to be through a different process. In necrosis, PARP is cleaved into two major fragments of 89 and 50 kDa, and two minor fragments of 35 and 40 kDa. However, during apoptosis PARP is cleaved into only two fragments of 89 kDa and 24 kDa that contain the active site and the DNA-binding domain of the enzyme respectively.
References: Schrieber, V., et al. 2006. Nat. Rev. Mol. Cell Biol. 7, 517. Parsons, J.L., et al. 2005. FEBS J. 272, 2012. Pascucci, B., et al. 2005. Nucleic Acid Res. 33, 280. Woon, E.C., and Threadgill, M.D. 2005. Curr. Med. Chem. 12, 2373. Chiarugi, A. 2002. Trends Pharmacol. Sci. 23, 122. Virag. L., and Szabo, C. 2002. Pharmacol. Rev. 54, 375. Burkle, A. 2001. BioEssays 23, 795. Davidovic, L. 2001. Exp. Cell Res. 268, 7. Tong, W.M., et al. 2001. Biochim. Biophys. Acta. 1552, 27. Ame, J.C., et al. 1999. J. Biol. Chem. 274, 17860. DAmours, D., et al. 1999. Biochem. J. 342, 249. Shieh, W.M., et al. 1998. J. Biol. Chem. 273, 30069. Mazen, A., et al. 1989. Nucleic Acids Res. 17, 4689. Alkhatib, H.M.., et al. 1987. Proc. Natl. Acad. Sci. USA 84, 1224. Kameshita, I., et al. 1984. J. Biol. Chem. 259, 4770.
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12 | Poly(ADP-ribosylation): PARP in DNA Repair and Apoptosis
Chapter Thirteen
Heterotrimeric G-Proteins: An Introduction

Guest Author: Stephen Graber, Ph.D., Department of Biochemistry and Molecular Pharmacology, West Virginia University, Morgantown, WV 26506
The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) mediate signaling from a large number of seven transmembrane spanning cell surface receptors, commonly known as G-protein coupled receptors (GPCRs), to a variety of intracellular effectors.
These signal transduction pathways control numerous essential functions in all tissues and are ubiquitous throughout the animal kingdom. G-proteins are also found throughout the plant kingdom, albeit it with a greatly reduced array of signaling elements and substantial differences in their mechanisms of action. G-proteins are composed of a 36-52 kDa -subunit, a 35-36 kDa -subunit and an 8-10 kDa -subunit. G-proteins were discovered independently by scientists studying the hormonal regulation of adenylyl cyclase and light induced changes of cyclic nucleotides in the visual system some thirty years ago. Remarkably, the scheme first proposed in 1981 for the flow of information in the visual system is still the basis for the now widely accepted mechanism of G-protein mediated signal transduction summarized in the figure. Agonist binding to a GPCR alters the receptors conformation allowing it to catalyze the exchange of GDP for GTP on the subunit of the G-protein coupled to the receptor. The binding of GTP produces a conformational change in the G-protein subunit resulting in the dissociation of the receptor-G-protein complex and freeing the and -subunits to interact with their target effectors. The slow intrinsic GTPase activity of the subunit hydrolyzes the bound GTP to GDP ending the interaction of the -subunit with its effector. The GDP bound subunit interacts strongly with -subunits and thus GTP hydrolysis presumably also terminates mediated signaling by promoting the association of the
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| Heterotrimeric G-Proteins: An Introduction
GDP-bound subunits with dimers. Ultimately signaling is controlled by these three interdependent association-dissociation cycles; interactions between receptor and G-proteins, between guanine nucleotides and G, and between G and G. In the years since their discovery an extraordinary richness of molecular detail has been uncovered and it is now clear that the basic process is fine-tuned by a complex and ever increasing number of additional components. Among the first levels of complexity to be appreciated was the abundance of distinct subtypes for each of the G-protein subunits. There are 35 genes for mammalian G-protein subunits; 16 encoding -subunits, 5 encoding -subunits and 14 encoding -subunits. Within the -subunit family alternative splicing brings the total number of distinct subunits to 23. The G proteins are usually classified by the nature of their - subunit, which are grouped into four general classes (s, i/o, q and 12) based on amino acid similarities. Most of the subunits are widely distributed across tissue types with the exception of the subunits mediating sensory transduction (vision, taste, and olfaction). Unfortunately, the functional role of specific subunits is not defined by their structural classifications. While there is some general discrimination among the major families of subunits for receptors and effectors, understanding the mechanisms that underlie signaling selectivity remains an important question. It is clear that various subunits are capable of interacting with the same effectors, that individual subunits are capable of modulating different effectors, and that individual receptors are capable of interacting with different subunits. Indeed, with the observation that human thyrotropin receptors interact functionally with members from each of the four major families of G-protein subunits, extreme caution should be adopted when generalizing about selectivity in these signaling pathways. The functional properties of the subunits are in part determined by the covalent attachment of various lipids. Reversible palmitoylation of N-terminal cysteines has been described for all subunits except transducin, while the nonreversible myristoylation of an N-terminal glycine is restricted to members of the i/o family. Although the precise functional significance of these modifications is unknown, they appear to
facilitate membrane association and functional interactions with other components of the signal transduction systems, especially the subunits and certain effector molecules. Several of the subunits are also substrates for covalent modification by bacterial toxins. Cholera toxin is capable of ADP-ribosylating members of the s family, inhibiting their ability to hydrolyze GTP and leaving them constituitively activated. Pertussis toxin ADP-ribosylates some, but not all, members of the i/o family uncoupling them from their cognate receptors and thereby inhibiting signaling mediated by such receptors. These toxins have been invaluable in identifying many G-protein mediated responses. Another family of compounds, the so-called receptor mimetics, including mastoparan and melittin, has been useful in studying G-protein mechanisms because of their ability to directly activate many subunits. A similar diversity and rich assortment of direct roles in signal transduction is now apparent for G-protein -subunits, once considered mere bystanders with a passive role as attenuators of activity. Individual dimers directly modulate (both positively and negatively) a large number of target molecules (see Figure) and also provide scaffolding functions that play a role in the activation of c-Src and ERK1/2 MAP kinase signaling pathways. The 5 and 14 subunits are ubiquitiously expressed (albeit at varying levels), with the exception of 1, which is restricted to retinal rods, and 3 that is preferentially expressed in retinal cones. Most, but not all, pairs of and subunits can be formed. Similarly much of the literature indicates redundant functions for most combinations of and despite noteworthy demonstrations of selectivity for particular combinations of with individual receptors and effectors. The subunits contain a CAAX box that directs a complex C-terminal processing, which results in the proteolytic removal of three amino acids and covalent attachment of either C15 or C20 iso-prenyl groups and carboxymethylation of the C-terminal cysteine. These lipid modifications have been shown to be important for interactions with membranes and the other components of the signaling pathways. For the most part the and subunits form 5 dimers that act as functional units, which are only separable under strongly denaturing conditions. However the 5 subunit, which shares just 50% sequence
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G-Protein signaling and various G and G effectors
identity with the highly homologous 1-4 subunits, associates in the absence of a subunit with several regulator of G protein signaling (RGS) protein family members, which contain a G-protein subunit-like (GGL) domain. In fact, although the 5 subunit does form functional dimers with individual subunits, there is as yet no proof that such dimers exist in vivo. As additional nuances of the many roles played by G-proteins are appreciated the initial view of a linear pathway from a receptor through G-proteins to intracellular effectors has given way to more complex models that begin to account for the numerous receptor and G-protein binding partners that are now known to modulate signaling at many levels. The above-mentioned RGS proteins are a case in point. Initially these proteins were shown to attenuate signaling by directly binding activated G protein subunits and accelerating their GTPase activity. The family has grown to include at least 37 members, some of which regulate the timing, intensity and duration of signaling by select GPCRs and others which seem to have roles unrelated to GPCRs and G proteins. Indeed, it is now known that the GPCRs themselves form both homo- and heterodimers and oligomers. The implications of such structures to G protein function and physiologic relevance is only beginning to be understood and is likely to significantly alter current understanding of the G-protein-receptor interface. On yet another
front receptor independent modes of G-protein signaling have become apparent. A yeastbased functional screen has identified 10 cDNAs known as activators of G-protein signaling (AGS) proteins which all activate the G-protein dependent pheromone response pathway in the absence of the pheromone receptor. As individual family members are studied in mammalian cells an array of alternative modes of G protein activation are beginning to emerge and include roles in glucocorticoid action and asymmetric cell division. It is also becoming clear that G-proteins function at subcellular locations other than the plasma membrane. In yeast, a G-protein subunit was found to activate a phosphatidylinositol 3-kinase at the endosome. G- protein subunits have been found to regulate intracellular vesicle budding at the Golgi via a protein kinase D and protein kinase C-dependent pathway. G-proteins have even been shown to signal in the cell nucleus where G-protein subunits bind and disrupt glucocorticoid receptor regulation of transcription. Even as these many additional G protein functions gain prominence the basic, fundamental, molecular mechanism by which the agonist occupied GPCR promotes the exchange of GDP for GTP on the G-protein subunit in a G-protein dependent process awaits complete elucidation. Clearly G-protein research will remain a rich and vibrant field of study for years to come.
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References: Birnbaumer, L. 2007 Biochim. Biophys. Acta 17, 756. Birnbaumer, L. 2007. Biochim. Biophys. Acta 17, 772. Blumer, J.B., et al. 2007. Pharm. Therap. 113, 488. Johnston, CA., and Siderovski, D.P. 2007. Mol. Pharmacol. 72, 219. Milligan, G., and Kostenis, E. 2006. Br. J. Pharmacol. 147, S46. Neitzel, KL., and Hepler, J.R. 2006 Sem. Cell Dev. Biol. 17, 383. Jones, M.B., et al. 2004. Mol. Interventions 4, 200.
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Chapter Fourteen
Tubulin Polymerization and Depolymerization: Exploiting the Dynamic Instability of Microtubules
Microtubules (MTs), rigid and hollow cylindrical structures of about 25 nm diameter, are composed of -and -tubulin dimers. They determine cell shape and play important roles in diverse processes such as cell division, cell motility and migration, cellular transport, and signal transduction. Both and tubulins exist in several isotypic forms and can undergo several post-translational modifications. In higher eukaryotes at least 14 tubulin isotypes have been reported that are expressed in a tissue specific manner.
MTs are polar structures with two distinct ends, a fast growing plus end and a slow growing minus end. In most cells, MTs are organized into a single array with their minus ends associated with a MT organizing center located near the nucleus, and their plus ends located toward the cells periphery near the plasma membrane. This gives the cell a defined polarity based on the inherent polarity of MTs. This polarity is utilized by the MT-associated motor proteins that move cargos to the minus or plus ends of cellular MTs. Tubulin dimers constantly polymerize and depolymerize, and MTs can undergo rapid cycles of assembly and disassembly. The first stage of MT formation, the nucleation phase, is slow. In the presence of Mg2+ and GTP, and tubulins join together in an end-to-end manner to form protofilaments with alternating and subunits. The second phase, also known as the elongation phase, proceeds rather rapidly. For tubulin heterodimerization and association of tubulins to form MTs, GTP must be bound to both and subunits. GTP bound to -tubulin is hydrolyzed to GDP during or immediately after polymerization. This weakens the
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| Tubulin Polymerization and Depolymerization
binding affinity of tubulin for adjacent molecules and favors depolymerization that contributes to the dynamic behavior of MTs. Heterodimers can add or dissociate at either end of a MT; however, there is greater tendency for addition to occur at the faster growing plus end where -tubulin
a
is exposed. MTs also undergo treadmilling, in which tubulin molecules bound to GDP are continually lost from the minus end and are replaced by the addition of GTP-bound tubulin molecules to the plus end of the same MT.
Tubulin
b
Colchicine
+ end
Microtubule
- end
+ Tau Protein Kinases + Nocodazole + Vinblastine Destabilized Microtubules
+ MAPs + Epothilones + Taxol
Mitotic Block
Signal Transduction: A short Overview of its Role in Health and Disease | Merck KGaA
Stabilized Microtubules Mitotic Block
Hyperphosphorylated Tau Proteins
Paired Helical Filaments (PHFs) Cell Death
Neurofibrillary Tangles (NFTs)
Neuronal Death
Cell Death
Tau phosphorylation and microtubule depolymerization in neuronal cell death
During the formation of MTs, the alternating elongation and shortening cycles provide dynamic instability that is critical for directing MTs towards target sites, such as kinetochores, focal adhesions, and migrating membranes. Dynamic instability is a tightly regulated phenomenon and is particularly critical for the remodeling cytoskeleton during mitosis. It is characterized by four important variables: the rate of MT growth, the rate of shortening, the frequency of transition from the growth state to shortening, and the frequency of transition from shortening to growth. The growth and shortening of a MT is dependent upon the rate of tubulin addition relative to the rate of GTP hydrolysis. Tubulin-bound GTP is hydrolyzed to tubulin-GDP + Pi and tubulin-GTP is added to the plus end almost simultaneously. However, when GTP-bound tubulin molecules are added more rapidly than GTP is hydrolyzed, the MT retains a GTP cap at its plus end and the growth continues. When the rate of polymerization declines, the GTP bound to tubulin at the plus end is hydrolyzed
to GDP and the GDP-bound tubulin dissociates, resulting in rapid depolymerization and shrinkage of MT. The inherent dynamic instability of MTs can be modified by the interactions with MT-associated proteins (MAPs) and MT- regulatory proteins. For example, MAPs can bind to MTs and increase their stability, while other proteins act to disassemble MTs, by increasing the rate of tubulin depolymerization. The best-characterized MAPs are MAP-1, MAP-2, and tau proteins. The activity of MAPs is tightly regulated by their phosphorylation state and altered phosphorylation state of MAPs has been positively linked to the pathogenesis of Alzheimers disease. Growth factor signals can activate protein kinases that catalyze phosphorylation of tubulin-binding domains of MAPs and allow them to detach from MTs. XMAP215, a highly conserved MAP of 215 kDa, plays an important role in controlling MT dynamics during cell cycle. It stabilizes the plus
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ends of MTs, promoting growth at the plus end and preventing catastrophic shrinkage. At the onset of mitosis, higher phosphorylation of XMAP215 results in increased MT instability causing them to disassemble. During the end of mitosis, protein phosphatase activity predominates as the MT array of interphase is re-established. Given their essential role in the formation of the mitotic spindle during cell division, MTs have been very attractive targets for cancer chemotherapy. Microtubules and their dynamics are affected by several chemically diverse groups of anti-mitotic agents that have been employed in cancer chemotherapy. In the brain tissue, assembly of microtubules is affected by phosphorylation status of Tau protein. Tau, a highly asymmetric and heat-stable protein, interacts with tubulins to stabilize MTs. The Tau protein is reported to be the predominant component of the paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) that are characteristic of pathological brain lesions in the Alzheimers disease. In their dephosphorylated state Tau promotes rapid and extensive MT polymerization, where as in their phosphorylated state their ability to promote MT assembly is diminished. The Tau protein, especially in Alzheimers brains, is found to be phosphorylated at many different sites. In vitro studies have shown that Tau is a substrate for a multitude of protein kinases, including CaM kinase II, casein kinase II, protein kinase A, MAP kinase (ERK2), cdk5/p35, and glycogen synthase kinase 3. It is believed that the phosphorylation of specific sites, rather than the overall extent of phosphorylation, is important in modulating the ability of Tau to bind MTs. Anti-mitotic agents that can selectively disrupt MT dynamics, either by targeting a specific tubulin isotype or a particular stage of cell division, have
great therapeutic value as chemotherapeutic agents. These agents exploit the difference in MT dynamics between rapidly dividing cancerous cells and normal cell populations. For example, drugs such as colchicine and colcemid bind tubulin and inhibit MT polymerization, thus blocking mitosis. On the other hand, agents such as paclitaxel (Taxol) stabilize MTs and prevent cell division. Hence, Microtubule-targeting antimitotic agents could be classified as microtubuledestabilizing agents (e.g., vinblastine, vincristine, colchicine etc.) or microtubule-stabilizing agents (e.g., paclitaxel, epothilones etc.). Paclitaxel is a highly complex molecule isolated from the bark of the yew tree. It binds to the -subunit of tubulin. It reportedly gains access to its binding sites by diffusing through small openings in the microtubule or fluctuations of the microtubule lattice. Each tubulin molecule has one paclitaxelbinding site. Once bound to the site on the interior of microtubule, it stabilizes the microtubule and increases microtubule polymerization. The suppression of spindle-microtubule dynamics caused by paclitaxel blocks the entry of the dividing cancer cells from metaphase into anaphase, which leads to apoptotic cell death.
References: Gallagher, B.M. Jr. 2007. Curr. Med. Chem. 14, 2959. Nogales, E., and Wang, H.W. 2006. Curr. Opin. Struc. Biol. 16, 221. Jordan, M.A., and Wilson, L. 2004. Nat. Rev. 4, 253. Yeh, I.T., and Luduena, R.F. 2004. Cell Motil. Cytoskeleton 57, 96. Hari, M., et al. 2003. Mol. Cancer Therap. 2, 597. Hadfield, J.A., et al. 2003. Prog. Cell Cycle Res. 5, 309. Nogales, E. 2001. Annu. Rev. Biophys. Biomol. Struct. 30, 397. Downing, K.H., and Nogales, E. 1998. Curr. Opin. Cell Biol. 10, 16. Jordan, M.A., and Wilson, L. 1998. Curr. Opin. Cell Biol. 10, 123. Wade, R.H., and Hyman, A.A. 1997. Curr. Opin. Cell Biol. 9, 12. Waterman-Storer, C., and Salmon, E.D. 1997. Curr. Biol. 7, 369.
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14 | Tubulin Polymerization and Depolymerization
Chapter Fifteen
APOPTOSIS: Receptor and Mitochondrial Gateways to Cell Death
The study of apoptosis has gained significant importance in human disease and its clinical management. Apoptosis is a highly regulated normal process in development and morphogenesis, however, failure to regulate apoptosis can lead to several diseases, including autoimmune disorders, neurodegenerative diseases, and cancer.
15
Apoptosis occurs when cells have sufficient time to organize and participate in their own demise. In response to specific signals, cells undergo apoptosis, which is manifested by a number of distinctive biochemical and morphological changes. Cells undergoing apoptosis exhibit shrinkage, membrane blebbing, chromatin condensation, and extensive nuclear fragmentation. Apoptosis is a carefully controlled programmed cell death that does not lead to any inflammation in the organism. On the other hand, necrosis, is a more frenzied cell death resulting from circumstances outside the cell and leads to edema and disruption of the plasma membrane. Many cells can be activated to undergo apoptosis following the interaction of selected ligands with cell surface receptors. The most well studied receptors are the Fas/APO-1 (apoptosis inducing protein 1) and tumor necrosis factor receptor 1 (TNFR1). Apoptosis mediated by both signaling cascades end in activation of caspases. Thus far fourteen caspases have been identified and are divided into three groups, which include initiator caspases (caspase 2, 8, 9, and 10), executioner caspases (caspase 3, 6, and 7), and caspases involved in cytokine processing (caspase 1, 4, 5, 11-14). Caspases are synthesized as single chain, ~45 kDa proenzymes that are activated by two cleavages. The first cleavage separates the
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| APOPTOSIS: Receptor and Mitochrondrial Gateways to Cell Death
N-terminal pro-domain and the second cleavage

separates the large (~p20) and the small (~p10) subunits that form a heterodimeric active caspase with two symmetrically arranged active sites at opposite ends of the molecule. Caspase activation is generally considered as the point of no return in apoptotic pathways. Activated caspases cleave key substrates required for normal cellular functions, such as the cytoskeletal proteins, nuclear proteins, and DNA repair enzymes. One of the major substrates of caspases is PARP (poly(ADP-Ribose) polymerase) whose cleavage by caspases renders it inactive and leads to cessation of DNA repair mechanisms. Caspases are activated either via the receptormediated (Fas ligand or TNF-mediated) pathway or via the mitochondrial pathway. The receptormediated pathway leads to the activation of procaspase-8. In the mitochondrial pathway, proapoptotic members of the Bcl-2 family associate with mitochondria and direct the release of cytochrome c (Cyt c) and other proteins, which activate procaspase-9. Fas-mediated death occurs much more rapidly than that triggered by TNFR1. When Fas interacts with Fas ligand (FasL), it bypasses the usual long sequence of signaling
enzymes and immediately activates Caspase-8 Action of Fas is mediated by Fas-associated death domain (FADD). Tumor necrosis factor (TNF) is another well known mediator of apoptosis. Its cellular response is mediated by TNFR1 that contains a death domain sequence localized on its carboxy terminus. TNFR1 interacts with a TNFR1-associated death domain (TRADD) to induce apoptotic changes in cells via downstream activation of caspases. A second family of signaling proteins utilized by TNFR are TRAFs (TNFR-associated factors). TRADD also interacts with FADD via its death domain. TNF activates NF-B exclusively through endogenous TNFR1 via interaction with TRAF2. Over expression of TRAF2 activates NF-B, but does not induce cell death. NF-B, consists of two subunits (p50 and p65) that exist in a complex with IB in resting cells. The signal from TRAF2 phosphorylates IB and causes the release of NF-B, which enters the nucleus to activate various genes carrying NF-B response element. Activation of Fas and TNFR also results in the stimulation of sphingomylinase that catalyzes the formation of ceramide and leads to apoptosis via JNK/SAPK cascade activation.
FAS and TNFR linked apoptotic cell death
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Until last decade, the role of mitochondria in apoptosis was rather obscure. This was due to the fact that mitochondria do not exhibit any prominent morphological changes during apoptosis. They contain all the components necessary to annihilate the cell by apoptosis, without giving any appearance of self-destruction. However, studies have shown that mitochondria undergo several major changes, including the changes in their membrane integrity, even before any classical signs of apoptosis appear. Susin, et al. (1998) have divided the apoptosis phenomenon into three phases - a pre-mitochondrial phase, which involves the activation of damage pathways; a mitochondrial phase, which involves the loss of mitochondrial functions; and a postmitochondrial phase, in which proteins released from the mitochondria activate caspases and nucleases to facilitate the ultimate cell demise. Bax and other pro-apoptotic members of the Bcl-2 family show some structural similarities with pore-forming proteins. Hence, it is believed that Bax can form transmembrane pores across the outer mitochondrial membrane, which leads to a loss of membrane potential. The localization of Bax has been shown to change from the cytosol to the mitochondria during apoptosis. Bcl-2 and other anti-apoptotic members of the Bcl-2 family are located in the outer mitochondrial membrane. Bcl-2 is especially enriched at contact sites where the inner and outer membranes come in close proximity. It acts on mitochondria to counteract the action of pore-forming Bax protein. A number of mechanisms have been described to highlight the mitochondrial involvement in apoptosis. High levels of cytosolic Ca2+ and excessive concentrations of reactive oxygen species are known to contribute to the opening of the mitochondrial permeability transition pore (PTP), which depolarizes the mitochondria. The PTP participates in the regulation of matrix Ca2+ levels, pH, and the volume. Adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC) are the two principal components of PTP. The VDAC operates at the inner and outer membrane contact sites and creates a channel that allows non-specific passage of ions and molecules smaller than 1.5 kDa. The opening of this channel in the inner membrane allows for equilibration of ions within the matrix and the intermembrane space. This dissipates the electrochemical gradient (m) and uncouples the respiratory chain, leading to the cessation of ATP production. The disruption
of the m is attributed largely to the damage of the inner mitochondrial membrane. This event is believed to occur even before DNA fragmentation indicating that mitochondrial injury is an early event in apoptosis. The use of cationic lipophilic dyes, such as DiOC6(3) has shown that a disruption of the m may even precede the activation of caspases. Several apoptosis inducing agents are known to trigger mitochondrial uncoupling that result in a diminution of m. In the low-conductance state, the opening of PTP is pH dependent, which permits the diffusion of only small ions and then closes spontaneously. However, in the high-conductance state, the channel is stabilized in an open position and allows water and larger molecules to enter the protein rich matrix, which results in mitochondrial swelling. Consequently, the inner membrane, which has a far greater surface area than the outer membrane, unfolds exerting pressure on the outer membrane. As a result, the outer membrane ruptures and causes the release of pro-apoptotic factors, such as apoptosis inducing factor (AIF) and Cyt c into the cytosol where Cyt c and apoptosis protease activating factor-1 (Apaf-1) act as cohorts in the activation of caspase-9. Cyt c, Apaf-1, ATP, and pro-caspase-9 come together to form a complex known as the apoptosome. Binding to Cyt c makes Apaf-1 more competent at binding pro-caspase-9. The N-terminus of Apaf1 has the caspase recruitment domain (CARD), which is shared by several caspases. Since Apaf-1 does not have any caspase activity, pro-caspase-9 is believed to be activated through autocatalysis in the apoptosome. Thus, Cyt c release from the mitochondria is considered as a key signal that initiates the irreversible events in cell death. The AIF, a 67 kDa nuclear encoded flavoprotein, is generally confined to the mitochondrial intermembrane space. AIF has been termed a fellow escapee by Waterhouse and Green (1999) that ensures eventual cell death. AIF is capable of activating caspase-3 and endonucleases independently of Cyt c. It induces a large-scale DNA fragmentation and a loss of mitochondrial transmembrane potential. A wide range of chemotherapeutic agents are known to induce AIF release from the mitochondria. On the other hand, anti-apoptotic agents, such as Bcl-2, inhibit the translocation of AIF. Another mitochondrial factor, second mitochondrial activator of caspases/direct inhibitor of apoptosis binding protein with low pI (Smac/DIABLO), has been identified that acts by binding to the inhibitor of
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15 | APOPTOSIS: Receptor and Mitochrondrial Gateways to Cell Death
apoptosis proteins (IAPs) and thereby potentiates apoptosis by relieving the inhibition of IAPs on caspases. Smac is an intermembrane protein that is released from mitochondria along with Cyt c and promotes Cyt c-dependent caspase activation. WOX1, a mitochondrial oxidoreductase, also migrates to the nucleus following induction of apoptosis. Phosphorylation of WOX1 at Tyr33 is considered to be essential for its cytotoxic effects in the nucleus. Endonuclease G, a 50 kDa nonspecific DNA/RNA nuclease, is also released from mitochondria during apoptosis and contributes to chromatin fragmentation. Despite volumes of data linking mitochondrial events to apoptotic cell death, some researchers believe that the release of Cyt c is only distal to caspase activation in Fas-activated apoptosis, and the mitochondrial signaling may not be essential for apoptosis. In their opinion, primary apoptosis signals are routed directly to caspase activation, and mitochondrial events only contribute to the amplification of cell death. These conclusions
may be based on the observation that caspase inhibitors, such as Z-VAD-FMK, block the release of Cyt c from mitochondria. It is of interest to mention here that both caspase-8, activated via the receptor-mediated pathway, and caspase-9, activated via the mitochondrial pathway, participate independently in the activation of pro-caspase-3, the active form of which is termed the central executioner of apoptosis. A point of clinical consideration may be the selective eradication of transformed cells by highly selective mitochondrial-specific chemotherapeutic agents, which may either prevent the decrease in m or block the Bcl-2-mediated stabilization of mitochondrial membranes. Some of the factors that mediate DNA damage in apoptosis are proteins that transmit apoptotic signals out of the nucleus and expedite apoptotic cell death. Nur77/TR3 is recognized as a nuclear messenger of apoptosis that translocates to the mitochondria and releases Cyt c. It binds to Bcl-2 and triggers a conformational change, which
Role of mitochondria in apoptotic cell death
64
exposes the pro-apoptotic BH3 domain. This converts Bcl-2 from an anti-apoptotic to a proapoptotic factor. Nur77/TR3 contains two nuclear localization and three nuclear export sequences. It is shown to heterodimerize with retinoid X receptor and is actively transported from the nucleus to the mitochondria in cells undergoing apoptosis. Research over the last few years have shown that although caspases are vital for the typical apoptotic morphology, caspase-independent cell death also co-exists with caspase-dependent death. It is shown that inhibition of caspase activities in Jurkat cells can still lead to cell death by a caspase-independent mechanism. Major differences between caspase-dependent and independent cell death are provided in the table below. Defects in p53 and bcl2 genes are associated with proliferative disorders and apoptosis. It is believed that p53, a key tumor-suppresser protein, accumulates when DNA is damaged and arrests the cell cycle at the G1 phase to allow extra time for repairs. However, if the repair process fails, p53 triggers apoptosis. When p53
is dysfunctional, apoptosis fails to occur. Also, an over expression of Bcl-2 retards normal apoptotic process. Anticancer drugs mediate their effect by triggering apoptosis. There is strong evidence to suggest that cancer cells can increase their resistance to anticancer drugs by increasing the expression of Bcl-2 that leads to inhibition of apoptosis. Inactivation of p53 also contributes to the initiation and progression of cancer. Hence, the p53 status becomes a strong determinant of response to treatment with anticancer agents. Genotoxic compounds such as 5-fluorouracil, etoposide, and adriamycin can increase p53 levels and cause growth inhibition in tumor cells. The mitochondrial (intrinsic) apoptotic pathway, activated by cytotoxic drugs ultimately leads to the activation of caspases that cause cell death in tumor cells. In addition, activation of the receptor-linked (extrinsic) apoptotic pathway leads to enhanced sensitivity of tumor cells toward cytotoxic agents. Hence, apoptosis is an important area of study where the ability to achieve a significant therapeutic index and differentiating normal cells from tumor cells may lower the threshold at which cell injury triggers apoptosis.
15 | APOPTOSIS: Receptor and Mitochrondrial Gateways to Cell Death
Differences Between Caspase-Dependent and Caspase-Independent Apoptosis

Caspase-Dependent Apoptosis Nucleus DNA Laddering Chromatin condensation Nuclear fragmentation Mitochondria Loss of membrane potential Outer membrane permeablization Plasma Membrane Phosphatidylserine translocates Annexin V positive Exclusion of vital dyes Cell Loss of proliferation capacity Apoptotic bodies present Cell detachment from matrix Phagocytosis of dying cells Loss of proliferation capacity No apoptotic bodies present Cells remain attached to matrix Unknown mechanism of disposal of dead cells Caspase-Independent Apoptosis No DNA Laddering Shrinkage of nucleus Marginalization of chromatin around nuclear membrane Membrane potential stays intact Swollen cristae Outer membrane swollen or absent Usually Annexin V negative Vital dye positive
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References: Ow, Y.L., et al. 2008. Nat. Rev. Mol. Cell Biol. 9, 532. Salvesen, G.S., and Riedl, S.J. 2008. Adv. Exp. Med. Biol. 615, 13. Belizrio, J.E., et al. 2007. Braz. J. Med. Biol. Res. 40, 1011. Ardehali, H. 2005. J. Bioenerg. Biomembr. 37, 171. Broker, L.e., et al. 2005. Clin. Cancer Res. 11, 3155. Ferrando-May, A. 2005. Cell Death Differen. 12, 1263. Green, D.R. 2005. Cell 121, 579. Hail. N. Jr. 2005. Apoptosis 10, 687. Orrenius, S., et al. 2004. Toxicol. Lett. 149, 19. Philchenkov, A. 2004. J. Cell. Mol. Med. 8, 432. Zhang, A., et al. 2004. Neuroembrology 3, 47. Liston, P., et al. 2003. Oncogene 22, 8568. Mayer, B., and Oberbauer, R. 2003. New Physiol. Sci. 18, 89. Ni, C-Z., et al. 2003. J. Mol. Recog. 3, 121. Finkel, E. 2001. Science 291, 624. Roberts, D.L., et al. 2001. J. Cell Biol. 153, 221. Shi, Y. 2001. Nat. Struct. Biol. 8, 394. Daugas, E., et al. 2000. FEBS Lett. 476, 118 Gorman, A.M., et al. 2000. Dev. Neurosci. 22, 348. Gottlieb, R.A. 2000. FEBS Lett. 482, 6. Shimizu, S., et al. 2000. J. Biol. Chem. 275, 12321. Srinivasula, S.M., et al. 2000. J. Biol. Chem. 275, 36152 Crompton, M. 1999. Biochem. J. 341, 233. Susin, S.A., et al. 1999. Nature 397, 441. Waterhouse, N.J., and Green, D.R. 1999. J. Clin. Immunol. 19, 378. Cai, J., et al. 1998. Biochim. Biophys. Acta 1366, 139. Reed, J.C., et al. 1998. Biochim. Biophys. Acta 1366, 127. Susin S.A., et al. 1998. Biochim. Biophys. Acta 1366, 151. Knudson, C.M., and Korsmeyer, S.J. 1997. Nat. Gen. 16, 358. Kroemer, G., et al. 1997. Immunol. Today 18, 44. Nagata, S. 1997. Cell 88, 355. Yin, C., et al. 1997. Nature 385, 637. Zhivotovsky, B., et al. 1997. Arch. Biochem. Biophys. 230, 481. Cuvillier, O., et al. 1996. Nature 381, 800. Hsu, H., et al. 1996. Cell 84, 299. Liu, X.S., et al. 1996. Cell 86, 147. Susin, S.A., et al. 1996. J. Exp. Med. 184, 1331. Wertz., I.E., and Hanley, M.R. 1996. Trends Biochem. Sci. 21, 359. Whyte, M., et al. 1996. Trends Cell Biol. 6, 245. Zamzami, N., et al. 1996. J. Exp. Med. 183, 1533. Cifone, M.G., et al. 1995. EMBO J. 14, 5859. Carston, D.A., and Lois, A. 1995. Lancet 346, 1009. Enari, M., et al. 1995. Nature 375, 78. Kischkel, F.C., et al. 1995. EMBO J. 14, 5579. Hannun, Y.A., et al. 1994. J. Biol. Chem. 269, 3125. Reed, J.C. 1994. J. Cell Biol. 124, 1. Smith, C.A. et al. 1994. Cell 76, 959. Wilson, K.P., et al. 1994. Nature 370, 270. Fisher, T.C., et al. 1993. Cancer Res. 53, 3321. Cohen, J.J., and Duke, R.C.. 1992. Annu. Rev. Immunol. 10, 267. Lane, D.P. 1992. Nature 358, 15. Thornberry, N.A., et al. 1992. Nature 356, 768.
Chapter Sixteen
p53: Choice of Response: Repair or Death
p53, a well-conserved phosphoprotein, is one of the best known tumor suppressors. Human p53 consists of 393 amino acids assembled into four structurally and functionally different domains: an acidic N-terminal region that contains the 42 amino acid transactivation domain followed by a hydrophobic proline-rich region (amino acids 6492), a central sequence-specific DNA-binding domain (amino acids 102292), a tetramerization domain (amino acids 324355), and a highly basic C-terminal region regulatory domain (amino acids 363393).
p53 is a sequence-specific nuclear transcription factor that binds to defined consensus sites within DNA as a tetramer and represses
16 | p53: Choice of Response: Repair or Death
transcription of a set of genes involved in cell growth stimulation, while activating a different set of genes involved in cell cycle control. It causes growth arrest before either DNA replication in the G1 phase or before mitosis in the G2 phase. This provides a window for DNA repair or elimination of cells with severely damaged DNA strands. Hence, p53 is considered as an important regulator of DNA repair that ensures genomic integrity. Agents that damage DNA induce p53 to become very stable by a post-translational mechanism, allowing its concentration in the nucleus to increase dramatically. In unstressed cells, p53 is latent and is maintained at low levels by targeted degradation mediated by MDM2. Through its binding to p53, MDM2 can shuttle p53 out of the nucleus into the cytoplasm for degradation. When normal mammalian cells are subjected to stress signals, such as hypoxia, radiation, or chemotherapeutic
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drugs, p53 is phosphorylated at multiple sites, including those involved in its binding to MDM2. This leads to its activation and blockage of its ubiquitin-dependent degradation. Activation of p53 can result in cell cycle arrest, presumably to allow for DNA repair before replication or mitosis.
In some cell types, however, p53 activation results in apoptosis as means of eliminating severely damaged cells. The final outcome of p53 activation depends on many factors, and is mediated largely through the action of downstream effector genes transactivated by p53.
p53
MDM2
MD
M2
p53
ATM Stress Signals ATR DNA-PK ATP
Ub
Ub Ub Ub Ub
p53
ADP
Ub
Ub
DNA Damage
Other Protein Kinases Proteasome

P P P P P
Activated CBP/p300 pCAF

Ac
p53
Ac
Ac
p21 gene expression

p21 Cdk Cyclin Cdk
Cell Cycle Arrest
Cell Cycle
p21
Cyclin
DNA damage and p53 activation
Human p53 is phosphorylated at least at 23 different sites by stress-activated protein kinases, DNA protein kinase (DNA-PK), casein kinase I and II, and cyclin-dependent kinases. Although the exact functions of specific phosphorylation at various sites is still controversial, evidence indicates that phosphorylation of p53 provides stability by promoting its dissociation from MDM2 and enhancing its transcriptional activity. Most of the p53 phosphorylation sites are clustered within the 40 amino acids at its N-terminus. ATM and ATR kinases promote phosphorylation of human p53 at Ser15 and Ser20, which are essential for the activation of p53 following DNA damage. DNAPK phosphorylates Ser15 within the critical Nterminal region of p53, which controls the interaction of p53 with the transcriptional apparatus and with the MDM2 protein. DNA-
PK also phosphorylates Ser9 and Thr18; however, phosphorylation at these sites is dependent upon the presence of full-length p53, but is independent of phosphorylation at other sites. Phosphorylation at Thr18 alters the structure of the amphipathic -helix with which MDM2 interacts. Studies have shown that when p53 co-localizes with DNA-PK and ssDNA, there is a 10-fold enhancement of p53 phosphorylation. Casein kinase I can also phosphorylate Ser9 and Thr18, however, these phosphorylations are dependent upon prior phosphorylation of Ser6 and Ser15. All types of tumor cells exhibit higher levels of p53 phosphorylation when compared to normal non-transformed cells. These phosphorylations offer greater stability to p53 regardless of p53 mutations.
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In spite of extensive studies on p53 phosphorylation, it is now known that phosphorylation is not the only mechanism that regulates activation of p53. Following cellular stress, p53 is shown to be acetylated by CBP/p300 at multiple lysine residues (Lys370, 372, 373, 381, and 382) and by pCAF at Lys320. The physiological relevance of p53 acetylation is still controversial, although acetylation does correlate well with increased cellular stress. Additional support for the role of acetylation comes from studies that show that increasing the level of p53 acetylation with deacetylase inhibitors prevents p53 degradation. Over-expression of MDM2 is also shown to effectively reduce p300-dependent p53 acetylation. p53 is shown to be either non-functional or mutated in most human cancers. The most common anomaly of p53 in human cancers is mutation of the p53 gene. A large number of mutations are caused by single base substitutions and about 30% of these mutations are reported to occur in hotspot codons. Functional p53 provides a protective mechanism against tumor growth and a loss of p53 function is a key step in the neoplastic cascade. In addition, the function of p53 is critical to the success of many cancer treatments since radiation and chemotherapy act in part by triggering cell suicide in response to DNA damage. A successful response to therapy is greatly reduced in tumors where mutant p53 is present, and these tumors are often very difficult to treat. The p53 network in normal, non-activated situations is non-functional, but is activated in cells as a response to various signals that take place in the carcinogenic process. Carcinogeninduced DNA damage, abnormal proliferative signals, hypoxia, and loss of cell adhesion are some of the most common signals that activate p53.
Phe19, Trp23, and Leu26 to fill up a complementary hydrophobic pocket of MDM2. The three amino acids are also essential for transactivation of p53. Binding of MDM2 to p53 antagonizes the transcriptional activity of p53 and blocks its acetylation and transactivation by interfering with p300/CBP. MDM2 is believed to function as an E3 ligase to ubiquitinate p53 and force its export from the nucleus to the cytoplasm where it is degraded in the proteasome. Some researchers believe that E3 ubiquitin ligase activity of MDM2 alone is not sufficient to trigger p53 degradation. This is due to the fact that MDM2 mono-ubiquitinates p53 at multiple sites, but does not catalyze the addition of polyubiquitin chains, which are essential for recognition by the proteasome. One possibility exists that mono-ubiquitination of p53 exposes a nuclear export signal; polyubiquitination and degradation can then proceed in the cytoplasm. Although p53-mediated transactivation is a nuclear event, p53 degradation occurs in the cytoplasm. Hence, the ubiquitin ligase function of MDM2 could serve as a cellular mechanism for turnover of p53-MDM2 complexes after their function is completed. It is generally agreed that the nuclear export signal of MDM2 is required for p53 degradation. Studies have shown that Leptomycin B, a blocker of nuclear export, also prevents nuclear-cytoplasmic shuttling of MDM2 and p53. p53, if sequestered in the cytoplasm, is resistant to degradation by MDM2. Import of p53 from the cytoplasm to the nucleus and export back to cytoplasm seems to be essential for its degradation, and the shuttling of MDM2 and p53 may be a mechanism to prevent their premature degradation. Malignant tumors, particularly breast tumors and soft tissue sarcomas, are reported to frequently overexpress MDM2. In breast cancer cells, overexpression of MDM2 is correlated with lack of p21 expression. However, overexpression of MDM2 in normal cells is known to cause G1 arrest. Hence, MDM2 induced by DNA damage in normal cells may have a protective role in preventing untimely cell cycle progression.
16 | p53: Choice of Response: Repair or Death
MDM2: The Regulator of p53 Degradation

MDM2, a p53-inducible phosphoprotein, binds to the N-terminus of the p53 and negatively regulates its activity. Mdm2 is a p53 responsive gene and its transcription can be activated by p53. Hence, in the presence of high levels of p53, MDM2 levels are also elevated. p53 interacts with MDM2 at
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References: Brooks, C.L., and Gu, W. 2006. Mol. Cell 21, 307. Lee, M.H., and Lozano G. 2006. Semin. Cancer Biol. 16, 225. Toledo, F., and Wahl, G.M. 2006. Nat. Rev. Cancer 6, 909. Watson, I.R., and Irwin, M.S. 2006. Neoplasia 8, 655. Bode, A.M., and Dong, Z. 2004. Nat. Rev. Cancer 4, 793. Luo, J., et al. 2004. Proc. Natl. Acad. Sci. USA 101, 2259. Soubeyrand, S., et al. 2004. Eur. J. Biochem. 271, 3776. Deb, S.P. 2003. Mol. Cancer Res. 1, 1009. Iwakuma, T., and Lazano, G. 2003. Mol. Cancer Res. 1, 993. Oren, M. 2003. Cell Death Differen. 10, 431. Dang, J., et al. 2002. Cancer Res. 62, 1222. Vousden, K.H., and Lu, X. 2002. Nat. Rev. Cancer 2, 594. Ito, A., et al. 2001. EMBO J. 20, 1331. Minamoto, T., et al. 2001. Oncogene 20, 3341. Prives, C., and Manley, J.L. 2001. Cell 107, 815. Hainaut, P., and Hollstein, M. 2000. Adv. Cancer Res. 77, 81. Vousden, K.H. 2000. Cell 103, 691. Craig, A.L., et al. 1999. Biochem. J. 342, 133. Zaika, A., et al. 1999. J. Biol. Chem. 274, 27474. Freedman, D.A., and Levine, A.J. 1998. Mol. Cell Biol. 18, 7288. Levine, A.J. 1997. Cell 88, 323. Sigalas, I., et al. 1996. Nat. Med. 2, 912.
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Chapter Seventeen
Ubiquitination and Proteasomal Degradation of Proteins
Proteasomes are large multi-subunit protease complexes, localized in the nucleus and cytosol that selectively degrade intracellular proteins. They play a major role in the degradation of many proteins involved in cell cycling, proliferation, and apoptosis.
A vast majority of short-lived proteins are degraded by the ubiquitin-proteasome pathway. They also participate in the removal of misfolded or aged housekeeping proteins. A protein marked for degradation is covalently attached to multiple molecules of ubiquitin (Ub), a highly conserved 76-amino acid (8.6 kDa) protein, which escorts it for rapid hydrolysis to the multi-component enzymatic complex known as the 26S proteasome. The proteolytic core of this complex, the 20S proteasome, contains multiple peptidase activities and functions as the catalytic machine. Unlike other proteases, the 20S proteasome ensures that virtually all peptide bonds within a protein substrate are susceptible to cleavage, by possessing multiple proteolytic activities in one proteolytic internal chamber. This core is composed of 28 subunits arranged in four heptameric, tightly stacked, rings (7, 7, 7, 7) to form a cylindrical structure. The -subunits make up the two outer, and the -subunits the two inner rings of the stack. The entrance of substrate proteins to the active site of the complex is guarded by the -subunits that only allow access for unfolded and extended polypeptides. The proteolytic activity is confined to the 1, 2, and 5-subunits. The proteolytic sites are formed by N-terminal threonine residues that face the central cavity of the 20S complex. The regulatory unit of the 26S proteasome is known as the 19S (PA 700) particle, and consists of at least 15 subunits that include
17 | Ubiquitnation and Proteasonmal Degradation of Proteins
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ATPases, a de-ubiquitinating enzyme, and polyubiquitin-binding subunits. ATPases function to continuously supply energy for selective degradation of target proteins. Their function includes recognition, deubiqutination, unfolding, and translocation of substrate proteins to the 20S complex. Energy is required for unfolding of proteins to allow them to penetrate the channel of -and -rings of the 20S proteasome. PA28 (11S regulator), a ring-shaped particle that associates with the 19S unit at both ends of the 20S proteasome, functions as an activator protein. It is composed of two homologous proteins, PA28 and PA28 that are reported to stimulate peptidase activities without affecting the degradation of large protein substrates.
In the ubiquitin-proteasome degradation pathway, Ub is first covalently ligated to target proteins by a multi-enzymatic system consisting of Ub-activating (E1), Ub-conjugating (E2), and the Ub-ligating (E3) enzymes. Ub molecules provide a recognition signal for the 26S proteasome. In the first step of reactions Gly76 of ubiquitin is activiated by E1. This step generates ubiquitinyl adenylate intermediate bound to E1. The E1 activates an Ub monomer at its C-terminal cysteine residue to a high-energy thiol ester bond that is then transferred to a reactive cysteine residue of the E2 enzyme in a transacylation reaction. The
final transfer of ubiquitin to -amino group of a reactive lysine residue of substrate proteins is brought about by the E3, the Ub-ligase enzyme. This final reaction forms an amide isopeptide bond between the carboxyl group of Gly76 of Ub and -amino group of lysine residues in proteins. More recently, an additional conjugation factor, E4, has been identified that binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjuction with E1, E2, and E3. Ubiquitinated protein is escorted to the proteasome where it undergoes final degradation and the ubiquitin is released and recycled. The unique and distinguishing feature of the proteasome is the presence of multiple peptidase activities that include chymotrypsin-like (cleavage after hydrophobic side chains), postglutamyl peptidase (cleavage after acidic side chains), and trypsin-like (cleavage after basic side chains) activities. It has been proposed that the intact protein substrates are first recognized by the 19S unit, which allows them to enter the proteasome cavity where PA28 stimulates their cleavage by peptidases. Another peptidase, tripeptidyl peptidase II (TPPII) has also been identified that copurifies with the 26S proteasome. TPPII is believed to participate in the degradation of extra-lysosomal polypeptides and may substitute for some metabolic functions of the proteasome, particularly in the absence of normal proteasome function.
A typical ubiquitination and proteasomal degradation cycle.
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The ubiquitin-proteasome pathway plays a major role in the breakdown of abnormal proteins that result from oxidative stress and mutations that might otherwise disrupt normal cellular homeostasis. The reactive oxygen species can promote partial unfolding of proteins, exposing their hydrophobic domains to proteolytic enzymes of the 20S complex. Rapid degradation of defective enzymes, as seen in diseases caused by metabolic abnormalities, also occurs in the proteasome. However, it is not known how the Ub system recognizes the abnormal state of these proteins. In the cell, proteasomes are located both in the nucleus and in cytoplasm. They constitute about 1% of total cell protein. Most proteasomes diffuse freely within cytoplasm and nucleoplasm. However, a small fraction is associated with different structures within the cell. In the nucleus, proteasomes are present throughout the nucleoplasm, but not in the nucleolus. They also associate with subnuclear domains called the PML nuclear bodies. In the cytoplasm they are associated with the outer surface of the endoplasmic reticulum and the cytoskeleton. The Ub-proteasome pathway has been implicated in several forms of malignancy, in the pathogenesis of several genetic diseases, and in the pathology of muscle wasting. It is also involved in the destruction of proteins that participate in cell cycle progression, transcription control, signal transduction, and metabolic regulation. Degradation of CDK activators and inhibitors by the proteasome complex regulates the progression of the cell cycle. It is believed that phosphorylation of various proteins, such as cyclin E, cyclin D, p27, IB, and STAT1 allows them to be ubiquitinated and marked for proteolysis by the proteasome complex. On the other hand, phosphorylation of certain other proteins, such as c-Fos and c-Jun, prevents their ubiquitination. This further indicates a direct involvement of the proteasome in cell proliferation and the cell cycling processes. Several elements of cell cycling process that are degraded in the Ub-proteasome pathway are potential targets for deregulation in tumors. For example, cyclins B, D1, and E that are rapidly degraded in this pathway are overexpressed in breast tumor cell lines. Alterations in proteasome activity in tumors
have also been linked to multi-drug resistance in mammalian cells. The study of Ub-dependent degradation of p53, a tumor suppressor gene product, has opened up a new arena of study of apoptosis and cancer. The accumulation of p53 is thought to occur mainly via the down-regulation of its degradation in the proteasome pathway. Human papiloma virus (HPV)-related cancers are linked to an up-regulation of p53 degradation in the Ub-proteasome pathway. Low levels of p27, an inhibitor of the CDK complex, reported in several common tumors, are shown to be due to its increased degradation in the proteasome complex. Another area currently under investigation is the mechanism by which nuclear de-ubiquinating enzyme, BAP1, binds to the breast cancer suppressor, BRCA1. Augmentation of the growth suppressive effects of BRCA1 are attributed to the overexpression of BAP1. Given the wide range of cellular substrates that are degraded by the ubiquitin-proteasome pathway, it has become an attractive target for therapeutic intervention in several diseases. Velcade (bortezomib), a proteasome inhibitor has been approved for clinical use in multiple myeloma patients. Although the ubiquitinproteasome pathway can be blocked at several steps, most inhibitors developed directly target the proteolytic activities of the 20S proteasome. Several distinct groups of compounds, designed to act as proteasome inhibitors, have helped immensely in understanding the biological role and importance of ubiquitin-proteasome pathway. These compounds block proteasome function without affecting the normal biological processes in the cell. The most notable of these is lactacystin (Calbiochem Cat. No. 426100) that acts as a covalent inhibitor of the chymotrypsin-like and trypsin-like activities of proteasome. Lactacystin targets the 20S proteasome by an irreversibly modifying the amino terminal threonine of -subunits. The active component of lactacystin is the clasto-lactacystin -lactone (Calbiochem Cat. No. 426102), which is a rearrangement product of lactacystin under aqueous conditions. It reacts with the hydroxyl group of the amino terminal threonine to make an ester adduct. It is shown to inhibit cell cycle progression and induce apoptosis in human monoblast cells. The table below highlights the mechanism of inhibition of a few selected proteasosme inhibitors.
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17 | Ubiquitnation and Proteasonmal Degradation of Proteins
Peptide aldehyde inhibitors, such as MG-132 and MG-115 are potent chymotrypsin-like activity blockers and are widely used in studying the role of the proteasome in various cellular processes. Vinyl sulfone compounds are irreversible, active site directed inhibitors of proteasosme. They modify the hydroxyl group of the amino terminal threonine. A major concern with use of vinyl sulfone inhibitors is their lack of specificity.
They are also known to inhibit other cysteine proteases. Another class of potent peptide-based inhibitors employs boronic acid functional group. They inhibit proteasome activity by forming a noncovalent complex. In these compounds a stable tetrahedral borane complex allows the peptide chain length of the boronic acid-based inhibitor to be truncated to a dipeptide with good retention of inhibitory activity.
Mechanism of Inhibition of Selected Proteasome Inhibitors

Proteasome Inhibitor Aclacinomycin Epoxomycin Mechanism of Inhibition Inhibits the chymotrypsin-like activity of the proteasome. Covalently binds to the 5 and 2 catalytic subunits of the 20S proteasome. Inhibits primarily the chymotrypsin-like activity of the proteasome. Covalently binds to the 5 subunit. Also inhibits cathepsin A and tripeptidyl peptidase II activities. Reversible inhibitor of the chymotrypsin-like activity of the proteasosme. Also inhibits cathepsins and calpains. Competitive inhibitors, largely specific for individual subunits of the 20S proteasome. Also inhibit intracellular cysteine proteases.
Lactacystin MG-132 Vinyl Sulfone Tripeptides
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Chapter Eighteen
Cancer Stem Cells: The Real Perpetrators in Cancer Growth and Progression
Stem cells, unspecified precursor cells, have the unique ability to self-renew and generate additional stem cells as well as to differentiate into various progenitor cells in response to appropriate signals. These functional properties have led researchers to explore opportunities for developing new strategies for tissue repair, replacement, and regeneration.
Stems cells are classified as either embryonic stem cells (ESC) or adult stem cells. ESC are derived from the inner cell mass of preimplantation embryos and are considered to be the most pluripotent. They can undergo infinite, undifferentiated proliferation in vitro
18 | Cancer Stem Cells: The Real Perpetrators in Cancer
and can also differentiate into a wide variety of somatic and extraembryonic tissues. Adult stem cells are unspecialized cells found in differentiated tissues that can self-renew and differentiate into mature cell types of the specific tissue. In contrast to ESC, adult stem cells can proliferate only for a limited number of cycles and their response to differentiation signals declines with each cycle.
Comparison of Embryonic and Adult Stem Cells

Embryonic Stem Cells Pluripotent Highly proliferative Potentially Tumorigenic Ethical Concerns Non-autologous Adult Stem Cells Multipotent Limited Proliferative Capacity Non-tumorigenic Less Controversial Autologous
The term cancer stem cells refers to a subpopulation of cancer cell that can self renew, generate diverse cells in the tumor mass, and sustain tumorigenesis. The origin of cancer itself is a highly controversial issue. Some researchers believe that cancer arise from cancer stem cells that originate as a result of mutational hits on
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normal stem cells or by the transformation of restricted progenitor cells or even the differentiated cells that acquire self-renewing capacity. These cancer stem cells drive tumor progression and recurrence. Another theory is based on the clonal evolution model, which suggests that some tumor cells acquire growth advantage and are selected and expanded. Some of these cells can subsequently produce more dominant aggressive type of cancer cells. However, both theories agree that tumors originate from a single cell that has acquired multiple mutations and becomes highly proliferative. Since both normal stem cells and cancer cells possess ability to self-renew, many
pathways that are classically associated with cancer are also involved in the regulation of normal stem cell development. For example, blocking of apoptosis by enforced expression of Bcl-2 can result in increased numbers of hematopoietic stem cells (HSCs). Signaling pathways associated with oncogenesis, such as the Notch, Sonic hedgehog (Shh) and Wnt pathways are also involved in the regulation of self-renewal of stem cells. Notch activation in HSCs by Jagged-1 increases the amount of primitive progenitor activity suggesting that Notch activation promotes HSC self-renewal and the maintenance of their multipotentiality.
Common Features Between Normal Stem Cells and Cancer Stem Cells
Normal Stem Cells Self Renewal Tissue-specific normal stem cells must self-renew throughout the life of the organism to maintain specific organs. Differentiation into phenotypically diverse mature cell types Give rise to a heterogeneous population of cells that compose the organ or the tumor, but lack the ability for unlimited proliferation (hierarchical arrangement of cells). Regulated by similar pathways in cancer stem cells. Some cells may be benign. Pathways that regulate self-renewal in normal stem cells are dysregulated Form a distinct population in tumors that likely cause disease relapse and metastasis. Cancer Stem Cells Cancer stem cells undergo selfrenewal to maintain tumor growth.
Shh, a secreted morphogen, has been implicated in several embryonic developmental processes. It displays inductive, proliferative, neurotrophic, and neuroprotective properties. Human HSCs exhibit increased self-renewal in response to Shh stimulation in vitro, albeit in combination with other growth factors. Shh signaling is required throughout embryonic development and is involved in the determination of cell fate and embryonic patterning during early vertebrate development. Shh also functions with other signaling molecules, such as the fibroblast growth factors and bone morphogenetic proteins, to mediate developmental processes. Mutations in any of the components of the Shh pathway can lead to congenital defects and diseases, including cancer. Hence, the Shh pathway has become a potential target for drug development for the treatment of cancers and degenerative diseases. Shh often works in concert with the Wnt signaling
protein to set embryonic development patterns. Wnt proteins are intercellular signalling molecules that regulate development in several organisms and contribute to cancer when dysregulated. The Wnt pathway uses -catenin to transduce its signals to the nucleus. The expression of Wnt proteins in the bone marrow indicates that they may have some regulatory influence on HSCs. Using highly purified mouse bone-marrow HSCs, Reya et al. (2001) have shown that overexpression of activated -catenin in long-term cultures of HSCs expands the pool of transplantable HSCs. In additon, over-expression of Axin, an inhibitor of Wnt signaling pathway, leads to inhibition of HSC proliferation and increased death of HSCs in vitro. Higher levels of -catenin are also reported to increase the proliferative capacity of cultured human keratinocytes suggesting that Wnt signaling promotes stem cell self-renewal. In spite of their tremendous capacity for self-
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Accumulation Self-renewal of Mutations Normal Stem Cells Signals Regulated Cell Division Cancer Stem Cells Dysregulated Cell Division Possible Effective Targets Self-renewal
Multipotent Progenitor Cells Mutated Progenitor Cells Signals Differentiation Normal Tissue (eg: Liver) Specific Cells Classical Chemotherapeutic Target
Hepatocytes
Kupffer Cells
Stellate Cells
Differentiated Cancer Cells
Dysregulated stem cell signaling in tumorigenesis
renewal, normal stem cells are generally quiescent and remain in the G0 stage. Due to the fact that stem cells can repair their DNA as they self-renew, they have the potential to accumulate more mutations, some of which may transform them into cancer stem cells. Irrespective of their origin (mutations in normal stem cells or by clonal evolution), cancer stem cells may be the ones that survive various bouts of chemotherapy. Also, any abnormality in the signaling processes during stem cell differentiation can lead to tumorigenesis. Cancer stem cells often renew themselves in a poorly regulated manner in contrast to normal stem cells, which are strictly regulated for selfrenewal. It is believed that stem cells are often the target of genetic events that are necessary for malignant transformation. Cancer researchers have concentrated on identifying the genetic alterations that transform cells into a cancer phenotype. If one views tumor as an abnormal organ, then the principles of normal stem cell biology can be applied to better understand how tumors develop.
Both normal stem cells and tumorigenic cells have extensive proliferative potential with ability to give rise to new, normal or abnormal, tissues. If we believe that most tumors have a clonal origin, then tumorigenic cancer cells must give rise to phenotypically diverse cell types, including cancer cells with indefinite proliferative potential and those with limited or no proliferative potential. This suggests that tumorigenic cancer cells undergo processes that are analogous to the selfrenewal and differentiation of normal stem cells. The role of stem cells in normal tissue turnover is barely understood, and even less is known about the role of stem cells in tumor growth and progression. A tumor mass can be considered as an abnormal organ that contains a variety of tumor cells, which are at different stages of differentiation. It is believed that in this abnormal organ only a limited number of cells possess the capacity to self-renew. For example, it has been shown that in leukemia and multiple myeloma only a small subset of cancer cells can lead to extensive proliferation. Park et al. (1971)
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showed that when mouse myeloma cells obtained from mouse ascites are separated from normal hematopoietic cells and are placed in clonal in vitro colony-forming assays, only 1 in 10,000 cancer cells go to form colonies. Also, when leukemic cells are transplanted in vivo, less than 4% of cells are able to form spleen colonies. These clonogenic leukemic cells can be described as leukemic stem cells. Bonnet and Dick (1997) demonstrated that human acute myeloid leukemia (AML) stem cells could be identified and purified as CD34+/CD38- cells. Although these cells represent only a very small proportion of AML cells they were the only cells capable of transferring AML from human patients to NOD/SCID mice. Several other studies have shown that in solid tumors cells are phenotypically heterogeneous and only a small proportion of these cells are clonogenic in culture and in vivo. These studies indicate that only a small number of cancer cells are actually tumorigenic and can be considered as cancer stem cells. For example, studies by Al-Haji et al, (2003) have shown that breast cancer cells that are CD44+/CD24- display stem cell properties are capable of generating tumors in NOD/SCID mice. More recently Ince et al., (2007) successfully transformed breast primary epithelial cells into highly tumorigenic BPLER cells, which were able to produce metastatic tumors in nude mice. Identification and selective destruction of cancer stem cells would certainly change the way cancer therapy is implemented. Currently, all phenotypically diverse cancer cells are treated in a manner that considers each cell to have unlimited proliferative and metastatic potential. One must note here that either a highly effective immune surveillance system in our body kills most cancer cells before they have any opportunity to form a tumor or most cancer cells are devoid of capability to form a new tumor. If the latter is truly the case, the appropriate therapy will be able to identify and destroy cancer stem cells before they proliferate and differentitate into different cancer phenotypes. Evaluation of success rates of most existing therapies is based on their ability to shrink solid tumors. However, they fail to eradicate solid tumors completely and any shrinkage is only transient. There exists a strong possibility that cancer stem cells are more resistant to chemotherapeutic agents than other tumor cells with limited proliferative potential.
Chemotherapeutic agents may cause complete regression of tumors, but might spare enough cancer stem cells to allow regrowth. Hence, chemotherapy directed only against cancer stem cells would be highly desirable.
References: Visvader, J.E., and Lindeman, G.J. 2008. Nat. Rev. Cancer 8, 755. Bapat, S.A. 2007. Semin. Cancer Biol. 17, 204. Campbell, L.L. and Polyak, K. 2007. Cell Cycle 6, 2332. Ince, T.A., et al. 2007. Cancer Cell 12, 160. Burkert, J., et al. 2006. J. Pathol. 209, 287. Costea, D.E., et al. 2006. Oral Dis. 12, 443. Houghton, J., et al. 2006. Semin. Cancer Biol. 17, 191 Luo, L., Han, J.C. 2006. Int. J. Hematol. 84, 123. Scadden D.T. 2006. Nature 441, 1075. Zhang, P., et al. 2006. Pathol. Int. 56, 485. Armanios, M., and Greider, C.W. 2005. Cold Spring Harb. Symp. Quant. Biol. 70, 205. Dean, M., et al. 2005. Nat. Rev. Cancer 5, 275. Weissman, I. 2005. JAMA 294, 1359. Christopherson, K.W. et al. 2004. Science 305, 1000. Ding, S., and Schultz, P.G. 2004. Nat. Biotech. 22, 833. Fu, M., et al. 2004. J. Cell Biol. 166, 673. Rattis, F.M., et al. 2004. Curr. Opin. Hematol. 11, 88. Al-Haji, M., et al. 2003. Proc. Natl. Acad. Sci. USA 100, 3983 Pardal R., et al. 2003. Nat. Rev. Cancer 3, 895. Orkin, S.H., and Morrison, S.J. 2002. Nature 418, 25. Reya, T., et al. 2001. Nature 414, 105. Taipale, J., and Beachy, P.A. 2001. Nature 411, 349. McKay, R. 2000. Nature 406, 361. Bonnet, D., and Dick, J.E. 1997. Nat. Med. 3, 730. Pierce, G.B. 1974. Am. J. Pathol. 77, 103. Park, C.H., et al. 1971. J. Natl. Cancer Res. Inst. 46, 411.
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Chapter Nineteen
Sonic Hedgehog Signaling
Mammalian Hedgehog proteins include Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh). They are involved in regulating pattern formation during embryonic development and in the maintenance of some tissue types in adults. Shh is expressed mainly in the epithelia in the tooth, hair, gut, bladder, urethra, vas deferens, and lung. Dhh is found in Schwann and Sertoli cell precursors and Ihh is expressed in gut and cartilage.
Shh is the best-characterized Hedgehog protein. It is synthesized as a 45 kDa precursor protein, which is then auto-catalytically cleaved to generate a 20 kDa N-terminal fragment that is responsible for all Hh biological activity, and a 25 kDa C-terminal fragment that contains the auto-processing unit. The N-terminal fragment of Shh contains palmitic acid and cholesterol as two lipid tethers, which allow it to remain associated with the plasma membrane. The cholesterol moiety is believed to be responsible for directing Hedgehog traffic in secretory cells. Shh, a secreted morphogen, has been implicated in several embryonic developmental processes. It is involved in neural tube patterning, ventral cell specifications, and development of leftright symmetry. In skin, Shh is involved in maintaining stem cell population. Shh displays inductive, proliferative, neurotrophic, and neuroprotective properties. Shh often works in concert with the Wnt signaling protein in setting embryonic patterns. The Wnt pathway uses -catenin to transduce its signals to the nucleus; however, the Shh pathway utilizes a 155 amino acid protein, Cubitus interruptus (Ci155) in Drosophila or Gli in mammals.
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These proteins act as transcriptional activators. Shh signaling is known to occur through a receptor complex associating two membrane proteins, Patched (Ptc) and Smoothened (Smo). Ptc is a twelve-pass membrane protein that acts as a receptor and binds Hedgehog ligand; Smo is a seven-pass membrane protein that acts as a signal transducer. In this regard, Smo displays homology to G-protein-coupled receptors that are usually associated with G-protein coupled receptor kinases. In the absence of a Shh, Ptc interacts with Smo and inhibits its activity. Under these conditions, Ci is targeted for proteolysis, which generates a truncated 75-amino acid residues form (Ci75), which acts as a transcriptional repressor. In vertebrates three Gli proteins (Gli1, Gli2, and Gli3) have been reported and despite several homologous regions, including a DNA-binding domain with five C2-H2 zinc fingers and a C-terminal transcription activation domain, these proteins have distinct activities and are not considered to be functionally equivalent. Shh binding to Ptc removes the inhibitory effect on Smo and allows Ci/Gli to enter the nucleus and act as a transcriptional activator. Smo action is mediated through a protein complex containing the kinesin-like protein Costal2 (Cos2), the Ser/ Thr kinase Fused (Fu) and Ci/Gli. Transcriptional activity of Ci/Gli is also regulated through its binding to Suppressor of Fu (Sufu), which is a negative regulator of hedgehog signaling in Drosophila as well as in vertebrates. It binds to all three Gli proteins with different affinities. Shh controls cell cycle progression by regulating the expression and activity of cyclins. It is also involved in expression of EGF and EGF receptor. Protein kinase A (PKA), casein kinase I (CKI) and glycogen synthase kinase 3 (GSK-3) play a significant role in regulating hedgehog signaling process. They all bind to Cos2, and phosphorylate homologous domains on Ci/Gli and Smo. Phosphorylation of Ci by PKA, CKI and GSK-3 is shown to be essential for the efficient processing of Ci155 to its transcriptional repressor form, Ci75. Inhibition of any of these kinases can lead to Ci155 accumulation. The role of phosphorylation in the regulation of vertebrate Gli proteins has not
yet been clearly defined, although PKA is shown to block vertebrate hedgehog signaling. Although Hh signaling is well studied during embryonic development, less is known about its role in adult tissues. Shh signaling is required throughout embryonic development and is involved in the determination of cell fate and embryonic patterning during early vertebrate development. During the late stage of development, Shh is involved in the proper formation of a variety of tissues and organs and it functions with other signaling molecules, such as the fibroblast growth factors and bone morphogenetic protein, to mediate developmental processes. Disruption of Shh in humans leads to holoprosencephaly (lack of development of forebrain in the embryo). Hh signaling is considerably down-regulated in adult tissues; however, up regulation of Hh signaling is reported in chemically injured tissues. Some studies have shown that Shh activity is retained by some cells in mature organs and dysregulation of activity in these cells leads to tumorigenesis. Mutations in any of the components of the Shh pathway can lead to congenital defects and diseases, including cancer. Loss of Ptc, over expression of Shh, and activating mutations of
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Gli have been reported in basal cell carcinomas, medulloblastoma, and breast carcinomas. Amplification of Gli has also been shown in malignant gliomas and osteosarcoma. Mutations in Smo and Sufu have also been associated with the formation of sporadic basal cell carcinoma and medulloblastoma. Hence, the Shh pathway has become a potential target for drug development for the treatment of cancers and degenerative diseases.
References: Aikin, R.A., et al. 2008. EMBO Reports 9, 330. Riobo, N.A., and Manning, D.R. 2007. Biochem. J. 403, 369. Athar, M., et al. 2006. Exp. Dermatol. 15, 667. Jiang, J. 2006. Cell Cycle 5, 2457. Simms-Mourtada, J., et al. 2006. Clin. Cancer Res. 12, 6565. Riobo, N.A., et al. 2006. Proc. Natl. Acad. Sci. USA 103, 4505. Macaron, N.C., et al. 2005. Arch. Dermatol. 141, 259. Benson, R.A., et al. 2004. Mol. Immunol. 41, 715. Magliano, M.P., and Hebrok, M. 2003. Nat. Rev. Cancer 3, 903. Watkins, D.N. et al. 2003. Nature 422, 313. Ruizi Altaba A., et al. 2002. Nat. Rev. Cancer 2, 361. Taylor, M.D., et al. 2002. Nat. Genet. 31, 306. Reinferberger, J., et al. 1998. Cancer Res. 58, 1798. Walterhouse, D.O., et al. 1999. Environ. Health Presp. 107, 167. Dahmane, N., et al. 1997. Nature 389, 876. Oro. A., et al. 1997. Science 276, 817. Roessler, E., et al. 1997. Hum. Mol. Genet. 6, 1847.
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Chapter Twenty
TGF- Signaling: Dual Role in Tumor Suppression and Oncogenesis
Transforming growth factor- (TGF-), a member of the TGF superfamily of proteins that includes activins, inhibins, and bone morphogenetic proteins (BMPs), regulates a wide array of cellular processes including cell differentiation, cellular senescence, immune response, wound healing, and apoptosis. TGF- signaling promotes growth and development during early embryogenesis.
However, in mature tissues many cells respond to TGF- with either a cytostatic or apoptotic response. TGF- signaling involves its binding to the TGF- receptor type II (TGF- RII), which allows it to recruit TGF- receptor type I (TGF- RI) and assemble it as a heterodimeric receptor complex. TGF- RII phosphorylates TGF- RI in the glycine-serine rich region (GS sequence) and activates the serine/threonine kinase activity of TGF- RI, which in turn phosphorylates receptor-linked Smad (Small mothers against decapentaplegic) proteins. To prevent any spontaneous phosphorylation of Smads, the inhibitor molecule FKBP12 binds to the GS region of the TGF- RI and blocks the access of TGF- RII to this domain. This inhibitory effect is removed upon TGF- binding to the receptor. The TGF- receptor complex is internalized by lipid-raft and clathrin endocytotic pathways. The clathrin endocytotic pathway is considered to be essential for activation of the TGF- signaling cascade. The lipid-raft pathway for TGF- receptor internalization negatively regulates TGF- signaling by inducing receptor complex degradation. The activated TGF- RI in the internalized complex phosphorylates specific Smad proteins. At least nine different Smad proteins
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have been reported in vertebrates that are classified as: (a) receptor-activated Smads (R-Smads): Smad1, Smad2, Smad3, Smad5, and Smad8; (b) co-mediator Smads: Smad4 and Smad10; and (c) inhibitory Smads (I-Smads): Smad6 and Smad7. R-Smads2 and 3 are involved in TGF- and activin signaling, whereas R-Smads 1, 5, and 8 are mediators of BMP signaling. R-Smads and Co-Smads are predominantly located in the cytoplasm and their activity is modulated by adaptor proteins, such as Smad anchor for receptor activation (SARA) and ELF. SARA is reported to associate with endosomal membranes via lipid-binding FYVE domains. It binds Smad2 and Smad3 and facilitates their interaction with TGF- receptors. Smad2 and Smad3 are directly phosphorylated by TGF- RI, which changes their conformation and releases these R-Smads from the receptor complex. The C-terminal phosphoserines of R-Smads are recognized by the Mad Homology 2 (MH2) domain of Smad4 that enables them to form a heterodimeric complex (R-Smad/Co-Smad). This complex then translocates to the nucleus where Smad proteins bind to their cognate DNA binding sites with low affinity. This binding is further enhanced in the presence of transcriptional co-activators. Both Smad3 and Smad4 bind to DNA sequences known as the Smad-binding elements (SBE); however, Smad2 participates in DNA-bound complexes via its interaction with Smad4. Genes for cyclin-dependent kinase inhibitors, p21 and p15, which mediate growth inhibitory processes, are up-regulated by TGF- stimulation. TGF- -induced growth inhibition is also achieved by down-regulation of c-Myc, which further amplifies the expression of p21 and p15. The transcriptional induction of inhibitory Smad6 and Smad7 is under the control of TGF-, which provides a negative feedback loop for attenuation of TGF- signaling. Smad6 antagonizes TGF- signaling by blocking the interaction of Smad2 and Smad4, whereas Smad7 negatively regulates TGF- signaling by recruiting Smurf1 or Smurf2 to the Smad7-TGF--receptor complex. It has also
been suggested that Smad7 blocks the activity of all R-Smads, but Smad6 preferentially blocks bone morphogenetic protein-activated Smads. Inhibitory Smads are also known to reside in the nucleus where they associate with Smurfs, a group of E3 ligases. Following TGF- stimulation, the Smad7-Smurf complex is exported to the cytoplasm where it targets TGF- receptors. By binding to TGF- RII, Smad7 inhibits recruitment and phosphorylation of R-Smads. In the nucleus, Smads are ubiquitinated by the E3 ligases and are exported to cytoplasm for proteasomal degradation. Even in the cytoplasm R-Smads can be ubiquitinated by Smurfs and degraded in the proteasome complex. This keeps the pool of R-Smads at low levels. Nuclear Smurfs can also ubiquitinate phosphorylated R-Smads, which can then be degraded in the nucleus. Smad shuttling between the cytoplasm and the nucleus is dependent on phosphorylation of R-Smads by TGF-R1 kinase in the cytoplasm and dephosphorylation by R-Smad phosphatases in the nucleus. However, a majority of activated R-Smad is believed to be recycled following its dephosphorylation. Apart from its role as a growth inhibitor and tumor suppressor, TGF- also promotes tumor metastasis during late stages of tumor development. TGF- also modulates cell invasion, immune regulation, and microenvironment modification that cancer cells exploit to their advantage. Dysregulated TGF- signaling has been implicated in the pathogenesis of human solid tumors. Several key proteins involved in tumor metastasis, such as Snail, Slug (zinc-finger transcriptional repressors) and Smad-interacting protein 1 (SiP1), are positively regulated by TGF-. Any disruption in TGF- signaling, either by mutational inactivation or by down regulation of expression of any of the signaling components involved, can lead to tumor development. Inactivating mutations in TGF- RII are observed in about 25% of all colon cancer cases, and a reduction in tumorigenicity and restoration of TGF--mediated growth arrest are reported in human colon cancer cell lines stably
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TGF- signaling cascade
transfected with wild-type TGF- RII. Mutations in Smad2 and Smad4 can also disrupt TGF- signaling, leading to tumor development. Smad4, initially identified as DPC4 (deleted in pancreatic carcinoma locus 4), undergoes biallelic loss in about 50% of all pancreatic cancers. Of current interests in preventing tumor growth and metastasis are the development of molecules for inhibition or sequestration of TGF-, blocking the kinase activity of TGF- RI, and inhibition of downstream Smad signaling.
References: Massagu, J. 2008. Cell 134, 215. Padua, D., et al. 2008. Cell 133, 66. Ross, S., and Hill, C.S. 2008. Int. J. Biochem. Cell Biol. 40, 383. Leivonen, S.K., Khri,V.M., 2007. Int. J. Cancer 121, 2119. Pennison, M., and Pasche, B. 2007. Curr. Opin. Oncol. 19, 579. Schmierer, B., and Hill, C.S. 2007. Nat. Rev. Mol. Cell Biol. 8, 970. Rider, C.C. 2006. Biochem. Soc. Trans. 34, 458. Verrecchia, F., et al. 2006. Autoimmunity Rev. 5, 563. Larsson, J., and Karlsson, S. 2005. Oncogene 24, 5676. Lin, H-K., et al. 2005. Oncogene 24, 5693. Mishra, L., et al. 2005. Oncogene 24, 5775. Liu, D., et al. 2004. EMBO J. 23, 1557. Martinez-Alvarez, C., et al. 2004. Dev. Biol. 265, 207. Wang, T., and Donahoe, P.K. 2004. Front. Biosci. 4, 619. Wolfraim, L.A., et al. 2004. N. Eng. J. Med. 351, 552. Shi, Y., and Massague, J. 2003. Cell 113, 685. Moustakas, A. 2002. J. Cell Sci. 115, 3355. Derynck, R., et al. 2001. Nat. Genet. 29, 117. Shi, Y. 2001. BioEssays 23, 223. Goggins, M., et al. 1998. Cancer Res. 58, 5329. Itoh, S., et al. 1998. J. Biol. Chem. 273, 29195. Wang, J., et al. 1995. J. Biol. Chem. 270, 22044.
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Chapter Twenty one
Angiogenesis: Its Role in Tumor Growth and Metastasis
The vascular system is formed through a combination of vasculogenesis and angiogenesis. In vasculogenesis, blood vessels are formed de novo by the assembly of angioblasts of mesodermal origin. Angiogenesis is the formation of new capillaries from preexisting vasculature by migration and proliferation of endothelial cells. Angiogenesis is a
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fundamental process required for a number of physiological and pathological events and is considered to be a key step in tumor growth, invasion, and metastasis.
Angiogenesis is required for proper nourishment and removal of metabolic wastes from tumor sites. Under physiological conditions, angiogenesis is a highly regulated phenomenon. The progression of angiogenesis is controlled by a delicate balance between the positive and negative regulators of this process. Although angiogenesis occurs only during embryonic development, wound healing, and the menstruation cycle, unregulated angiogenesis is seen under pathological conditions such as tumor growth, diabetic retinopathy, and psoriasis. Angiogenesis is not only a prerequisite for tumor growth, but also a major factor affecting the metastatic spread of malignant cells. Angiogenesis is a multi-step process that begins with
| Angiogenesis: Its Role in Tumor Growth and Metastasis
the degradation of basement membrane at post-capillary
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venule. During angiogenesis, the assembly of a vascular network occurs in several sequential steps that include: (a) Release of proteases from activated endothelial cells (b) Degradation of basement membrane surrounding the existing blood vessel
(c) (d) (e) (f)
Migration of endothelial cells to the tumor Proliferation of endothelial cells Canalization and branching Creation of new basement membrane and enrollment of pericytes for vascular stability (g) Fusion of newly formed blood vessels (h) Establishment of blood flow
Basic process in tumor angiogenesis.
Angiogenic growth factors such as the vascular endothelial growth factor (VEGF), acidic and basic fibroblast growth factors, tumor necrosis factor- (TNF-), angiogenin, and others secreted by tumors, endothelial cells, and supporting cells accelerate the process of angiogenesis. These factors act as autocrine or paracrine growth factors to produce angiogenesis. The human VEGF, a 45 kDa homodimeric glycoprotein, is one of the most potent angiogenic factors involved in endothelial cell proliferation, migration, and secretion of matrix metalloproteinases (MMPs). It also serves as an important survival factor for endothelial cells and inhibits apoptosis. VEGF belongs to a family that includes VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (PLGF). VEGFA
is the key regulator of angiogenesis, whereas VEGFC and VEGFD are involved in the regulation of lymphatic angiogenesis. VEGFB plays a significant role in embryonic angiogenesis. In humans four prominent isoforms of VEGFA are recognized, VEGF121, VEGF165, VEGF189, and VEGF206. In addition, two other less frequent splice variants, VEGF145 and VEGF183, have also been reported. The properties of native VEGF are very similar to VEGF165. Of all the different isoforms of VEGF, the VEGF165 has the highest bioavailability and biological potency. VEGF action is mediated by VEGF receptor-1 (VEGFR-1) and VEGFR2. VEGFR-1 (180 kDa, Flt-1) expression is upregulated by hypoxic conditions in the inner core of tumor mass. VEGFR-1 binds mainly to VEGFA, VEGFB and PLGF. Activation of VEGFR-1 leads to endothelial cell proliferation, migration,
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and survival, induction of MMPs, and vascularbed specific release of growth factors. VEGFR-2 (200 kDa, KDR) is a major mediator of mitogenic and angiogenic permeability enhancing effects of VEGF. It is also reported to mediate survival signals in endothelial cells. VEGFR-3 (Flt-4) binds to VEGFC and VEGFD and is primarily associated with lymphogenesis. Basic fibroblast growth factor (bFGF) is another important proangiogenic factor that acts on a smooth muscle cells, pericytes, fibroblasts, and endothelial cells. It is also a potent mitogen and chemoattractant for endothelial cells and is expressed by hypoxic tumor cells. It is reported to upregulate expression of both urokinse-type plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI) by endothelial cells. bFGF is also reported to increase cellmatrix adhesion that enhances the potential for invasion of the extracellular matrix by endothelial cells. Another group of proteins known as the angiopoietins (Ang-1 and Ang-2) are endothelial growth factors that bind to the tyrosine kinase receptor Tie-2 and contribute to blood vessel formation during angiogenesis. Ang-1 is involved in vessel maturation and integrity. It recruits pericytes and mediates the interaction between endothelial cells and the basement membrane. Some tumor cells also express Ang-1, which induces angiogenic sprouting and chemotactic migration of endothelial cells. On the other hand, Ang-2 is considered as a Tie-2 antagonist that counteracts the stabilizing action of Ang1. Ang-2 is not widely expressed under normal physiological conditions and its spatial and temporal expression is closely regulated. It is expressed by endothelial cells only in regions undergoing vascular remodeling. In addition to its role in angiogenesis, Tie-2 is also involved in maintaining the long-term population and quiescent status of hematopoietic stem cells in the bone marrow. Dormant tumors secrete inhibitory factors such as angiostatin, endostatins, thrombospondins, and tissue inhibitors of MMPs that prevent tumors from switching to the angiogenic phenotype and arrest the growth of tumors. For angiogenesis to occur, proangiogenic molecules have to overcome the effect of these anti-angiogenic factors. To trigger angiogenesis, either the production of
proangiogenic factors must increase or the level of inhibitors must decrease. Although antiangiogenic molecules may not be present in sufficient quantities to block angiogenesis at the local site, but they may have a longer half-life in circulation, thereby favoring turning off the angiogenic switch at sites distant from the primary tumor. Surgical removal of the primary tumor may result in less abundance of these anti-angiogenic factors, enabling tumor-produced pro-angiogenic factors to dominate the angiogenic switch and drive neovascularization of distant tumors. Tumor-induced angiogenesis begins with the dissolution of the basement membrane surrounding a preexisting blood vessel, a process aided by MMPs produced by tumor cells and supporting cells. The non-mitotic endothelial cells then migrate towards the tumor through the disintegrated tissue extracellular matrix. The dissolution of extracellular matrix also facilitates the release of sequestered angiogenic factors. An augmentation in MMP activity is positively linked to the increase in metastatic and angiogenic potential of tumors. Upregulation of MMP-2, MMP-7, MMP-9, and stromelysin-3 (MMP-11) mRNA has been reported during tumor invasion and metastasis. Most tumors persist for years without any angiogenic activity, incapable of growing beyond 2 to 3 mm in size. However, when they switch to the angiogenic phenotype they grow rapidly. Avascular, microscopic tumors proliferate and grow at a rate similar to rapidly growing tumors. However, in the dormant stage, the rate of tumor cell proliferation is balanced by apoptosis of tumor cells. The acquisition of angiogenic phenotype results in a diminution in the apoptotic rate and a shift in balance in favor of cell proliferation. During angiogenic neovascularization a selective expression of adhesion receptor integrin v3 has also been reported. The binding of integrin v3 to the receptor provides a specific signal that enhances the survival of angiogenic endothelial cells. This signal is also linked to a decrease in p53, p21WAF1 and Bax expression and an increase in Bcl-2 expression, thereby facilitating a switch to the angiogenic phenotype. The obligatory neovascularization, a rather uncommon process under normal conditions, for tumor growth and metastasis makes angiogenesis
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a prominent target for therapeutic intervention. Most of the antitumor agents used in cancer therapy are cytotoxic in nature, designed mainly to prevent tumor growth. However, to be useful, an ideal therapeutic agent should exhibit selectivity and minimal acute or chronic toxicity. Angiogenic inhibitors could prove to be useful agents in this arena. Their selective effect on vasculature may not only have an overall reduced toxicity, they may also be able to overcome drug resistance commonly seen in solid tumors. Any specific inhibitor of angiogenesis will be well tolerated by tumor patients because under physiological conditions there is minimal, if any, angiogenesis (e.g., wound healing). Several modes of treatment, separately or combined, are under consideration and include agents that act directly on the tumor cells to prevent the release of angiogenic agents, drugs that inactivate already released angiogenic molecules, and agents that obliterate endothelial cell response to angiogenic stimulators. Contrary to the above, some questions have been raised relating to the efficacy of anti-angiogenic agents. Blocking the action of any one specific agent may induce tumors to over express another activator; also, it is not clear whether anti-angiogenic agents will reduce the size of the tumor or simply inhibit further tumor growth.
References: Das, CR., and Choong, P.F. 2008. J. Drug Target. 16, 449. Martin, V., et al. 2008. Histol. Histopathol. 23, 773. Adams, R.H., and Alitalo, K. 2007. Nat. Rev. Mol. Cell Biol. 8, 464. Lemieux, C., et al. 2005. Blood 105, 1523. Wahl, M.L., et al. 2005. J. Cell. Biochem. 96, 242. Ferrara, N., et al. 2003. Nat. Med. 9, 669. Plank, M.J., and Sleeman, B.D. 2003. J. Theor. Med. 5, 137. Bhushan, M. and Griffiths, C.E. 2002. Br. J. Dermatol. 147, 418. Liekens, S., et al. 2001. Biochem. Pharmacol. 61, 253. Terman, B.I., and Stoletov, K.V. 2001. Einstein, Q. J. Biol. Med. 18, 59. Beck, L., and DAmore, P.A. 1997. FASEB J. 11, 365. Gastl, G., et al. 1997. Oncology 54, 177. OReilly, M.S., et al. 1997. Cell Physiol. Biochem. 88, 277 Pluda, J.M. 1997. Sem. Oncol. 24, 203. Hashimoto, K., et al. 1997. Nippon Hin. Gakk. Zasshi 88, 852. Tanigawa, N., et al. 1997. Cancer Res. 57, 1043. Twardowski, P., and Gradishar, W.J. 1997. Curr. Opin. Oncol. 9, 584. Uhr, J.W., et al. 1997. Nature Med. 3, 505. Yu, A.E., et al. 1997. Drugs Aging 11, 229. Bicknell, R., and Harris, A.L. 1996. Curr. Opin. Oncol. 8, 60. Petruzzeli, G.J. 1996. Head and Neck 18, 283. Stromblad, S., et al. 1996. J. Clin. Invest. 98, 426. Folkman, J. 1995. In: Molecular Basis of Cancer (Mendelsohn, J., et al. Eds.), Philadelphia, W.B. Sanders, pp 206232. Holmgren, L., et al. 1995. Nat. Med. 1, 149. Brooks, P.C., et al. 1994. Cell 79, 1157. OReilly, M.S., et al. 1994. Cell 79, 315. Weinstat-Saslow, D., and Steeg, P.S. 1994. FASEB J. 8, 401. Polette, M., et al. 1993. Invasion Metastasis 13, 31. Hahnel, E., et al. 1993. Int. J. Cancer 55, 771. Folkman, J., and Ingber, D. 1992. Sem. Cancer Biol. 3, 89. Hori, A., et al. 1991. Cancer Res. 51, 6180. Ausprunk, D.H., and Folkman, J. 1977. Microvasc. Res. 14, 53.
This chapter is dedicated to the memory of Professor Judah Folkman who founded the modern field of angiogenesis. His revolutionary approach to cancer therapy has evolved into one of the most exciting scientific fields of modern era.
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Chapter Twenty two
NF-B Activation: A Link Between Chronic Inflammation and Cancer
Inflammation is a normal physiological response to injury or infection, where the inflammatory response isolates the damaged area, mobilizes effector cells and molecules to the site of damage, and ultimately promotes healing.
The inflammatory response includes the transcriptional activation of several pro-inflammatory genes, which leads to the release of pro-inflammatory cytokines and chemokines, adhesion molecules, and reactive oxygen and nitrogen species. Simultaneously, antiinflammatory pathways are also activated that serve as counter regulatory measures to keep the inflammatory response in check. The inflammatory response can be either acute or chronic. Acute inflammation may last only for a few days and can be easily treated with non-steroidal anti-inflammatory drugs (NSAID). However, chronic inflammation can last for several weeks or months and can cause severe tissue damage. Inflammation resulting from chronic exposure to infectious and non-infectious agents is an important factor in tumor development. Production and release of free radicals at the site of inflammation can be helpful in eliminating invading pathogens and foreign bodies, but these free radicals can also damage healthy epithelial and stromal cells, which may eventually lead to malignancy. In addition, excessive lipid peroxidation, protein nitration, and DNA damage caused by free radicals can activate pathways that lead to the transcriptional activation of several proto-oncogenes. Certain pro-inflammatory cytokines that are encoded by the target genes of nuclear factor B (NF-B) are also linked to tumor development and progression. The inflammatory link to carcinogenesis is also supported by the fact that the NF-B signal transduction pathway
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is shown to be dysregulated in a variety of human cancers, especially those of lymphoid cell origin. Several human lymphoid cancer cells are reported to have either mutations or amplifications of genes encoding NF-B transcription factors. NF-B, discovered in the laboratory of David Baltimore in 1986, plays an important role in regulating inflammatory and autoimmune responses, cell proliferation, and apoptosis by regulating the expression of genes encoding inflammatory cytokines, cell adhesion molecules, and cyclooxygenase-2 (COX-2). NF-B is known to be activated by over 450 different activators, including oxidative stress, mitogens, bacteria, and mediators of apoptosis. The activation of NF-B occurs either via the classical pathway, which is triggered by bacterial and viral infections and pro-inflammatory cytokines, or by the alternate pathway, which is activated by members of the Tumor Necrosis Factor (TNF) family. These two pathways switch on different sets of genes and therefore mediate different immune functions. Five members of the NF-B family have been identified: NF-B1 (p50/p105), NF-B2 (p52/ p100), RelA (p65), RelB, and c-Rel that associate to form various homodimeric or heterodimeric combinations. They share a highly conserved Rel homology domain (RHD), which is responsible for DNA binding, dimerization, and interaction with IBs. The p50/RelA (p65) heterodimer is the major NF-B complex in most cells. The activity of NF-B is tightly regulated by its interaction with inhibitory IB proteins. In most resting cells, NF-B is sequestered in the cytoplasm in an inactive form associated with inhibitory molecules such as IB, IB, IB, p105, and p100. This interaction blocks the ability of NF-B to translocate to the nucleus and bind to DNA. Following exposure to pro-inflammatory cytokines, UV light, reactive oxygen species, or bacterial and viral toxins, the NF-B signaling cascade is activated, leading to the proteasomal degradation of IB. This allows the translocation of unmasked NF-B from the cytoplasm to the nucleus where it binds to NF-B response elements in target genes and regulates their transcription. Among the NF-B regulated genes involved in managing the response to inflammation are those encoding for iNOS, COX-2, TNF, IL-1, IL-6, IL-8, and others. Some of these molecules have
also been linked to carcinogenesis. Studies have shown that NF-B is constitutively active in many types of human tumors. The activation of NF-B by extracellular inducers depends on the phosphorylation and subsequent degradation of IB proteins. Activation of NF-B is achieved through the action of a family of serine/threonine kinases known as IB kinase (IKK). IKK contains two catalytic subunits (IKK and IKK) and a regulatory/adapter protein IKK (also known as NEMO). IKK activity and NF-B activation are largely dependent on the integrity of NEMO and IKK. Cells devoid of IKK can still show normal induction of NF-B-DNAbinding in response to stimuli. IKK and IKK phosphorylate IB proteins and the members of the NF-B family. All IB proteins contain two conserved serine residues within their N-terminal region, which are phosphorylated by IKK. IKK and IKK share about 50% sequence homology and can interchangeably phosphorylate Ser32/36 of IB and Ser19/23 of IB. These phosphorylation events lead to the immediate polyubiquitination of IB proteins and rapid degradation by the 26S proteasome complex. In the nucleus, recruitment of NF-B to its target genes and regulation of NF-B-mediated transcriptional activation are mediated mainly by phosphorylation and acetylation of NF-B, which enhance its DNA binding activity. Several protein kinases, including PKA, PKC and casein kinase II directly phosphorylate p65 (at Ser276, Ser311, Ser529, respectively). However, others like PI 3-K/Akt and NIK (NF-B-inducing kinase) phosphorylate IKK, which in turn phosphorylates p65 at Ser536. Reversible acetylation of NF-B can also determine its active or inactive state. p300 and CBP acetyltransferases play a major role in the acetylation of p65, principally targeting Lys218, 221, 310 for modification. Acetylated NF-B is active and is resistant to the inhibitory effects of IB. Deacetylation of p65 by histone deacetylase 3 (HDAC3) promotes its binding to IB and leads to rapid export of deacetylated NF-B from the nucleus into the cytoplasm. One of the target genes activated by NF-B is that encoding IB. Newly synthesized IB can enter the nucleus, remove NF-B from DNA, and export the complex back to the cytoplasm to restore its original latent state.
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TLRs Viruses Bacterial LPS Proinammatory Cytokines
TNF
Cytokines CD40L BAFF LTR
FADD
TNFR TRAF2 TRADD
RIP
RIP NIK
PIP2
PIP3 AKT PDK1 mTOR SCF/ TrCP
RTK PI-3K IKK (NEMO) IKK AKT IB Ub IB

Degradation
IKK IKK IKK
p50
p65
p100 Rel-B
(NF-B)
Ub
p52 Proteasome
Rel-B
(NF-B)
Nuclear Import
Nuclear Export
22 | NF-kB Activation: A Link Between Chronic Inflammation and Cancer
Nuclear Import
NF-B
ac dea etyla cet tio yla n tio n
NF-B IB HDAC3
Pro-inammation Survival (anti-apoptotic) Angiogenesis Chemokines Adhesion molecules Enzymes (COX-2, iNOS)
p300 (CBP) NF-B
NF-B responsive gene transcription

Classical and alternate pathways of NF-B activation
Unlike normal cells, in most cancer cells NF-B is constitutively active and resides in the nucleus. In some cases, this is due to chronic stimulation of the IKK pathway, while in others the gene encoding IB may be defective. Several key processes, such as self-sufficiency in growth signals, insensitivity to growth inhibitors, evading apoptosis, unlimited replicative potential, tissue invasion, and sustained angiogenesis, are all enhanced following NF-B activation. Hence, designing antitumor agents to block NF-B activity may have great therapeutic value. Researchers have been exploring possibilities of interfering with NF-B activation and its binding to DNA. They include inhibition
of NF-B dimerization, inhibition of NF-B activation, use of proteasome inhibitors that block IB degradation, and inhibition of NF-B nuclear translocation. Since IKK and IKK are shown to only phosphorylate proteins involved in NF-B signaling, they are considered as important targets for the development of chemotherapeutic agents. NSAIDs and corticosteroids are shown to mediate anti-inflammatory effects via inhibition of NF-B activation and are shown to reduce the incidence of cancer. Certain natural compounds such as curcumin, derived from turmeric, are also potential chemotherapeutic agents and act by blocking NF-B activation.
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References: Naugler, W.E., and Karin, M. 2008. Curr. Opin. Genet. Develop. 18, 19. Waes, C. V. 2007. Clin. Cancer Res. 13, 1076. Aggarwal, B. B., et al. 2006. Biochem Parmacol. 71, 1397. Liu, S.F., and Malik, A.B. 2006. Am. J. Physiol. 290, L622. Karin, M., and Greten, F.R. 2005. Nat. Rev. Immunol. 5, 749. Ohshima, H., et al. 2005. Mutation Res. 591, 110. Wu, J-T., and Kral, J.G. 2005. J. Surg, Res. 123, 158. Bouwmeester, T., et al. 2004. Nat. Cell. Biol. 6, 97. Chen, L.F., and Greene, W.C. 2004. Nat. Rev. Mol. Cell Biol. 5, 392. Hayden, M.S., and Ghosh, S. 2004. Genes Dev. 18, 2195. Karin. M., et al. 2004. Nat. Rev. Drug Disc. 3, 17. Hussain S.P., et al. 2003. Nat. Rev. Cancer 3, 276. Chen, L.F., et al. 2002. EMBO J. 21, 6539. Garg, A., and Aggarwal, B.B. 2002. Leukemia 16, 1053. Ghosh, S., and Karin M. 2002. Cell 109, S81. Gilmore, T., et al. 2002. Cancer Lett. 181, 1. OByrne, K.J., and Dalgleish, A.G. 2001. Br. J. Cancer 85, 473. Gilmore, T.D. 1999. Oncogene 18, 6842. Verma, I.M., et al. 1995. Genes Dev. 9, 2723. Sen, R., and Baltimore, D. 1986. Cell 46, 705.
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Chapter Twenty three
Tumor Metastasis: Role of Cell Adhesion and Matrix Metalloproteinases
Tumor invasion and metastasis largely depend on the interaction between the tumor cells, the extracellular matrix (ECM), and the blood vessels surrounding the tumor. The ECM, in addition to providing structural compartmentalization, is a dynamic matrix of structural proteins, growth factors, and latent enzymes that undergo constant remodeling.
Matrix metalloproteinases (MMPs) maintain the integrity of ECM by removing undesired proteins; however, over-expressed MMPs play a critical role in tumor invasion and metastasis. Tumor invasion requires a decreased affinity between tumor cells or between tumor cells and the ECM that facilitates their release from the primary tumor mass. When tumors progress towards greater malignancy, cells within tumors develop greater ability to detach and invade surrounding tissues. Within a primary tumor mass, cell-cell adhesion is mediated by E-cadherin in combination with - and -catenin and other cytoplasmic components that link E-cadherin to the cytoskeleton. E-cadherin acts as the main suppressor of epithelial tumor invasion. Decrease or loss of E-cadherin expression is described in many human carcinomas. A single mutation of the E-cadherin gene can transform an adenoma into a carcinoma. Expression of E-cadherin is shown to be moderate in invasive carcinomas without metastases. However, E-cadherin expression is shown to be significantly elevated in metastases compared to the primary tumor. Aberrant N-glycosylation of E-cadherins can also lead to increased motility and a reduction in adhesion between tumor cells resulting in release of these cells from the primary tumor mass.
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These cells can ultimately lead to metastasis. An overall decrease in the neural cell adhesion molecule (NCAM) level has been observed in colon carcinoma, pancreatic cancer, and astrocytoma. Loss of NCAM closely correlates with poor prognosis. A loss of NCAM function is also shown to be associated with lymph node metastasis. Galectin-9, a member of animal lectins, shows an inverse relationship with metastasis. It blocks binding of tumor cells to vascular endothelium and the extracellular matrix. It is also shown to block lung metastasis in mice using B16F10 melanoma and Colon26 cancer cells. Other examples include activated leukocyte cell adhesion molecule (ALCAM) that is over expressed in metastatic human melanoma cell lines, but not in non-metastatic cell lines.
and metastasis are highly complex, yet wellcoordinated, processes and can be summarized under the following scheme: (a) release of tumor cells from primary tumor mass (b) adhesion of tumor cells to extracellular matrix and basement membrane (c) hydrolytic activities (proteases and endoglycosidases) of tumor cells for destruction of extracellular matrix and basement membrane (d) migration of tumor cells through the degraded matrix and into the blood circulation and interaction of tumor cells with platelets followed by the activation of platelets to express P-selectin, ICAM, and other adhesion proteins (e) selectin and ICAM-dependent tumor cell adhesion to endothelial cells followed by extravasation and formation of metastatic deposit (f) formation of metastatic deposit.
For metastasis to occur, tumor cells must cross the basal membrane and stroma to gain access to the blood stream. Those tumor cells that escape the immune system leave the blood vessels to form new colonies from the primary tumor. Invasion
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The proteolytic degradation of the ECM by tumor cells requires the action of highly specialized MMPs that are expressed in a cell- or tissuespecific pattern. They also play important roles in wound healing, angiogenesis, embryogenesis, and in pathological processes such as tumor invasion and metastasis. About 26 different types of MMPs have been identified and classified based on their protein domain structures. They are characterized by the presence of a zinc ion in the active site, which is required for their catalytic activity. All MMPs sequenced to date have at least three domains in common. The pro-domain contains a highly conserved segment of eight amino acids that folds over to cover the catalytic site and helps maintain the inactive conformation following the release of MMPs. Cleavage of the pro-domain destabilizes the inhibitory interaction between the unpaired cysteine in the sequence and the active site zinc atom. The catalytic domain contains the conserved structural metal-binding sites consisting of 106 to 119 residues. MMPs also have a highly conserved zinc-binding active site domain containing 52 to 58 amino acids. The zinc-binding domain contains three histidine residues that occupy three of the coordination sites of the active site Zn2+. Additional domains, such as transmembrane and gelatin-binding domains, have also been reported on some MMPs. In addition to these, the hemopexin like domain found in all MMPs (except MMP-7) plays a role in substrate specificity. The activation of MMPs is dependent mainly on urokinase-type (u-PA) and tissue-type plasminogen activators (t-PA) that cleave plasminogen into active plasmin. The activity of these plasminogen activators is regulated by the levels of PAI-1 and PAI-2 that bind to the uPA receptor on the cell membrane and prevent the activation of uPA. Plasmin initiates an activation cascade by cleaving proMMP-1 and -3 into MMP-1 and -3, respectively. A major control point in the regulation of active enzyme is inhibition of the active form by the TIMP family of inhibitors (21 - 28 kDa). TIMPs regulate the function of MMPs either by inhibiting active MMPs or by controlling their activation process. They form tight, noncovalent inhibitory complexes with MMPs (Kd = 10 - 50 pM). During inhibition, the terminal amino
group of the TIMP fills the fourth coordination site of the active site zinc. Tumor growth beyond a few millimeters in diameter requires angiogenesis, the growth of new blood vessels to bring oxygen and nutrients for tumor growth. ECM degradation by MMPs releases growth factors, such as VEGF and bFGF that stimulate endothelial cells to form new blood vessels. These growth factors also promote the synthesis and release of collagenases and plasminogen activators by the endothelial cells of blood vessels surrounding the tumor. MMPs facilitate tumor cell invasion and metastasis by at least three distinct mechanisms: (a) by eradicating physical barriers to invasion through degradation of collagens, laminins, and proteoglycans in the ECM; (b) by modulating cell adhesion and enabling cells to form new cellto-cell and cell-to-matrix attachments, while breaking the existing ones; and (c) by acting on ECM components and other proteins to expose hidden biological activities, such as release of angiostatin from plasminogen. In normal adults, MMP expression is very low except in rapidly remodeling tissue, such as during wound healing and in menstrual endometrium. Many control elements, such as secretion of MMPs in their latent form and the presence of TIMPs, tend to keep MMPs inactive in the ECM. MMP activity is also controlled by PAIs that diminish the activity of uPA and tPA. A discussion on the role of these various inhibitors in tumor progression is beyond the scope of this brief article. From the foregoing account, it is evident that increased expression and activation of MMPs are necessary for ECM degradation. In tumor cell invasion and metastasis, a tightly controlled balance between active proteases and their inhibitors appears to be essential for preventing invasion. A wide variety of MMP inhibitors have been designed and developed for use as cytostatic agents that show promise as chemotherapeutics in patients with malignant tumors. Monitoring changes in the activity of MMPs and TIMPs offer great prognostic value in determining the outcome of chemotherapy.
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References: Gloushankova, N.A. 2008. Biochemistry (Moscow) 73, 742. Fukumaru, K., et al. 2007. J. Dermatol. 34, 746. Nobumoto, A., et al. 2008. Glycobiology 18, 735. Jeschke, U., et al. 2007. Anticancer Res. 27, 1969. Crnic, I., et al. 2004. Cancer Res. 64, 8630. Lon, C.L.T., et al. 2000. Am. J. Pathol. 156, 769. Forget, M.A., et al. 1999. Can. J. Physiol. Pharmacol. 77, 465. Streuli, C. 1999. Curr. Opin. Cell Biol. 11, 634. Kleiner, D.E., and Stetler-Stevenson, W.G. 1999. Cancer Chemother. Pharmacol. 43 (suppl.), S42. Kugler, A. 1999. Anticancer Res. 19, 1589. Murphy, G., and Gavrilovic, J. 1999. Curr. Opin. Cell Biol. 11, 614. Westermarck, J., and Kahari, V.M. 1999. FASEB J. 13, 781. Blavier, L., et al. 1999. Ann. N.Y. Acad. Sci. 878, 108. Chintala, S.K., and Rao, J.S. 1999. Int. J. Dev. Neurosci. 17, 495. Ellerbroek S.M., and Stack, M.S. 1999. BioEssays 21, 940. Glinsky, G.V. 1998. Cancer Metast. Rev. 17, 177. Toi, M., et al. 1998. Breast Cancer Res. Treat. 52, 113. Nol, A., et al. 1997. Invasion Metastasis 17, 221. Hakomori, S. 1996. Cancer Res. 56, 5309. Mannori, G., et al. 1995. Cancer Res. 55, 4425. Matrisian, L.M. 1992. BioEssays 14, 455. Springman, E.B., et al. 1990. Proc. Natl. Acad. Sci. USA 87, 364.
Chapter Twenty four
Nitric Oxide and Nitric Oxide Synthases
Nitric oxide (NO), a highly reactive, diffusible, and unstable radical, plays an important role in the regulation of a wide range of physiological processes, including cellular immunity, angiogenesis, neurotransmission, and platelet aggregation. Free NO is a transient species with a half-life of only about five seconds. Hence, most studies on NO actions are based on the activity of nitric oxide synthases (NOS).
NO produced is capable of diffusing across the cell membrane and react with a variety of targets. In vivo, NO is involved in three main chemical reactions: activation of soluble guanylate cyclase (sGC), rapid reaction with hemoglobin, and oxidation by superoxide. NO reaction with sGC is one of the best-studied signaling pathways wherein activated sGC catalyzes the conversion of GTP
24
to cyclic-GMP. By this mechanism NO regulates smooth muscle tone and the local blood flow. Reaction of NO with O2 in aqueous solutions produces the relatively unreactive nitrate and nitrite ions. However, NO can rapidly react with superoxide to produce highly reactive peroxynitrite (ONOO-). Almost all biological effects of NO are achieved either directly or through other reactive nitrogen intermediates. Nitric oxide synthases (NOS) are hemoproteins that catalyze the oxidation of arginine to NO and citrulline. They exist in three isoforms: (a) a soluble constitutively expressed enzyme found in high concentrations in the brain (bNOS; nNOS; or NOS-1); (b) an inducible enzyme (iNOS or NOS-2) that is associated with the cytotoxic function of macrophages and (c) a constitutively expressed endothelial membrane bound enzyme (eNOS, NOS-3).
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| Nitric Oxide and Nitric Oxide Synthases
These three isoforms exhibit similarities in their structure and mechanism of action. Calmodulin (CaM) is required for the activity of all three isoforms of NOS. The activation of the constitutively expressed isoforms requires Ca2+- dependent binding of calmodulin to the
Enzyme nNOS iNOS eNOS Gene NOS1 NOS2 NOS3 Number of Residues 1429-1433 1144-1153 1203-1205
enzyme. However, in case of iNOS, calmodulin is irreversibly bound to the enzyme and the activity of the enzyme is regulated by its rate of synthesis rather than by Ca2+ concentration. In the absence of CaM, iNOS is highly unstable.
Subcellular Localization Brain: mainly soluble skeletal muscle: mainly particulate Mainly soluble Mainly particulate
Regulation Ca2+/CaM Cytokine-inducible Ca2+independendent Ca2+/CaM
All NOS isoforms are single polypeptides that differ in their rates of NO synthesis, Ca2+dependence, cytochrome c reduction, and NADPH utilization. For their catalytic activities, NOS isoforms require three distinct domains: (a) a reductase domain, (b) a calmodulinbinding domain, and (c) an oxygenase domain. The reductase domain contains the flavin adenine dinucleotide (FAD) and flavin adenine mononucleotide (FMN) moieties. The oxygenase domain, which contains the binding sites for heme, tetrahydrobiopterin, and arginine, catalyzes the conversion of L-arginine to citrulline and NO. The maximal rate of NO synthesis is established by the intrinsic maximum ability of the reductase domain to deliver electrons to the heme domain. The nNOS is expressed in the nerve tissue (nNOS) and in skeletal muscle (nNOS). The nNOS differs from nNOS by the insertion of 34 amino acid residues between the calmodulinand flavin-binding domains. The nNOS form consumes NADPH and reduces cytochrome c at approximately half the rate of nNOS and its degradation rate is relatively slower than nNOS. The functionality of nNOS requires a rapid, localized production of NO and a timely end of biosynthesis. Hence, its primary regulation is mediated through the obligatory binding of calmodulin, which occurs in response to transient increases in intracellular Ca2+. The binding of calmodulin to nNOS triggers the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain. This initiates the catalytic activity of the enzyme. The physiological concentrations of NO generated by nNOS are in the picomolar range. The N-terminal
region of nNOS also has a unique binding site for a highly conserved protein called PIN (protein inhibitor of NOS) that inhibits its catalytic activity by destabilizing homodimer formation. The binding of PIN occurs at amino acids 163-245 of nNOS. When nNOS is phosphorylated by CaM kinase, the interaction between PIN and nNOS is disrupted. Thus CaM kinase may potentially augment NOS activity by blocking PIN repression of nNOS. Improper regulation of nNOS has been implicated in a number of neurologic diseases including neurodegenerative diseases such as Huntingtons disease and Parkinsons disease. A strong association of nNOS with neurofibrillary tangles and plaques has been demonstrated by immunolabeling studies. The iNOS is expressed in a variety of cells and is localized mainly in the cytoplasm. It binds tightly to CaM at very low Ca2+ levels, hence the enzyme activity is unaffected by changes in intracellular Ca2+ levels. The complete Ca2+- independence of iNOS requires interactions of CaM with both the CaM-binding motif and the flavoprotein domain. This property is important in assuring a longlasting release of NO. The iNOS is capable of producing large amounts of NO when induced by mediators such as TNF-, IL-1, IL-2, IL-6, and -IFN. The amount of NO released by iNOS is in the nanomolar range. The iNOS-induced release of NO plays an important role in the macrophage-mediated response to infections, where NO acts as an effector molecule to kill the invading organisms. This toxic effect is achieved via the production of highly reactive nitrogen and oxygen intermediates. The induction of iNOS has also been implicated in increased blood flow
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to the tumor site resulting in tumor growth. The cytotoxic effects of NO against cancer cells have also been reported. Overexpression of iNOS has also been implicated in septic shock. Here excessive generation of NO, in response to infection, leads to a serious drop in blood pressure and subsequent dysfunction of multiple organs.
eNOS is inhibited by its interaction with caveolin. Following agonist activation, eNOS dissociates from caveolin and translocates to the cytosol. The eNOS is generally activated in response to an increase in intracellular Ca2+ following stimulation with receptor-dependent agonists such as acetylcholine. However, shear stress can also activate this enzyme, independently of Ca2+ Although, iNOS is easily induced and expressed level changes, via a tyrosine kinase-dependent in macrophages during host defense mechanisms, mechanism involving its phosphorylation and higher expression of iNOS is also observed in a redistribution. Low concentrations of NO in variety of human cancer cells. This up-regulation endothelial cells prevent their apoptotic death and is associated with angiogenesis and higher tumor maintain their integrity during inflammation and grade. Several studies have shown that levels of atherosclerosis. Distorted production of NO in HIF-1 are elevated under the influence of NO, endothelial cells has been linked to hypertension, which leads to higher expression of vascular hypercholesterolemia, diabetes, and even cardiac endothelial growth factor (VEGF) and angio- failure. genesis in the tumor.
Nitric Oxide Donors

The eNOS is mainly expressed in vascular endothelial cells and cardiomyocytes. The NO produced by eNOS in endothelial cells acts as a vasodilator and regulates blood flow and blood pressure. In cardiac cells, eNOS is expressed in response to shear stress, exercise, chronic hypoxia, and cardiac failure. In endothelial cells, eNOS is localized in specialized plasmalemmal pockets known as caveolae, which contain a scaffolding protein, caveolin. Acylation of eNOS by either myristate or palmitate targets it to caveolae, which facilitates enzyme activation following receptor stimulation. In a resting cell, Researchers have always sought relatively stable NO donors with potential therapeutic value. In considering the biological or therapeutic value of any NO donor, one must take several factors into consideration. One must be able to control the rate and the amount of NO released; by-products of the reaction should have minimal side effects; and the release of NO should not be affected by common biochemical factors. Additionally, the quantities of NO delivered must be large enough to achieve the desired physiological effects. It may be possible to accomplish these objectives through local administration of NO-releasing
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| Nitric Oxide and Nitric Oxide Synthases
compounds, such as a carotid artery infusion to reverse cerebral vasospasm or transurethral administration to treat impotency. To effectively use NO-based drugs, development of NO-based pro-drugs, with protective groups attached may be necessary to obtain a targeted release of NO.
Synthetic chemical reagents that release NO continuously over a period of time, under physiological conditions, have been in use for a long time in clinical management of cardiovascular diseases. Among the most widely used NO donors are organic nitrates (e.g., glyceryl trinitrate) and nitrites, and furoxane derivatives. These donors require thiols as a cofactor for generating NO and can use endogenous sources of thiols. Here NO is first transferred to the thiol and is then released from the S-nitrosothiol. Hence, the thiol derivative becomes a true NO donor. Therefore, under conditions of thiol depletion (as in extreme oxidative stress), Another series of NO donors known as the NOR additional doses of organic nitrates do not provide compounds has been developed. These are nonthiol based compounds and they release NO any NO activity. spontaneously under physiological conditions in A number of NO donors have been developed a rate-controlled manner. Their by-products do to augment the action of intracellularly released not exhibit any significant biological activity. The NO, which are also known to stimulate guanylate pattern of NO release is very similar to that of the cyclase activity. Some of the known natural NOC series of compounds and follows the firstcarriers of NO are S-nitrosoglutathione and order kinetics. The half-life of NOR compounds S-nitrosocysteine. Upon conversion of thiols to varies from a few minutes to few hours. Therefore, S-nitrosothiols, NO is released by a homolytic one can select the appropriate NOR compound to break of the S-N bond (2 RS-NO RS-SR + 2 NO). control the release of NO. NOR compounds are The bioaction of S-nitrosothiols is reported to be relatively stable in anhydrous organic solvents. similar to that of NO in most cases. However, the use of S-nitrosothiols has a disadvantage in that BNN series of NO donors are photo-labile caged they are extremely unstable to be used as long compounds that release two molecules of NO term NO donors. S-Nitroso-N-penicillamine when exposed to UV light (~300 nm; xenon lamp (SNAP) offers somewhat higher stability when or laser flash light) for a brief period. The NO compared to other nitoso compounds. Although release is completed in about 20 seconds following the release of NO is spontaneous, it is somewhat exposure to UV light, allowing a controlled sudden sustained. Glyco-SNAPs ar more stable analogs burst of NO in the cell. of SNAP and the release of NO can be monitored over a period of 24 to 30 hours. Maragos, et al. (1991) developed a versatile group of stabilized NO-amine complexes known as NOC compounds. These donors release NO
spontaneously without the influence of any cofactors. They act as intramolecular zwitterions, stabilized with an intramolecular hydrogen bond through dispersion of the negative charge, which prevents protonation. These compounds, when dissolved in aqueous medium, such as buffer, plasma, or cell culture medium, dissociate to form two NO molecules and one molecule of the corresponding amine. The NO release follows the first-order kinetics and the half-life of NO release varies from a few minutes to several hours. Another important point to note here is that NO is released from NOC compounds at much higher rate at lower pH values. Therefore, it is advantageous to prepare a slightly alkaline solution of NOC, which can be then added to the sample buffer or to the culture medium. The alkaline stock solution is stable, at the most, for a day.
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References: Fitzpatrick, B., et al. 2008. Nitric Oxide 19, 217. Ekmekcioglu S., et al. 2007. Curr. Cancer Drug Targets 5, 103. Lancaster, J.R. Jr., and Xie, K. 2006. Cancer Res. 66, 6459. Lechner M., et al. 2005. Semin. Cancer Biol. 15, 277. Govers. R., and Rabelink, T.J. 2001. Am. J. Physiol. Renal Physiol. 280, F193. Al-Sadoni, H., and Ferro, A. 2000. Clin. Sci. 98, 507. Fitzhugh, A.L., and Keffer, L.K. 2000. Free Radic. Biol. Med. 28, 1463. Janero, D.R., et al. 2000. Free Radic. Biol. Med. 28, 1495. Mori. M., and Gotoh, T. 2000. Biochem. Biophys. Res. Commun. 275, 715. Palmer, L.A., et al. 2000. Mol. Pharmacol. 58, 1197. Titheradge, M.A. 1999. Biochim. Biophys. Acta 1411, 437 Yamamoto, T., and Bing, R.J. 2000. Proc. Soc. Exp. Biol. Med. 225, 200. Stuehr, D.J. 1999. Biochim. Biophys. Acta 1411, 217. Moncada, S. 1999. J. R. Soc. Med. 92, 164. Michel, T. 1999. Braz. J. Med. Biol. Res. 32, 1361. Namiki, S., et al. 1999. Bioorg. Med. Chem. 7, 1695. Petit, C., et al. 1999. Braz. J. Med. Biol. Res. 32, 1407. Wang, Y. et al. 1999. Crit. Rev. Neurobiol. 13, 21. Jaffrey, S.R., and Snyder, S.H. 1996. Science 274, 774. Anggard, E. 1994. Lancet 343, 1199. Hrabie, J.A., et al. 1993. J. Org. Chem. 58, 1472. Bartsch, H., et al. 1992. Pharmacogenetics 2, 272. Maragos, C.M., et al. 1991. J. Med. Chem. 34, 3242. Moncada, S., et al. 1991. Pharmacol. Rev. 43, 109.
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Chapter Twenty five
From Physiology to Pathology: Maintenance of the Critical Balance by Antioxidants

Guest Author: Harry Ischiropoulos, Ph.D., University of Pennsylvania, School of Medicine, Philadelphia, PA 19140
Oxygen metabolism, although essential for life, imposes a potential threat to cells because of the formation of partially reduced oxygen species.1,2 One electron reduction of oxygen produces superoxide whereas two electron reduction produces hydrogen peroxide. Therefore, electron flow through oxygen, utilizing processes such as the mitochondrial electron transport chain, flavoproteins, cytochrome P450 and oxidases, is tightly coupled to avoid partial reduction of oxygen.3
Normal cellular homeostasis is a delicate balance between the rate and magnitude of oxidant formation and the rate of oxidant elimination. Oxidative stress can, therefore, be defined as the pathogenic outcome of the overproduction of oxidants that overwhelms the cellular antioxidant capacity. Experimental support for oxidative stress as a mediator of cell death was provided recently by the finding that PC12 cells die following downregulation of Cu/Zn superoxide dismutase.4 Antioxidant defenses fall into two categories; enzymatic and nonenzymatic.1-3 Superoxide dismutases are metalloproteins that dismutate the superoxide radical (O ) to hydrogen peroxide (H O )
2 2 2
25 | From Physiology to Pathology: Maintenance of Critical Balance by Antioxidants
and molecular oxygen. Three types of superoxide dismutases are found in eukaryotic cells; Cu/Zn superoxide dismutase, predominantly located in the cytosolic fractions; Mn superoxide dismutase, located in the mitochondria, and EC superoxide dismutase, which is found in the extracellular space.1 Catalase, a heme protein located predominantly inperoxisomes and the inner mitochondrial membrane, catalyzes the conversion
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of H2O2 to H2O. In mammalian cells, the conversion of H2O2 to H2O is also accomplished by the reaction with glutathione catalyzed by glutathione peroxidases, a family of cytosolic
selenoenzymes. Non-enzymatic defenses include small molecules such as membrane associated -tocopherol, ascorbate and glutathione.
Organization of the antioxidant network
Biochemistry of Reactive Species: Free Radicals vs. Oxidants

The term free radicals has been equated with reactive species or oxidants. By definition, a radical is a molecule possessing an unpaired electron. Superoxide, nitric oxide, hydroxyl, alkoxyl and alkyl-peroxyl (lipid) are radicals. However, with the exception of hydroxyl radical none of these radicals are strong oxidants. Thus, not all radicals are strong oxidants and not all oxidants are radicals. A critical function of reactive species is immunological host response. Generation of reactive species and strong oxidants by inflammatory cells is essential for killing invading microorganisms. However, experimental evidence has implicated reactive species in the pathogenic
mechanism of several diseases. It is, therefore, important to understand the biochemical pathways for the induction of oxidative stress by reactive species. The most reasonable biochemical hypothesis is the reactive species-mediated modification of critical cellular targets. Iron-sulfur enzymes are direct targets for superoxide and toxicity can be derived from the inactivation of these enzymes.1 Hydrogen peroxide at low M levels does not react with many biological targets at an appreciable rate. However, the reaction of hydrogen peroxide with reduced divalent redox active metals such as iron can lead to the formation of strong oxidants. This reactivity of hydrogen peroxide may be important in biological oxidations of proteins and lipids that take place at the sites of metal binding.
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Divalent redox active metals can also catalyze the formation of the highly reactive hydroxyl by the metal-catalyzed Haber-Weiss reaction.5,6 O2 + Fe3+ O2 + Fe2+ H2O2 +Fe2+ OH + -OH + Fe3+ However, hydroxyl radical reacts with almost all biological targets at rates exceeding 109 M-1sec-1 and therefore its diffusion distance inside a cell is minimal. Thus, in order for hydroxyl radical to cause toxicity it must be formed within a few Angstroms from a biological target. An alternative pathway of superoxide toxicity is the formation of peroxynitrite by the reaction with nitric oxide.7 Nitric oxide is synthesized by nitric oxide synthases and mediates important physiological functions such as vasorelaxation, platelet aggregation, long term potentiation, and immune responses.8-11 The principal biological target of nitric oxide is guanylate cyclase and/or other iron-containing heme proteins. Nitric oxide is a radical but a weak one-electron oxidant. Since both NO and O2 are radicals they react rapidly to form peroxynitrite: NO + O2 ONOO-
carbon dioxide.12,15 Whereas the Zn-S and Fe-S centers will be oxidized, the last two reactivities will promote nitration of tyrosine residues on proteins. Protein nitrotyrosine has been detected in human diseases and experimental models of disease that do not involve an inflammatory process.7
Cellular Responses to Reactive Species: Time and Magnitude of Exposure

The flux and the time of exposure are critical factors in determining the outcome of oxidative stress. Aging can be considered the result of a continuous exposure to a low flux of reactive species over the life span. Although the antioxidant networks maintain the critical balance towards physiology, a few reactive species escape the surveillance of the antioxidant network and react with biological targets. Oxidation of biological targets will not necessarily translate to expression of a phenotype because repair processes may sustain normal physiologic function. However, as the frequency of oxidation of biological targets increases (and possibly as repair processes slow), detection of oxidized proteins, lipids and even DNA becomes apparent with aging and other reactive-species mediated pathologies.16-18
Severe oxidative stress results in necrotic cell death. Generation of reactive species during hyperoxia (breathing of >95% oxygen) or The second order rate constant of the reaction reperfusion of an ischemic tissue leads to tissue between nitric oxide and superoxide is 6.7 x 109 necrosis.19-20 A moderate exposure to reactive M-1sec-1 which is nearly three times faster than the species can also result in cell death that usually reaction of superoxide with superoxide dismutase occurs 20-24 hours after the initial insult. In most (2.9 x 109 M-1sec-1) and nearly thirty times faster cases delayed cell death resembles apoptosis since than the reaction of NO with heme proteins. This DNA fragmentation and other features of apoptosis implies that the formation of peroxynitrite can are evident. It is not clear how reactive species can out-compete the major scavenging pathways for induce delayed cell death or apoptosis. Potential NO and O2. Peroxynitrite is not a free radical pathways that once altered by reactive species will but a strong one or two electron oxidant and lead to delayed cell death include energy sources nitrating agent.12-15 Although peroxynitrite can (mitochondria, activation of Poly- ADP ribosyl oxidize most biological molecules similar to synthase), ionic homeostasis, signal transduction the hydroxyl radical, the rate constants of the and membrane structural integrity.4,21,22 biological oxidations of peroxynitrite are 10,000 fold slower than the rate of hydroxyl radical. Overall, the inherent ability of cells to withstand This implies that peroxynitrite will diffuse much oxidative stress is dependent upon several further than the hydroxyl radical and will react factors: their antioxidant capacity, the ability with selective targets. The targets are determined to sustain metabolic requirements by deriving for the most part by the rate by which they react energy from alternate pathways, efficiency to with peroxynitrite. The fastest reactions for repair oxidatively modified biomolecules, and peroxynitrite presently are the reactions with availability and utilization of trophic support. Zn-S and Fe-S centers with metalloproteins and
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Targets of reactive oxygen species
Reactive Species and Signal Transduction

Recent evidence has suggested that reactive species can be utilized in signal transduction events.23-27 Signal transduction for the most part is viewed either as a specific interaction between proteins or events mediated by second messenger molecules such as Ca2+ and cyclic nucleotides. Nitric oxide can be clearly considered a signal transducing molecule because it specifically activates guanylate cyclase. However, except for nitric oxide, specific targets that can be utilized in signal transduction are not known for other reactive species. Moreover, the steady state levels of reactive species such as superoxide and hydrogen peroxide are under the control of enzymatic pathways. For example, the steady state levels of superoxide in superoxide dismutase deficient E. coli is 5 x 10-7 M (taking into account scavenging by glutathione) whereas in superoxide dismutase proficient E. coli the levels are 2 x 1010 M.28 Therefore, the lack of specificity and the low intracellular levels creates difficulty in explaining how reactive species can be utilized in signal transduction. The answer to this question in part can be found in the biochemical reactivities and the cellular targets for reactive species. Superoxide, nitric
oxide and peroxynitrite react with Fe-S and Zn-S centers. Fe-S and Zn-S centers are not only found in enzymes regulating bioenergetics but also in transcription factors and in iron regulatory proteins. Peroxynitrite is also a nitrating agent that nitrates tyrosine residues in proteins. Nitration alters the pKa of tyrosine residues and interferes with the ability of tyrosine kinases to phosphorylate.29,30 The activity of different kinases, transcription factors and ion channels is redox sensitive and is dependent on a critical cysteine residue which can be modified by reactive species. Finally, reactive species can indirectly induce signal transduction events by inducing mitochondrial Ca2+ release and lipid peroxidation. These signaling pathways may be critical in mediating apoptosis or delayed cell death.
How to Detect and Quantify Reactive Species

The short half life of most reactive species in biological systems does not permit for their direct detection and quantification.3,5,6,23 Therefore, detection of reactive species relies on indirect measurement of modified targets. If you will, consider reactive species as sharks. Their presence
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in biological systems is therefore determined by the bite marks formed on critical cellular targets. In simple in vitro assays, the task of detection and quantification of reactive species is relatively well established. However, as one moves from the simple test tube assay, to cells in culture, to isolated organs, to whole animals or humans, the difficulty in detecting these bite marks increases exponentially. The ability to detect and quantify reactive species is a function of the amount of modified molecules present at a given time and the sensitivity of the assay. Biological targets that have been utilized for detection of oxidative modification include lipids, proteins, thiols, and DNA. Reactive species react with more than one biological target and since the concentration of biological targets varies among cells, it is difficult to predict which target will be preferentially modified. Therefore, in more complex systems, it may be necessary to measure more than one end-point modification of biological targets. For example, measurement of the reduced to oxidized glutathione ratio will reflect a degree of oxidative stress, but will not be useful in elucidating potential pathways responsible for the oxidation. In some models interference with the formation of the potential reactive species maybe useful in elucidating the reactive pathways. Another method for detecting reactive species is the use of reporter compounds that are oxidized by reactive species to either chromogenic, fluorescent, luminescent or Electron Paramagnetic Resonance products. These probes have been utilized in cells, isolated organs and whole animal models and fall in two categories: cell permeable and non-permeable compounds. Intracellular detection requires a substrate that has a reasonably fast rate of reaction with reactive species and can be delivered at high enough concentrations to outcompete antioxidant and scavenging pathways. Extracellular detection represents the fraction of reactive species that are either generated very close to cell membrane or escape the antioxidant and scavenging networks and have not been reacted with cellular targets. This implies that the magnitude of the stress inside the cell could be significantly higher compared to what is measured extracellularly.
My apologies to all the scientists that over the years have made significant contributions to this field, but their work is not cited here due to space limitations. References: 1. Fridovich, I., 1986. Arch. Biochem. Biophys. 247, 1. 2. McCord, J.M., and Fridovich, I. 1988. Free Rad. Biol. Med. 5, 363. 3. Freeman, B.A., and Crapo, J.D. 1982. Lab. Invest. 47, 412. 4. Troy, C.M., et al. 1996. J. Neurosci. 16, 253. 5. Buettner, G.R. 1993. Arch. Biochem. Biophys. 300, 535. 6. Halliwell, B., and Gutteridge, J.M.C. 1990. Trends Biochem. Sci. 15, 129. 7. Beckman, J. 1996. J. Chem. Res. Toxic. 9, 836. 8. Furchgott, R.F. 1996. JAMA 276, 1186. 9. Murad, F. 1996. JAMA 276, 1189. 10. Ignarro, L.J., et al. 1990. Annu. Rev. Pharmacol. Tox. 30, 535. 11. Moncada, S., et al. 1991. Pharmacol. Rev. 43, 109. 12. Koppenol, W.H., et al. 1992. Chem. Res. Toxicol. 5, 834. 13. Radi, R., et al. 1991. J. Biol. Chem. 266, 4244. 14. Crow, J.P., et al. 1995. Biochemistry 34, 3544. 15. Ischiropoulos, H., et al. 1992. Arch. Biochem. Biophys. 298, 431. 16. Ames, B.N., et al. 1995. Biochim. Biophys. Acta 127, 165. 17. Stadtman, E.R. 1992. Science 257, 1220. 18. Imlay, J.A., and Linn, S. 1992. Science 240, 1302. 19. Turrens, J.F., et al. 1982. Arch. Biochem. Biophys. 217, 411. 20. McCord, J.M.N. 1985. N. Engl. J. Med. 312, 159. 21. Dawson, V.L., and Dawson, T.M. 1995. Adv. Pharmacol. 34, 323. 22. Szabo, C., et al. 1996. Proc. Natl. Acad. Sci. USA 93, 1753. 23. Lander, H.M. 1997. FASEB J. 11, 118. 24. Suzuki, Y.J., et al. 1997. Free Rad. Biol. Med. 22, 269. 25. Meyer, M., et al. 1993 EMBO J. 12, 2005. 26. Pantopoulos, K., et al. 1994. Trends Cell Biol. 4, 82. 27. McConkey, D.J., and Orrenius, S. 1994. Trends Cell Biol. 4, 370. 28. Imlay, J.A., and Fridovich, I.J. 1991. J. Biol. Chem. 266, 6957. 29. Gow, A., et al. 1996. FEBS Lett. 385, 63. 30. Chance, B., and Gao, G. 1994. Environ. Health Persp. 10, 29.
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Chapter Twenty six
Lipid Signaling
Bioactive lipids, generated during the remodeling of membrane lipids by activated lipases, serve as intra- and extracellular mediators in cell signaling. They play important roles in a variety of processes involving cell-cell communication, inflammation, host-defense mechanisms, and ischemiareperfusion. Imbalances in lipid signaling pathways can lead to autoimmune disorders, allergies, atheroscleorosis, cancer and other diseases.
There are three major pathways involved in lipid signaling. Ceramide, a second messenger, has emerged as a potentially important pleiotropic signal transducer in apoptosis as well as in cell proliferation and differentiation. All sphingolipids contain ceramide as the basic hydrophilic component. Ceramide is generated either through de novo synthesis mediated by ceramide synthase or through hydrolysis of membrane sphingomyelin by an acid or neutral sphingomyelinase. Acid and neutral sphingomelinases differ in their ion dependence, pH optima, and cellular localization. Studies have shown that the activation of a non-specific lipid scramblase during apoptosis induces the flipping of sphingomyelin from the cell surface to the cytoplasm side of the plasma membrane where it is
26
cleaved by neutral sphingomyelinase to generate ceramide. The production of ceramide induces blebbing of the plasma membrane and aids in rapid engulfment by phagocytes. Neutral sphingomyelinase-released ceramide has also been shown to be essential for capping of L-selectin in lymphocytes. Some evidence exists indicating that acid sphingomyelinase deficient
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cells have defects in apoptotic signaling pathways. Sphingomyelin is usually rapidly broken down in the late endosomes and lysosomes. Hence, in acid sphingomyelinase deficiency, sphingomyelin may be kinetically trapped in lysosomes and disrupt endocytic trafficking of raft-associated
cell surface signaling molecules. Defects in acid sphingomyelinase have also been linked to lysosomal storage disease known as Niemann-Pick disease, which results in progressive enlargement of liver and spleen.
Organization of lipid signaling pathways
Sphingomyelinase can be activated by a variety of extracellular signals including tumor necrosis factor- (TNF-), -interferon (-IFN), interleukin-1 (IL-1), and ionizing radiation. The development of exogenous cell-permeable analogs of ceramide has facilitated the understanding of intracellular effects of ceramide. Ceramide is reported to play an important role in the activation of NF-B, protein kinase C, phospholipase A2 (PLA2), JNK/SAPK, and p42 MAP kinase. A ceramide-activated protein phosphatase (CAPP), belonging to the PP2A serine/threonine phosphatase family, has also been reported and linked directly to the role of ceramide in apoptosis. Ceramide is also involved in the action of another major pathway in lipid signaling the synthesis of eicosanoids via the activation of cyclooxygenase (COX or prostaglandin endoperoxidase H synthase) and PLA2. The second major pathway involves the activation of PLA2. Activation of PLA2 and generation of arachidonic acid is a major step in the downstream synthesis of prostaglandins, thromboxanes, and
leukotrienes. PLA2 is found in a wide variety of cells and its expression is stimulated by the action of a number of extracellular signals including the TNF-, IL-2, IL-6, and several inflammatory signals. The secreted PLA2 requires millimolar levels of calcium for its activation. The activated PLA2 then translocates to the membrane where it hydrolyzes glycerophospholipids at the sn -2 position yielding free fatty acid (arachidonic acid) and lysophospholipid. The latter can yield platelet activating factor (PAF), another important second messenger, by the action of an acetyltransferase. Two major types of PLA2 are found in cells: the cytosolic form (cPLA2) and the secretory form (sPLA2). cPLA2, an 85 kDa enzyme, preferentially hydrolyzes phospholipids containing arachidonate at the sn-2 position and provides free arachidonic acid for the synthesis of eicosanoids. cPLA2 is found in a variety of cells where it acts as a receptor-regulated enzyme that can mediate agonist-induced arachidonic acid release. It is activated by low levels of Ca2+. sPLA2, following its release from cells, plays an important role in inflammation and in anti-microbial defense.
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However, excessive activity of sPLA2 has been shown to result in tissue damage and is linked to organ failure associated with septic shock. Arachidonic acid undergoes a stepwise catalysis to yield reactive intermediates, PGG2 and PGH2, which serve as precursors of prostaglandins, prostacyclins, and thromboxanes. Prostaglandin, the 20-carbon polyunsaturated molecule, generated by the action of cyclooxygenase (COX), functions in an autocrine or paracrine manner. Several effects of prostaglandins are mediated by a series of G-protein coupled cell surface receptors. Their effects include vasodilation, smooth muscle contraction, inhibition of platelet aggregation and several others. COX are bifunctional hemoproteins that catalyze both the bisoxygenation of arachidonic acid to form PGG2 and the peroxidative reduction of PGG2 to form PGH2. Hence, COX has two different active sites. On one side, it has the cyclooxygenase active site, and on the opposite side is an entirely separate peroxidase site, which is required to activate the heme groups that participate in the cyclooxygenase reaction. The enzyme complex is a dimer of identical subunits, two cyclooxygenase active sites and two peroxidase active sites. COX-1 is a constitutive enzyme associated with the endoplasmic reticulum. It is responsible for maintaining normal physiologic function and is considered as a housekeeping enzyme. COX-2 is an inducible enzyme mainly associated with the nuclear envelope and is primarily associated with inflammation. Several cytokines and growth factors increase the expression of COX-2, mainly at inflammatory sites, producing prostaglandins, which mediate inflammation, pain, and fever. Increased expression of COX-2 has also been associated with increased incidence of colon and breast cancers. Non-steroidal anti-inflammatory drugs (NSAIDs) exert anti-inflammatory and analgesic effects through the inhibition of prostaglandin synthesis by blocking COX activity. Traditional NSAIDs inhibit prostaglandin formation through the inhibition of both COX-1 and COX-2. Inhibition of COX-1 is not necessary for anti-inflammatory and analgesic effects, but is thought to account for much of the toxicity of traditional NSAIDs. Based on structural differences in the active sites of these two isozymes, several new drugs have been developed that specifically inhibit only COX-2 activity. COX-2 selective inhibitors
have the potential to provide the traditional benefits of NSAID with significantly reduced incidence of endoscopic ulcers. They allow the preservation of COX-1 activity, which is essential in maintaining prostaglandins that are important for normal platelet function and protection of the gastrointestinal mucosa and still inhibit COX-2 to reduce inflammation and other pathologic processes. Due to the consideration of inflammation as a causative factor in Alzheimers disease there has been an upsurge of interest in COX-2 inhibitors as possible candidates for the treatment of this disease. Lipoxygenases (LOX) belong to a heterogenous family of lipid-peroxidizing enzymes and are involved in the biosynthesis of mediators of inflammation. Based on their regiospecificity during interaction with substrates, LOX have been classified as 5-, 8-, 12-, and 15-LOX. They insert oxygen at carbon 5, 8, 12 or 15 of arachidonic acid, respectively, forming 5S-, 8S-, 12S-, or 15S hydroperoxyeicosatetraenoic acid (5-, 8-, 12-, or 15-HPETE). HPETEs can be further reduced by glutathione peroxidase to the hydroxy forms (5-, 8-, 12-,15-HETE), respectively. 5-LOX is a dioxygenase that catalyzes the incorporation of molecular oxygen into arachidonic acid (oxygenase activity), producing HPETE and then forms the unstable epoxide LTA4 (LTA4 synthase activity). This is followed by the insertion of molecular oxygen at position C5, converting LTA4 to either 5(S)-hydroxy-6-trans-8,11,14cis-eicosatetranoic acid (5-HETE) or leukotrienes. Hydrolytic attack of LTA4 by leukotriene A4 hydrolase yields LTB4, a potent neutrophil chemoattractant and stimulator of leukocyte adhesion to endothelial cells. LTA4 can be conjugated with glutathione to form LTC4 by the action of LTC4 synthase. 5-LOX pathway has been implicated in the development and progression of human cancers. Hence, 5-LOX inhibitors have been sought for their chemopreventive effects. Inhibition of 5-LOX activity is shown to block prostate cancer cell proliferation. 12-LOX exists in three distinct forms: the platelet-type, the leukocyte-type, and the epidermal form. The platelet-type 12-LOX converts arachidonic acid to 12-(S)-HETE. The leukocyte-type 12-LOX metabolizes arachidonic acid or linoleic acid to either 12(S)- HETE or 15(S)-HETE. The epidermal form of 12-LOX converts arachidonic acid to 12-HETE and 15-HETE. 12-LOX has been shown to be involved in both cancer cell proliferation and survival.
26
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Inhibition of 12-LOX blocks cell proliferation and induces apoptosis in carcinosarcoma cells. 8-LOX is expressed in the skin after irritation or treatment with tumor promoters. Compared with other LOX enzymes, 8-LOX has received little attention for its role in carcinogenesis and cancer growth. 15-LOX exists as two isozymes, 15-LOX1 and 15-LOX-2. It converts arachidonic acid to 15-HPETE which is then reduced by glutathione peroxidase to 15-HETE. The preferred substrate for 15-LOX-1 and 15-LOX-2 are linoleic acid and arachidonic acid, respectively. The15- LOX-1 product, 13-S-HODE, is reported to enhance cell proliferation and potentiate the mitogenic response to EGF in different cell types. 15-LOX has also been implicated in the pathogenesis of atherosclerosis.
require Ca2+ for their activation. The binding of Ca2+ translocates PKC to the membrane where it interacts with DAG to transform into a fully active enzyme. All PKC isoenzymes, with the exception of and , are activated by diacylglycerol (DAG) that acts by increasing the affinity of PKC for Ca2+ and helps in full activation of PKC without a net increase in Ca2+ concentration.
References: Wymann, M.P., and Schneiter, R. 2008. Nat. Rev. Mol. Cell Biol. 9, 162. Van Echten-Deckert, G., and Herget, T. 2006. Biochim. Biophys. Acta 1758, 1978. Kuhn, H. 2005. Expert Rev. Cardiovas. Ther. 3, 1099. Brune, K., and Hinz, B. 2004. Scand. J. Rheumatol. 33, 1. Zhao, L., and Funk, C. D. 2004. Trends Cardiovasc. Med. 14, 191. Cremesti, A.E., et al. 2002. FEBS Lett. 531, 47. Funk, C.D. 2001. Science 294, 1871. Johnson, A.J., et al. 2001. Adv. Enzyme Regul. 41, 221. McGeer, P.L., and McGeer, E.G. 2001. Neurobiol. Aging 22, 799. Schnitzer, T.J. 2001. Am. J. Med. 110 (Suppl 1), S46. Shureiqi, I., and Lippman, S.M. 2001. Cancer Res. 61, 6307. Sillence, D.J. 2001. BMC Cell Biol. 2, 24. Thomas, T., et al. 2001. NeuroReport 12, 3263. Weggen, S., et al. 2001. Nature 414, 212. Zhang, Y., et al. 2001. J. Biol. Chem. 276, 11 775. Crofford, L.J., et al. 2000. Arthritis Rheum. 43, 4. Fournier, D.B., et al. 2000. J. Cell Biochem. 77, 97. Herget, T., et al. 2000. J. Biol. Chem. 275, 30344. Sugaya, K., et al. 2000. Jpn. J. Pharmacol. 82, 85. Malisan, F., and Testi, R. 1999. FEBS Lett. 452, 100. Yamamoto, S., et al. 1999. Adv. Exp. Med. Biol. 447, 37. Jaffrezou, J.P., et al. 1998. FASEB J. 12, 999. Martin, T.F.J. 1998. Annu. Rev. Cell Dev. Biol. 14, 231. Tomiuk, S., et al. 1998. Proc. Natl. Acad. Sci. USA 95, 3638. Anderson, K.M., et al. 1996. Anticancer Res. 16, 2589. Roberts, M. 1996. FASEB J. 10, 1159. Serhan, C.N., et al. 1996. FASEB J. 10, 1147. Spiegel, S., and Milstein, S. 1995. J. Membr. Biol. 146, 225. Obeid, L.M., and Hannun, Y.A. 1995. J. Cell. Biochem. 58, 191. Banno, Y., et al. 1994. FEBS Lett. 340, 185. Hannun, Y.A. 1994. J. Biol. Chem. 269, 3125. Piomelli, D. 1993. Curr. Opin. Cell Biol. 5, 274. Bielawska, A., et al. 1993. J. Biol. Chem. 268, 26226. Dobrowsky, R.T., and Hannun, Y.A. 1992. J. Biol. Chem. 267, 5048. Nishizuka, Y. 1992. Science 258, 607. Whatley, R.E., et al. 1990. Prog. Lipid Res. 29, 45. Mene, P., et al. 1989. Am. J. Physiol. 256, F375.
The third major pathway in lipid signaling involves phosphoinositide-specific phospholipase C (PLC) that generates two ubiquitous second messengers - diacyglycerol (DAG) and inositol trisphosphate (IP3). PLC is reported to exist in three major forms - , , and . All mammalian PLC isoforms contain four common domains catalytic domain, C2 domain, plekstrin homology domain, and EF-hand domain. The PLC is activated by tyrosine kinases, while the PLC is regulated by the -subunit of the Gq family of G-proteins as well as through the -subunits of the pertussis toxin-sensitive G-protein. Binding of a hormone or other affector molecule to the membrane receptor results in the activation of PLC via a G-protein-dependent phenomenon. The activated PLC hydrolyzes phosphatidylinositol4,5-bisphosphate (PIP2) to produce DAG and IP3. The IP3 binds to the IP3 receptor on the endoplasmic reticulum and causes the release of endogenous Ca2+ that binds to the cytosolic PKC and exposes the phospholipid binding site. The regulatory domain of PKC contains a Ca2+ binding site, designated the C2 region, that is found only on , , and -isozymes. The PKC isozymes , , , , , and lack the C2 region and do not
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Chapter Twenty seven
Calcium Signaling
Guest Author: Michael L. Berridge, Ph.D., Bubraham Institute, Cambridge, UK CB2 3ES
The divalent cation calcium (Ca2+) is used by cells as a second messenger to control many cellular processes including muscle contraction, secretion, metabolism, neuronal excitability, cell proliferation, and cell death.
The cytosolic level of Ca2+ in resting cells is kept low (10 - 100 nM), but stimulation results in the level increasing into the 500 to 1000 nM range required to activate sensors such as calmodulin and troponin C. The cell has access to two sources of signal Ca2+, entry from the external medium and release from internal stores.1-3 These Ca2+ ON mechanisms are balanced by Ca2+ pumps which constitute the OFF mechanisms responsible for removing the Ca2+ signal.4 These ON and OFF mechanisms are often organized to produce brief spikes and waves of calcium. Cells may avoid the cytotoxic effects of calcium by employing this oscillatory mode of calcium signaling.
Ca2+ ON Mechanisms
Plasma Membrane Ca2+ Channels
The plasma membrane has a variety of Ca2+ entry channels that are characterized by their mechanisms of activation.
27
Voltage-operated channels (VOCs)

A family of channels that open in response to membrane depolarization to mediate the selective entry of Ca2+.5 The multiple types (L, T, N, P/Q and R) are classified on the basis of their kinetics and pharmacological properties: L-type (long-lasting, activated by high voltage, sensitive to Verapamil and 1,4-dihydropyridines such as Nifedipine. The non-skeletal muscle L channels are blocked by FS-2 and Calciseptine)
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N-type (neuronal, transient, activated by high Intracellular Ca2+ Channels voltage, sensitive to -conotoxin GVIA) The endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) has two families of intracellular P/Q-type (long-lasting, low-voltage activated, channels responsible for releasing Ca2+ from this sensitive to -agatoxin and funnel web spider internal store.1,8 toxin, FTX-3.3) R-type (activated by high voltage)
Inositol 1,4,5-trisphosphate (InsP3) Receptors:
Agonists acting through G-protein-linked or T-type (transient, low-voltage activated, tyrosine kinase-linked receptors stimulate phospholipase C (PLC) to generate the second sensitive to mibefradil) messenger InsP3, which then diffuses into the cytoplasm to release stored Ca2+ by binding to Receptor-operated channels (ROCs) Ca2+ channels that are opened by the binding of InsP3 receptors.1,9 The enzyme PLC can be activated specific agonists usually neurotransmitters such by Pasteurella multocida toxin and inhibited by U-73122 and Neomycin. The InsP3 receptor can as glutamate (the NMDA receptor) or ATP. be activated by Adenophostin A, Furanophostin, Store-operated channels (SOCs) and Ribophostin, but is inhibited by Heparin, Many cells have SOCs in the plasma membrane Xestospongin and 2-APB. that are opened by the emptying of the internal stores.6 Just how empty stores communicate Ryanodine Receptors (RYR) with the membrane SOCs is still unclear, current Originally described in muscle cells, these proposals include a calcium influx factor (CIF) RYRs are also found in neurons and other cell or information transfer through a direct protein- types.9,10 Cyclic ADP ribose is a putative second messenger for regulating the activity of RYRs.11,12 protein interaction.7 Ryanodine also binds to these receptors and can
Many cellular processes are regulated by the second messenger Ca2+ which is derived from two separate sources. The ON mechanisms depend upon Ca2+ entry through channels in the plasma membrane or Ca2+ release through ryanodine receptors (RYRs) or inositol trisphosphate receptors (InsP3Rs). The OFF mechanisms remove Ca2+ from the cytoplasm using pumps. Also illustrated are the sites of action of some of the products capable of effecting these ON and OFF mechanisms.
An overview of the ON and OFF mechanisms and modulation of intracellular calcium levels
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initiate release by locking the Ca2+ channel in an open configuration. RYR-induced Ca2+ release is activated by Caffeine and 4-Chloro-m-cresol but is inhibited by Dantrolene. The RYR channel is modulated by various associated proteins such as the FK506 binding protein 12 (FKBP12).9 Bastadins can modulate Ca2+ release by interacting with the RYR/FKBP12 complex.13
a variety of factors including calmodulin, acidic phospholipids, and protein kinases A and C (PKA and PKC). The internal stores have a sarco/ endoplasmic reticulum Ca2+ ATPase (SERCA) whose activity is inhibited by agents such as thapsigargin, cyclopiazonic acid and BHQ.8 The mitochondria also play an important role in Ca2+ signaling in that they sequester Ca2+ rapidly during the recovery phase and then release it back slowly when the cell is at rest. They are particularly effective when they lie close to the Ca2+ release channels.18 Uptake of Ca2+ into the mitochondria is inhibited by Ru360. If the mitochondrion accumulates too much Ca2+ it forms a permeability transition pore, which collapses the transmembrane potential leading to the release of cytochrome c and the initiation of apoptosis. The apoptotic regulatory proteins that function as death antagonists (Bcl-2 and Bcl-XL) or death agonists (Bax, Bak, and Bad) may act by modulating the way the mitochondrion handles Ca2+.
Sphingolipid Ca2+ release-mediating protein of the ER (SCAMPER):

This putative SCAMPER channel is activated by Sphingosine-1-phosphate.
Calcium-Induced Calcium Release (CICR)

The InsP3Rs and the RYRs are sensitive to Ca2+ through both positive and negative feedback effects. A positive feedback process of Ca2+induced Ca2+ release (CICR) is of critical importance in generating and shaping Ca2+ signals. Once the channels open a microdomain of Ca2+ begins to build up and the channels then close as the positive feedback effect is replaced by a negative feedback response. An important function of InsP3 and cyclic ADP ribose is to increase the Ca2+ sensitivity of the intracellular channels such that the cytoplasm becomes an excitable medium capable of generating Ca2+ spikes and waves.
Ca2+-Binding Proteins
Cells have a variety of Ca2+-binding proteins functioning either as buffers to shape the cellular response or as sensors to carry out the messenger role of Ca2+.
Elementary and Ca2+ Signaling
Global
Aspects
of
When cells are stimulated, the global Ca2+ signal often appears as repetitive spikes, often organized into regenerative waves which can sometimes take on complex spiral patterns as described in Xenopus oocytes.2,14 These global responses are built up from the elementary events represented by the opening of either single or localized groups of InsP3, and RYR channels.15 Sensitive imaging techniques have begun to reveal these elementary events, e.g. Ca2+ sparks in muscle cells and puffs in Xenopus oocytes.15-17 Regenerative Ca2+ spikes and waves are produced by coordinating these elementary events through the process of CICR.
Ca2+ Buffers
Both the cytoplasm and the lumen of the ER/SR have proteins capable of buffering Ca2+. As Ca2+ is pumped into the lumen of the ER/SR, it is buffered by storage proteins such as calsequestrin and calreticulin, which have a low affinity (Kd in the mM range), but a high capacity (approximately 50 Ca2+ ions bound/molecule). When Ca2+ enters the cytosol it is rapidly buffered by proteins such as calbindin, calretinin, and parvalbumin. More than 90% of the Ca2+ entering the cell is bound to these buffers with the small remainder representing the stimulus-evoked elevation of Ca2+ responsible for activating the Ca2+ sensors.19
27 | Calcium Signaling
Ca2+ OFF Mechanisms

The Ca OFF mechanisms depend on pumps that remove the Ca2+ signal during the recovery from stimulation.4 The surface membrane has a Na+/Ca2+ exchanger (found mainly in excitable cells) and the ubiquitous plasma membrane Ca2+ ATPase (PMCA). The latter is regulated by
2+
Ca2+ Sensors
Ca2+ sensors such as the EF-hand proteins, annexins20 and the S100 proteins mediate the intracellular effects of Ca2+. The EF hand proteins such as troponin C (TnC) and calmodulin (CaM), which have a characteristic
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Ca2+-binding domain between two helices (named after the E and F -helices of parvalbumin), are the major Ca2+ sensors. TnC functions in skeletal and cardiac muscle whereas CaM has a much more general role. CaM exerts some of its effects by stimulating a multifunctional CaM-dependent protein kinase II (CaMKII) or a CaM-dependent protein phosphatase known as calcineurin.21 The function of CaM can be antagonized by W-5, W-7, W-12 and W-13. The immunosupressant drugs FK506 and Cyclosporin A can inhibit Calcineurin. The annexins represent a heterogeneous family that share a common property of interacting with membranes in a Ca2+-dependent manner.20 Since they have a low affinity for Ca2+, their action seems to be restricted to domains near membranes where Ca2+ channels create localized high elevations of Ca2+. Annexins have been implicated in the control of phospholipase A2, cytoskeletal reorganization, vesicle movement and some may function as Ca2+ channels.
Metabolism
Glycogen breakdown in liver cells is controlled by a calcium-dependent activation of phosphorylase.
Neuronal Excitability
The excitability of neurons can be modulated through calcium-dependent effects on ion channels (e.g. potassium channels) and ionotropic receptors (e.g. AMPA receptors). Some of these effects of calcium are long lasting, such as longterm potentiation (LTP) or long-term depression (LTD), and have been implicated in learning and memory.
Cell Proliferation
Calcium plays an important role both in fertilization22 and in controlling cell proliferation.23 In the case of lymphocyte activation, the immunosuppressant drug cyclosporin A acts by inhibiting the calcium-dependent transfer of information from the T cell receptor to the nucleus.24
Cell Death
Elevated levels of calcium, especially if maintained for long periods, can be cytotoxic.25 Calcium has been implicated in both necrosis and apoptosis.
Signaling Functions of Calcium

As indicated in the figure, calcium functions as a second messenger to regulate a great variety of cellular processes.
Manipulation and Measurement of Intracellular Calcium

A large number of reagents have been developed to manipulate and to measure intracellular levels of calcium.
Contraction
Excitation-contraction coupling in skeletal and cardiac muscle depends upon the release of calcium by RYRs located on the sarcoplasmic reticulum. Pharmacomechanical coupling in smooth muscle is controlled by the release of calcium from either InsP3Rs or RYRs depending on the muscle type.
Calcium Ionophores
One of the simplest methods of artificially raising the level of intracellular calcium is to use ionophores such as A23187 and Ionomycin.
Secretion
During stimulus-secretion coupling, calcium acts either to release preformed materials by exocytosis (e.g., transmitter release at synaptic endings) or to stimulate the ionic mechanisms responsible for fluid secretion (e.g., in exocrine glands).
Caged Pounds
A number of calcium signaling components are available as caged compounds. They are introduced into cells as inactive precursors and then released by illumination with near UV light. Some of these caged compounds have been
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produced as membrane-permeant acetoxymethyl (AM) esters. The InsP3 receptor can be activated using either Caged InsP3 or Caged GPIP2. Sudden increases in intracellular calcium can be obtained using a variety of caged calcium compounds.
Calcium Probes
Intracellular calcium is measured by introducing a variety of either single-wavelength (e.g., QUIN 2, FLUO 3) or dual-wavelength indicators (FURA 2, INDO 1).
Calcium Buffers
It must be remembered that the calcium probes mentioned above act by binding calcium and thus function as buffers, especially if large amounts are loaded into cells.19 Both EGTA and BAPTA can be used to increase the normal buffering capacity of the cell.
References: 1. Berridge, M.J. 1993. Nature 361, 315. 2. Clapham, D.E. 1995. Cell 80, 259. 3. Simpson, P.B., et al. 1995. Trends Neurosci. 18, 299. 4. Carafoli, E. 1994. J. Hypertension 12 (Suppl. 10), S47. 5. Catterall, W.A. 1998 Cell Calcium. 24, 307. 6. Putney, J.W., Jr., and McKay, R. R. 1999 BioEssays 21, 38. 7. Berridge, M.J. 1995. Biochem. J. 312, 1. 8. Pozzan, T., et al. 1994. Physiol. Rev. 74, 595. 9. Mackrill, J. J. 1999. Biochem. J. 337, 345. 10. McPherson, P.S., and Campbell, K.P. 1993. J. Biol. Chem. 268, 13765. 11. Galione, A., and White, A. 1994. Trends Cell Biol. 4, 431. 12. Lee, H.C. 1994. Mol. Cell Biochem. 138, 229. 13. Pessah, I.N., et al. 1997. Am. J. Physiol. 272, C601. 14. Amundson, J., and Clapham, D. 1995. Curr. Opin. Neurobiol. 3, 375. 15. Bootman, M.D., and Berridge, M.J. 1995. Cell 83, 675. 16. Yao, Y., et al. 1995. J. Physiol. 482, 533. 17. Niggli, E. 1999. Annu. Rev. Physiol. 61, 311 18. Rizzuto, R., et al 1993.Science 262, 744. 19. Neher, E., and Augustine, G.J. 1992. J. Physiol. 450, 273. 20. Swairjo, M.A., and Seaton, B.A. 1994. Annu. Rev. Biophys. Biomol. Struct. 23, 193. 21. Schulman, H. 1993. Curr. Opin. Cell Biol. 5, 247. 22. Stricker, S.A. 1999. Develop. Biol. 211,157. 23. Berridge, M.J. 1995. BioEssays 17, 491. 24. Crabtree, G.R., 1999 Cell 96, 611. 25. Trump, B.F., and Berezesky, I.K. 1995. FASEB J. 9, 219.
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Chapter Twenty eight
Cytokine and Chemokine Signaling
Cytokines, a group of small secreted proteins, are involved in mediation and regulation of immunity, inflammation, and hematopoiesis. They are produced de novo in response to an immune stimulus and act over short period of time at very low concentrations.
Cytokine action is initiated by binding to specific membrane receptors, which then signal the cell via second messengers to alter cellular behavior, including gene expression. Cytokines can increase or decrease expression of membrane proteins, including cytokine receptors, cell proliferation, and secretion of effector molecules. Cytokines also include lymphokines (cytokines of lymphocytic origin), monokines (cytokines of monocyte origin), chemokines (cytokines with chemotactic properties), and interleukins (cytokines made by one leukocyte and acting on other leukocytes). They may have an autocrine, paracrine, or endocrine
28
function. Several cytokines have therapeutic potential, either alone or when delivered along with any chemotherapeutic agent. Cytokines share many properties with hormones and growth factors. They mediate communication among cells in the immune system through binding to specific receptors on target cells. Their biological actions vary widely depending upon the type of target tissue involved. They are endowed with anti-proliferative properties and regulate the synthesis of acute phase proteins following tissue injury, trauma, inflammation, and sepsis. Receptors for a large number of cytokines have been cloned and shown to be membrane-spanning glycoproteins with their amino termini in the extracellular space. Unlike receptors for growth factors,
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cytokine receptors generally lack identifiable catalytic activity. The major diagnostic feature of the cytokine receptors is the presence, in the extracellular region of the receptor, of a domain containing multiple cysteine residues and a conserved amino acid motif, WSXWS (Trp-SerX-Trp-Ser) that functions in the recognition and binding of the ligand. The cytokine family of signaling molecules includes several interleukins, a variety of growth and colony-stimulating factors, ciliary neurotrophic factor, interferons, and several other molecules that exhibit pleiotropic effects on cell differentiation, tissue development, and
homeostasis. Interleukins comprise the largest group of cytokines that stimulate immune cell proliferation and differentiation. For example, interleukin 1 (IL-1) activates T cells; IL-2 stimulates proliferation of antigen-activated T and B cells; IL-4, IL-5, and IL-6, stimulate proliferation and differentiation of B cells; Interferon (IFN-) activates macrophages; and IL-3, IL-7 and Granulocyte Monocyte Colony-Stimulating Factor (GM-CSF) stimulate hematopoiesis. Different cytokines may carry out similar or even over-lapping functions. This property of cytokines ensure that hematopoietic system has sufficient flexibility to prevent any hematopoietic failure.
Role of cytokines in hematopoietic cells organization.
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Members of the hematopoietin family of cytokine receptors, which include several interleukins and GM-CSF, generally have two subunits, one cytokine-specific and one signal transducing. For example, GM-CSF receptor has a unique subunit that specifically binds to GM-CSF and a shared subunit signal transducer that increases cytokine-binding affinity. Cytokine binding promotes dimerization of the and subunits, which then associate with cytoplasmic tyrosine kinases to phosphorylate proteins, which activate transcription. The IL-2R subfamily of receptors (e.g., receptors of IL-2, IL-4, IL-7, IL-9, and IL-15) has a common signal-transducing chain and each member has a unique cytokine-specific
chain. Trimeric IL-2 and IL-15 receptors share an IL-2R chain. Monomeric IL-2R has low affinity for IL-2, dimeric IL-2R has intermediate affinity, and trimetric IL-2R binds IL-2 with high affinity. The Interferon family receptors have the conserved cysteine residues, but not the Trp-SerX-Trp-Ser sequence that is seen in interleukins. Most cytokine receptors such as those for IL-3, GM-CSF, and the interferons lack intrinsic kinase activity. They are thought to transmit their regulatory signals primarily by the receptorassociated JAK (Janus kinase) family of tyrosine kinases. Ligand binding to the receptor results in JAK activation that phosphorylates cytoplasmic
Origin and Biological Role of Various Cytokines

Interleukins/ Interferons IL1- and - Macrophages and other antigen presenting cells (APCs) IL-2 IL-3 IL-4 Activated TH1 cells, NK cells Activated T cells TH2 and mast cells Co-stimulation of APCs and T cells, inflammation and fever, acute phase response, hematopoiesis Proliferation of B cells and activated T cells Growth of hematopoietic progenitor cells B cell proliferation, eosinophil and mast cell growth and function, IgE and class II MHC expression on B cells IL-5 IL-6 IL-7 IL-8 IL-9 IL-10 TH2 and mast cells Activated TH2 cells, APCs, other somatic cells Thymic and marrow stromal cells Macrophages, other somatic cells T cells Activated TH2 cells, CD8+ T and B cells, macrophages IL-11 IL-12 IL-13 INF- and - Stromal cells B cells, macrophages TH2 cells Macrophages, neutrophils and some somatic cells INF- Activated TH1 and NK cells Eosinophil growth and function Acute phase response, B cell proliferation, thrombopoiesis T and B lymphopoiesis Chemoattractant for neutrophils and T cells Hematopoietic and thymopoietic effects Inhibits cytokine production, promotes B cell proliferation and antibody production, suppresses cellular immunity, mast cell growth Synergisitc hematopoietic and thrombopoietic effects Proliferation of NK cells, INF- production, promotes cell-mediated immune functions IL-4-like activities Antiviral effects, induction of class I MHC on all somatic cells, activation of NK cells and macrophages Induces of class I MHC on all somatic cells, induces class II MHC on APCs and somatic cells, activates macrophages, neutrophils, NK cells, and promotes cell-mediated immunity. Has antiviral effects. Produced by Major Biological Role
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28 | Cytokine and Chemokine Signaling
STAT (signal transducer and activator of transcription) proteins. Following phosphorylation on tyrosine residues, STATs are dimerized (resulting from phosphotyrosine-SH2 domain association). This dimerization is accompanied by translocation of STAT to the nucleus resulting in DNA binding to specific response elements and stimulation of gene transcription. Chemokines belong to a family of structurally related, low molecular weight (8 to 10 kDa), glycoproteins of approximately 70 to 80 amino acid residues. Most chemokines fall into two subfamilies (a) CXC chemokines that contain a single amino acid between the first and second cysteine residues; (b) CC chemokines that have adjacent cysteine residues. Most CXC chemokines are chemoattractants for neutrophils, whereas CC chemokines generally attract monocytes, lymphocytes, basophils, and eosinophils. Within the CXC subfamily, the chemokines can be further divided into (a) CXC chemokines with characteristic ELR (glutamic acid-leucinearginine) sequence immediately preceding the first cysteine residue near the amino terminus, and (b) CXC chemokines lacking the ELR domain. The ELR containing chemokines, such as IL-8, act primarily as chemoattractants and activators of neutrophils. The ELR lacking chemokines, such as RANTES, eotaxin etc., chemoattract and activate monocytes, B and T lymphocytes, NK cells, basophils, and eosinophils. In addition to these two major groups there are two other small subgroups. The C group contains only one member, lymphotactin, which lacks one of the cysteines in the four-cysteine motif, but shares homology at its carboxyl terminus with the CC chemokines. The other subgroup is the C-X3-C subgroup, which has three amino acids separating the first two cysteine residues. Fractalkine/neurotactin belongs to this subgroup. They are tethered directly to the cell membrane via a long mucin stalk and are involved in adhesion and migration of leukocytes.
Chemokines exert their effect through chemokine receptors, which belong to a superfamily of seven transmembrane loops and transduce their signals through heterotrimeric G Proteins. The chemokine receptors that bind CXC chemokines are designated CXCRs and the receptors that bind CC chemokines are designated CCRs. Various CXCRs and CCRs exhibit some overlapping ligand specificities. CXCR-1 and CXCR-2 share about 77% amino acid sequence homology. IL-8 binds to both receptors with high affinity and induces rapid elevation of cytosolic Ca2+ levels. CXCR-1 exhibits much higher specificity for IL-8, however, CXCR-2 has broader specificity and binds with high-affinity to other ELR motif containing chemokines including GRO, and . Those chemokines that lack the ELR motif do not exhibit any affinity for CXCR-2. CXCR-3, which is highly expressed by IL-2- activated T lymphocytes, binds IP-10 (INF- inducible protein of 10 KDa) and Mig (monokine induced by INF- ) with high affinity and mediates Ca2+ mobilization and chemotaxis. It does not bind ELR-containing CXC chemokines. CXCR-4, also known as fusin, is reported to be a necessary cofactor for entry of T cell-tropic HIV viruses into CD4+ cells. PBSF/SDF-1 chemokines act as ligands for CXCR-4. CCR-1, the first identified CC chemokine receptor, is expressed on monocytes and neutrophils and binds macrophage inflammatory protein-1 (MIP-1, RANTES, and monocyte chemoattractant protein-3 (MCP-3) with high affinity. CCR-2A and CCR-2B are expressed on monocytes and specifically bind MCP-1 and MCP-3. CCR-3 exhibits high affinity for eotaxin. CCR-4, originally cloned from a human immature basophilic cell line, is also expressed in T cells and IL-5-primed basophils. It mediates the effects of RANTES, MIP-1, and MCP-1. CCR-5, the most recent entry to the group of CC receptors exhibits about 48 to 75% amino acid sequence homology with other CC receptors. It is expressed in primary adherent monocytes, but not in neutrophils or eosinophils. It mediates the activities of RANTES, MIP-1, and MIP-1.
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Selected Chemokines: Receptors and their Expression

Chemokine Eotaxin 1 Fractalkine I-309 IL-8 GRO I-TAC MIF-1 Alternate Names CCL11 CX3CL1 CCL1 CXCL8 CXCL1; Growth-related protein CXCL11; Interferon-inducible T cell chemoattractant CCL3; Macrophage migration inhibitory factor 1 CCR9 MCP-1 CCL2; Monocyte chemoattractant protein-1 CCR10 MCP-3 CCL7; Monocyte chemoattractant protein-3 CCR3 MDC MIP-3 MIP-3 CCL22; Macrophage-derived chemokine CCL20; Macrophage inflammatory protein 3 CCL19; Macrophage inflammatory protein 3 SDF-1/PBSF CXCL12; Pre-B cell growth stimulating factor; Stromal cellderived factor 1 SDF-1/PBSF CXCL12; Pre-B cell growth stimulating factor; Stromal cellderived factor 1 TARC CCL17; Thymus and activationregulated chemokine CCR8 Activated T cells, NK cells TECK CCL25; Thymus expressed chemokine CCR9 CCR11 Dendritic cells, thymocytes. Expressed in various organs CCR4 Activated T cells, basophils, dendritic cells. CXCR4 T cells, monocytes CXCR4 CCR7 CCR6 Activated T cells, B cells, immature dendritic cells Activated T cells, B cells, immature dendritic cells T cells, monocytes CCR4 CCR2 CCR2 CCR5 Activated T cells, monocytes, dendritic cells Dendritic cells, thymocytes Activated T cells, basophils, monocytes, dendritic cells, NK cells Skin T cells Activated T cells, basophils, monocytes, dendritic cells Activated T cells, eosinophils, basophils, dendritic cells Activated T cells, basophils, dendritic cells Known Receptor CCR3 CX3CR1 CCR8 CRCR1 CXCR2 CXCR3 Activated T cells, eosinophils, basophils, dendritic cells Activated T cells, monocytes, NK cells Activated T cells, NK cells Neutrophils, NK cells Neutrophils, NK cells Activated T cells, NK cells Receptor Expression
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References: Margolin, K. 2008. Expert Opin. Biol. Ther. 8, 1495. Callewaere, C., et al. 2007. J. Mol. Endocrinol. 38, 355. McInnes, C.J., et al. 2005. J. Virol. 79, 11205. Kieseier, B.C., et al. 2002. Brain 125, 823. Lotem, J., and Sachs, L. 2002. Oncogene 21, 3284. McCubrey, J.A., et al. 2000. Leukemia 14, 9. Paukku, K., et al. 2000. Biochem. J. 345, 759. Haan, S., et al. 2000. Biochem. J. 345, 417. Baserga, R., et al. 1999. J. Cell Biochem. (Suppl.). 32-33, 68. Platanias, L.C., et al. 1999. Exp. Hematol. 27, 379. Feng, Y., et al. 1996. Science 272, 872. Kitaura, M., et al. 1996. J. Biol. Chem. 271, 7725. Power, C.A., et al. 1995. J. Biol. Chem. 270, 19495. Reddy. G.P. 1994. J. Cell Biochem. 54, 379. Sadowski, H.B., et al. 1993. Science 261, 1739. Canalis, E. 1992. J. Clin. Endocr. Metab. 75, 1. Lee, J., et al. 1992. J. Biol. Chem. 267, 16283. Miyajima, A., et al. 1992. Annu. Rev. Immunol. 10, 295. Holmes, W.E., et al. 1991 Science 253, 1278. Koj, A., et al. 1991. Biomed. Biochim Acta 50, 421. Takishima, K., et al. 1991. Proc. Natl. Acad. Sci. USA 88, 2520. Bazan, J. 1990. Proc. Natl. Acad. Sci. USA 87, 6934. Ullrich, A., and Schlessinger, J. 1990. Cell 61, 203. Sibley, D.R., et al. 1988. Endocr. Rev. 9, 38.
Chapter Twenty nine
Collagens and Other Extracellular Matrix Proteins
The extracellular matrix (ECM) is composed of fibrillar and nonfibrillar components. The two main groups of macromolecules that form ECM are fibrous proteins and glycosaminoglycans. The fibrous proteins can be grouped as (i) structural proteins, such as collagens and elastin and (ii) adhesive proteins, which include fibronectin and laminin.
Collagen, the most abundant protein in the body is predominantly synthesized by fibroblasts; however, epithelial cells may also synthesize small amounts of collagen. The collagen super family includes over 20 different types of collagen with at least 38 distinct polypeptide chains. Of these types I, II, and III are the most abundant and are involved in the formation of fibrils. Type IV collagen forms a two-dimensional reticulum and is a major component of the basal lamina. All collagens contain non-collagenous domains, which perform functions that are distinct from those of the collagen domains. For example, endostatin, a fragment released from type XVIII collagen has been shown to block angiogenesis and reduce tumor growth. In addition to providing the mechanical resistance and flexibility in multicellular organisms, collagens also act as signaling molecules defining cellular shape and behavior. The communication between collagens and cells is achieved by cell surface receptors, such as integrins, discoidin domain receptors, and glycoprotein VI. Upon collagen binding they trigger a variety of signaling processes that are independent of each other.
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Other Extracellular Matrix Proteins
Properties and Distribution of Major Collagen Types

Collagen Types I II III Chains [1(I)]2 [(I)] [1(II)]3 [1(III)]3 Anchor Fibronectin Fibronectin Fibronectin Cell Surface Receptor Integrin Integrin Integrin Associated Proteoglycans Chondroitin and dermatan sulfates Chondroitin sulfate Heparan sulfate and heparin Localization in Tissues (and cells) Skin, tendon, bone (fibroblasts) Cartilage, vitreous humor (chondrocytes) Skin, muscle, frequently found with type I collagen (quiescent hepatocytes, epithelial; fibroblasts) All basal lamina (all epithelial cells, endothelial cells, regenerating hepatocytes)
IV
[1(IV)2 [2(IV)]
Laminin
Laminin receptors
Heparan sulfate and heparin
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Collagens contain a Gly-X-Y repeating structure. The Glycine residue is necessary in every third position to ensure close packaging. Although any amino acid can be present at position X or Y, proline is more frequently found in the X position and hydroxyproline in the Y position. Due to their ring structure, both proline and hydroxyproline stabilize the helical conformation of polypeptide chains. This triplet of amino acids allows collagen chains to twist into a helical structure (-helix) forming a super helix. Each collagen molecule contains 3 chains twisted around each other to form a triple helix. These 3 chains may either be identical or different depending on the type of collagen. After collagens are secreted in the form of pro-peptides, they are converted to collagen molecules by specific extracellular proteolytic enzymes. The collagens then assemble in the extracellular space to form collagen fibers. Collagen fibers strengthen and help in the organization of the matrix, whereas elastin fibers provide structural flexibility.
Collagens are synthesized as longer precursor proteins called procollagens. Collagen fibers begin to assemble in the endoplasmic reticulum and Golgi complexes. Here the signal sequence is removed and specific proline and lysine residues are hydroxylated. Prolyl hydroxylase converts proline residues to hydroxyproline in the endoplasmic reticulum. Lysine residues in collagen are also frequently converted to hydroxylysines by lysyl hydroxylase. The OH groups of these modified amino acids help in stabilizing the collagen triple helix by forming hydrogen bonds between polypeptide chains. Following completion of this processing, procollagens are secreted into the extracellular space where extracellular enzymes remove the pro-domain. Subsequently, collagen molecules polymerize to form collagen fibrils. Oxidation of specific lysine residues by lysyl oxidase forms reactive aldehydes that are involved in the formation of specific cross-links between two chains. This process helps in stabilizing collagens in the fibril. In type IV collagen, found in basal lamina, the Gly-X-Y repeats are frequently interrupted by non-helical sequences that allow greater flexibility than seen in fibril-forming collagens.
Mitochondrion
Lysyl oxidase Golgi

Collagen Polymerization
Secretory Vesicle
Prodomain Cleaved
Collagen Fibrils
a-chains
Endoplasmic Reticulum
ne b ra em M ma Plas
Processing and packaging of collagen
Figure: Synthesis and Secretion of Collagen (a) 3 a-chain forms procollagen helix. (b) Procollagen moves from the endoplasmic reticulum to the Golgi apparatus. (c) Procollagen is processed and packed into secretory vesicles. (d) Secretory vesicles fuse with plasma membrane. (e) Procollagen is secreted into extracellular space. (f) Prodomain region is cleaved off by extracellular proteases. (g) Collagen undergoes polymerization to form fibrils. (h) Lysyl oxidase produces aldehydes to enhance cross-linking and give rigidity.
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The critical role of collagens is evident from a wide spectrum of diseases resulting from hundreds of known mutations in 22 genes for different collagen types. Defects in the synthesis and degradation of collagen and elastin contribute to a number of diseases. Some of the well-known collagen related diseases include osteogenesis imperfecta (brittle bone disease), many chondrodysplasias (abnormally short and deformed limbs), some cases of osteoporosis, and several subtypes of the Ehlers-Danlos syndromes.
The adhesive proteins assist cells to attach to the extracellular matrix. For example, fibronectin, a large dimeric glycoprotein, promotes the attachment of fibroblasts and other cells to the matrix in connective tissue. It interacts with a wide variety of proteins, including collagen, heparin, fibrin, gelatin, DNA and cell-surface receptors of integrins. Fibronectins can also regulate the shape of cells and organize cytoskeleton. Fibronectins bind extracellular matrix proteins through a tripeptide arginineglycine-aspartic acid (RGD) sequence.
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Other Extracellular Matrix Proteins
Selected Collagen Disorders and Their Clinical Implications

Type of Collagen Disorder Ehler-Danlos Type I and II Clinical Symptoms Mild to severe hyperextensibility, premature birth often with hernias, hyperextensible skin, hyperextensible joints leading to osteoarthritis. Asymptomatic except for pain and joint laxity Repeated arterial rupture leading to massive hematomas or sudden death; thin transparent skin, very fragile tissues. Several scoliosis, poor wound healing, joint dislocations, hyperextensible skin and joints, diminished muscle mass, and retinal detachment. Pronounced joint hypermobility, Moderate bruising, round faces, short stature, multiple dislocations of large joints. Hypermobile joints, very extensible skin. Molecular or Biochemical Defect Unknown defects of collagen or other matrix proteins leading to abnormal collagen fiber formation. Unknown Mutation within the type III collagen gene that result in production of a defective collagen molecule. Reduced hydroxylysine content in type I collagen of skin and bone due to defective lysyl hydroxylase activity. Abnormal accumulation of procollagen in tissues due to defective and lower enzymatic cleavage of the precursor to its mature form Poisoning with -aminopropionitrile that forms tight complex with lysyl oxidase and blocks the transformation of lysyl side chains into aldehydes. Extremely fragile collagen. Null -I(I) allele possibly due to a defect in mRNA splicing, premature stop codon, or frame shift mutation. Reduced collagen synthesis for the affected allele. Deletions or substitutions in one of the 1(I)-chain genes leading to destabilized collagen helix. The triple helix structure is disrupted near the end, exposing it to excessive hydroxylation and glycosylation. Collagen is partly unfolded at body temperature and cannot form fibrillar arrays. Reduced hydroxyproline resulting in defective propyl hydroxylase activity. Insufficient hydroxylation of collagen takes place which lead to less thermostabile collagens.
Ehler-Danlos Type III Ehler-Danlos type IV (ecchymotic or arterial)
Ehler-Danlos Type VI (ocular)
Ehler-Danlos Type VII (arthrochalasis multiplea congenita)
Lathyrism
Mild Osteogenesis Imperfecta
Prominent frontal bones and narrow mandible, loss of hearing after age 20, radiological osteoporosis. Multiple intrauterine fractures, death within one week of birth usually from diminished respiratory reserve, skeletal deformities arising from multiple fractures due to brittle bones.
Lethal Osteogenesis Imperfecta
Scurvy
Poor wound healing, deficient growth, capillary weakness.
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By facilitating the migration of immune cells to the site of injury fibronectins play an important role in wound healing. They bind to integrins on platelets via RGD domains and cause localization of platelets to the site of injury. They also bind to fibrin and assist in blood clotting. Other adhesive molecules, such as laminin, promote the attachment of epithelial cells to the basal lamina. At least 12 different types of laminins are present in mammals. Each laminin is a heterotrimer composed of different , , subunits. Each subunit contains at least 12 repeats of EGF-like domains. The number of EGF repeats varies between different species. Laminins also stimulate spreading of many cell types and promote the outgrowth of neurites in culture. Glycosaminoglycans (GAGs) generally form a highly hydrated, gel-like substance, in which fibrous proteins are embedded. GAGs are heteropolysaccharides consisting of long unbranched polysaccharides containing a repeating disaccharide unit. The disaccharide units contain either N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc) and an uronic acid such as glucuronate or iduronate. GAGs are located either on the cell surface or in ECM. They exhibit high degree of viscosity and low compressibility, which makes them suitable for lubricating joints. The majority of GAGs are linked to core proteins and form proteoglycans (mucopolysaccharides). This linkage involves a specific trisaccharide consisting of two galactose and a xylulose residue (GAG-GalGalXyl-OCH2-protein). Major types of GAGs are dematan sulfate, heparin, keratan sulfate, hyaluronan, and chondroitin sulfate. Aggrecan, a large aggregating chondroitin sulphate proteoglycan is found mainly in cartilage. It may account for up to 10% of the dry weight of cartilage. It is considered as a spacefilling proteoglycan and its primary function appears to maintain a high level of hydration in cartilage ECM. Aggrecan is a monomer consisting of a protein backbone of 210 to 250 kDa to which chondroitin sulfate and keratan sulfate are attached. Individual monomers can interact
with hyaluronic acid to form high molecular weight aggregates. The presence of chondroitin sulphate chains on aggrecan helps in generating an osmotic swelling pressure, which may result in up to 75% water content in the articular cartilage. The osmotic swelling is primarily due to the glycosaminoglycan chains attached to the aggrecan core. During the resting phase, osmotic swelling is reported to be at its maximum. However, during loading when body weight compresses the cartilage water is squeezed out. When the load is removed and the compressive force is normalized and maximum swelling is restored.
References: Robins, S.P. 2007. Biochem. Soc. Trans. 35, 849. Donahue, T.R., et al. 2006. Hernia 10, 478. Avery, N.C., and Baily, A.J. 2005. Scand. J. Med. Sci. Sports 15, 231. McAlinden, A., et al. 2005. J. Biol. Chem. 278, 42200. Robert, L., et al. 2001. J. Soc. Biol. 195, 125. Primorac, D., et al. 2001. Croat. Med. J. 42, 393. Myllyharju, J., and Kivirikko, K.I. 2001. Ann. Med. 33, 7. Danen, E.H., and Yamada, K.M. 2001. J. Cell Physiol. 189, 1. Colognato, H., and Yurchenco, P.D. 2000. Dev. Dynamics 218, 213. Takala, T.E., and Virtanen, P. 2000. Scand. J. Med. Sci. Sports 10, 321. Buckwalter, J.A., and Mankin. H. J. 1998. Instr. Course Lect. 47, 477. Leahy, D.J., et al. 1996. Cell 84, 155. Dickinson, C.D., et al. 1994. J. Mol. Biol. 236, 1079. Upholt, W.B., et al. 1993. Experientia 49, 384.
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Chapter Thirty
Integrins: Molecular Adhesives in Cell Survival and Cell Death
The ability of cells to adhere to other cells and to the extracellular matrix (ECM) is important for growth, development, apoptosis, inflammation, migration, tumorigenesis, and immune responses. Amongst all the various molecules involved in the adhesion process, integrins are considered as the most important group.
Signal transduction through integrins occurs in two directions - moving from the extracellular microenvironment into the cell (outside-in signaling) and from the cytoplasm to the extracellular domain of the receptor (inside-out signaling). Integrins are heterodimeric cell surface receptors composed of a variable -subunit of 150-170 kDa and a conserved 95 kDa -subunit. There are 18 known and 8 -subunits that can combine to form at least 24 heterodimers. They contain a large extracellular domain responsible for ligand binding, a single transmembrane domain, and a cytoplasmic domain. The exact combination of various - and -subunits dictates the binding specificity of integrins to different ECM components. Although both subunits are required for adhesion, the binding specificity primarily depends on the extracellular region of the -subunit. The structural similarities between -subunits of various integrins are remarkable. Their extracellular domains contain seven homologous repeats of 30 to 40 amino acids spaced by stretches of 20-30 amino acids. The three or four repeats, which are in the extreme extracellular region, are involved in ligand binding. The -subunits of all integrins share the five amino acid motif, GFFKR, located directly under the transmembrane region. The exact function of this motif has not been
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Integrin signaling cascade
elucidated. The -subunits contain tandem repeats of four cysteine-rich regions that are essential for maintaining the tertiary structure of integrins. The recognition site for most integrins that bind the ECM consists of an RGD (arginine-glycineaspartic acid) sequence. Integrins bind to their ligands with low affinity and this binding occurs only when a certain minimum number of integrins are present at specific points known as focal contacts. In resting cells integrins are diffused over the cell surface and lack sufficient adhesive force. In response to specific stimuli they cluster in focal contacts and their combined affinities create a region on the cell surface, which presents sufficient adhesive capacity to adhere to the ECM. This allows cells to bind to a large number of matrix molecules simultaneously while maintaining their ability to explore their environment without losing all attachments. Any stronger binding to their ligands will cause an irreversible binding to the matrix, depriving them of their motility. Upon binding to ECM molecules, integrins undergo a conformational change that allows the intracellular domain of their -subunit to interact with focal-adhesion proteins such as talin and -actinin. Integrin-mediated cell attachment is essential for survival signaling in many types of normal cells and offers protection against a variety of apoptotic stimuli. Epithelial and endothelial cells that are
largely dependent on integrin-mediated cell attachment for their survival undergo apoptosis upon loss of integrin-mediated cell attachment. Although integrins do not possess any enzymatic activity or a kinase domain, they initiate signaling through association with non-receptor kinases, such as focal adhesion kinase (FAK) and the Src family of kinases. These kinases in turn activate several downstream survival pathways, such as PI 3-kinase, Akt, and MAPK/ERK. Integrins interact with a number of regulatory adaptor molecules, such as paxillin, and with the structural proteins, vinculin and talin that couple integrin to the actin cytoskeleton. Paxillin, a highly conserved 68 kDa multi-domain adaptor molecule, is located at the interface of the plasma membrane and the actin cytoskeleton. Together with integrins it participates in the regulation of cell adhesion and motility. In spite of being a relatively small molecule (559 amino acids), paxillin does contain several protein binding modules, which allow it to bind a variety of structural and signaling molecules. Paxillin contains an amino terminus and a carboxyl terminus, which consists of four LIM domains (LIM 1-4) arranged in tandem. LIM domains are double zinc-finger motifs of about 50 amino acids, which anchor the protein at the plasma membrane. The exact mechanism of paxillin recruitment to focal adhesions is not clearly understood; however, several paxillin LIM-domain binding partners have been identified and include several kinases
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and phosphatases. Paxillin is an important target of tyrosine kinases that are activated as a result of integrin signaling or by growth factor stimulation of cells. FAK in association with Src phosphorylates paxillin at Tyr31 and Tyr118. Although tyrosine phosphorylation of paxillin is not absolutely essential for localization to focal adhesions, it does generate functional SH2-binding sites for the Crk family of SH2-SH3 adaptor proteins. These adaptor proteins bind to p130CAS. In addition to tyrosine phosphorylation, paxillin is also phosphorylated on Ser188/190 at the amino terminal region and at several serines in LIM domains. This phosphorylation is brought about by PKC as well as by p21-activated kinase (PAK). Upon integrin ligation to the ECM, paxillin associates with FAK and induces a conformational change that facilitates FAK tyrosine phosphorylation and its activation. Upon activation, FAK combines with Src, which phosphorylates paxillin and p130CAS. Both paxillin and p130CAS serve as scaffolds for the recruitment of various adaptors and signaling intermediates. FAK does not contain an SH2 or SH3 domain; hence it localizes at focal adhesions by binding to talin and paxillin via its C-terminal region, known as the Focal Adhesion Targeting (FAT) sequence. FAK contains several tyrosine residues that are phosphorylated in response to growth factors and cell spreading. FAK auto-phosphorylates on Tyr39, which allows it to bind to the SH2 domain of Src. Src phosphorylates a number of tyrosine residues on FAK, particularly Tyr567, 576 that are important for regulation of its kinase activity. Src also phosphorylates Tyr925, which serves as a docking site for the SH2 domain of Grb2 and links integrin signaling to ERK activation. FAK also contains two proline rich regions that bind to the SH3 domain of p130CAS, a large adaptor protein that can bind to other adaptor proteins such as Crk and Nck. Phosphorylation of tyrosine residues on p130CAS by the FAK-Src complex allows it to serve as a docking site for the SH2 domains of Crk and Nck. In addition to phosphorylating FAK and p130CAS, Src also phosphorylates paxillin and tensin, which are involved in regulating the adhesion.
It is well known that loss of adhesion from the ECM can trigger apoptosis in normal cells and that integrin ligation can rescue these cells. In some cells integrins are known to activate transcription of the Bcl-2 gene and enhance the activity of MAPK, JNK, PI-3 kinase, and Akt. Cancer cells deprived of contact with the matrix die rather than circulate and colonize distant sites. Hence, integrins have a significant effect on the progression of malignant tumors. Integrin-mediated cell attachment has been shown to modulate cancer cell responses to chemotherapeutic agents. Hence, elucidation of the molecular mechanisms contributing to defective adhesion is important in developing new drugs for treatment of various diseases. More recently integrins have also been linked to the progression of many inflammatory and autoimmune disorders. Hence, integrins have become attractive drug targets even in these areas. Drugs that antagonize integrin 2b3 (e.g. Abciximab), L2 (Efalizumab), and 41 (Natalizumab) are already approved for acute coronary syndromes, psoriasis, and multiple sclerosis, respectively.
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References: Takada, Y., et al. 2007. Genome Biol. 8, 215. Vanderslice, P., and Woodside, D.G. 2006. Expert Opin. Invest. Drugs 15, 1235. Stupak, D. G. 2005. Cell Death Diff. 12, 1021. Hsia, D.A., et al. 2003. J. Cell Biol. 160, 753. Lewis, J. M. et al. 2002. Proc. Natl. Acad. Sci. USA 99, 3627. Rust, W.L., et al. 2002. J. Biomed. Biotechnol. 2, 124. Wade, R., et al. 2002. Oncogene 21, 96. Aoudjit, F., and Vuori, K. 2001. Oncogene 20, 4995. Zamir, E., and Geiger, B. 2001. J. Cell Sci. 114, 3583. Giancotti, F.G. 2000. Nat. Cell Biol. 2, E13. Gilmore A.P., et al. 2000. J. Cell Biol. 149, 431. Parise, L.V., et al. 2000. Semin. Cancer Biol. 10, 407. Damiano, J.S., et al. 1999. Blood 93, 1658. Etzioni, A. 1999. Lancet 353, 341. Liu, S., et al. 1999. Nature 402, 676. Hemler M.E. 1998. Curr. Opin. Cell Biol. 10, 578. Schlaepfer, D.D., et al. 1998. Mol. Cell. Biol. 18, 2571. Vuori, K. 1998. J. Membr. Biol. 165, 191. Frisch, S.M., and Ruoslahti, E. 1997. Curr. Opin. Cell Biol. 9, 701. Hungerford, J.E., et al. 1996. J. Cell Biol. 135, 1383. Wary, K.K., et al. 1996. Cell 87, 733. Hynes, R.O. 1992. Cell 69, 11.
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Chapter Thirty one
Diabetes, Obesity, Dyslipidemia, and Hypertension
Diabetes, a chronic metabolic disorder, affects about 5% of the population in the industrialized nations and accounts for over $200 billion in medical costs. In the year 2005, diabetes, directly and indirectly, accounted for about 3 million deaths worldwide.
Type l diabetes often manifests in childhood and may result from autoimmune destruction of -cells. Type II diabetes, a more widespread metabolic disorder, generally manifests after the age of 40 and involves progressive development of insulin resistance leading to overt hyperglycemia. Insulin is the major hormone that counters the concerted action of a number of hyperglycemia generating hormones. It enhances glucose uptake in muscle and adipose tissue and reduces gluconeogenesis and lipolysis. Insulin resistance, caused by obesity and higher levels of free fatty acids, can result in elevated fasting and postprandial glucose levels and predispose individuals to the risk of type II diabetes. Action of insulin on target cells is mediated via its interaction with insulin receptor (IR), a heterotetrameric glycoprotein consisting of two extracellular -subunits (135 kDa) and two transmembrane -subunits (95 kDa). IR functions as an allosteric enzyme in which the -subunit inhibits the tyrosine kinase activity of the -subunit. Insulin binding to the -subunits results in the stimulation of the tyrosine kinase activity of the -subunits. The kinase domains of the -subunits are juxtaposed to the -subunits, which permit autophosphorylation of Tyr1158, Tyr1162, and Tyr1163, the first step in receptor activation. IR transphosphorylates tyrosine residues on several immediate substrates, including insulin receptor substrate (IRS) proteins 1-4, Shc, Grb-2 associated binder-1 (Gab1), and
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APS adapter protein, all of which provide specific docking sites for other signaling proteins containing SH2 domains. These events lead to the activation of downstream signaling molecules, including PI 3-kinase (PI 3-K). The four IRS proteins exhibit a high degree of homology. IRS1-knockout mice exhibit growth retardation and impaired glucose tolerance due to resistance to insulin and insulin-like growth factor-1 (IGF1). IRS-2-knockout mice show severe insulin resistance in the liver and peripheral tissues and develop overt type II diabetes. In addition to tyrosine phosphorylation, both IR and IRS proteins undergo serine phosphorylation by PKC, GSK-3, Akt, and mTOR, which attenuate insulin signaling by blocking insulin-stimulated tyrosine phosphorylation. This serves as a negative feedback loop for insulin signal transduction and allows crosstalk with other pathways that may mediate insulin resistance. IRS-3 and IRS-4 share common features of the IRS family. However, IRS-3 is smaller and has fewer phosphorylation sites. Both IRS-3 and IRS-4 can be phosphorylated by insulin and IGF-1 receptor tyrosine kinases. A negative regulatory role has been suggested for both IRS-3 and IRS-4. PI 3-K plays a critical role in the metabolic actions of insulin. Inhibitors of class 1a PI 3-K, such as LY 294002 block most metabolic actions of insulin, including stimulation of glucose transport and glycogen and lipid synthesis. Activated PI 3-K phosphorylates PIP2 to generate PIP3, which then enlists PI 3-K-dependent kinase (PDK1) and Akt from the cytoplasm to the plasma membrane. This leads to conformational changes in Akt, allowing it to be phosphorylated on Thr308 and Ser473 (for Akt1) or Thr309 and Ser474 (for Akt2) by PDK1 and mTORC2, respectively to achieve full activation. Akt phosphorylates GSK-3 and inactivates it, which then allows the activation of glycogen synthase to proceed. GSK-3 has been implicated in the multi-factorial etiology of skeletal muscle insulin resistance in obese animal models and in obese type II diabetics. Overexpression and hyperactivity of GSK-3 in skeletal muscle of obese type 2 diabetics has been linked with an impaired ability of insulin to activate glucose disposal and glycogen synthase. Selective inhibition of GSK-3 in insulin-resistant skeletal muscle tissue is shown to improve insulin-stimulated glucose transport.
In the overall scheme of glucose homeostasis in the body, skeletal muscle and adipose tissue are two major glucose utilizing tissues in the post-absorptive state and glucose uptake by these tissues plays an important role in determining the glycemic state. Any alteration in insulin-stimulated glucose uptake can lead to derangements in whole body glucose disposal. Mammalian cells possess two types of glucose carriers (a) the Na+-glucose co-transporter and the (b) facilitative glucose transporter. The Na+glucose co-transporter transports glucose against its concentration gradient by coupling its uptake with the uptake of Na+. It is largely expressed in epithelial cells of the small intestine and in kidney. The facilitative glucose carriers of the glucose transporters (GLUT) family accelerate the transport of glucose down its concentration gradient by facilitative diffusion. Members of the GLUT family are expressed in a tissue- and cellspecific manner and exhibit distinct kinetic and regulatory properties that reflect their specific functional roles. Each GLUT operates most efficiently at different levels of blood glucose. GLUT1, a widely expressed isoform, provides cells with their basal glucose requirements. It plays a unique role in transporting glucose across epithelial and endothelial barrier tissues. GLUT2 is a high Km isoform expressed in hepatocytes, pancreatic -cells, and the basolateral membranes of intestinal and renal epithelial cells. It acts as a high-capacity transport system to allow the flux of glucose into or out of these cells in a non-ratelimiting manner. In the liver GLUT2 mediates the bi-directional transport of glucose by hepatocytes. GLUT3 is a low Km isoform that is responsible for glucose uptake into neurons. GLUT4 is mainly expressed in insulin sensitive tissues, such as muscle and adipose tissue, and its expression changes in response to insulin. It is responsible for increased glucose disposal in these tissues in the postprandial state and is vital to whole-body glucose homeostasis. The GLUT5, expressed in small intestine, is involved in the trans-cellular transport of glucose by absorptive epithelial cells. GLUT7 is present in the endoplasmic reticulum (ER) membrane that allows the flux of free glucose out of the lumen of ER following the action of glucose-6-phosphatase on glucose 6-phosphate. GLUT6 and GLUT8 appear to recycle in a dynamin-dependent manner between internal membranes and the plasma membrane in rat adipose cells.
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Insulin receptor activation and its downstream effects.
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In the unstimulated state, GLUT4 vesicles are sequestered inside cells, and their translocation is arrested. Activation of Akt is closely linked to the translocation of GLUT4 vesicles to the cell membrane where they participate in the transport of glucose. Although activation of PI 3-K is required for GLUT4 trafficking, it is considered to be inadequate to produce GLUT4 translocation. It has been suggested that the insulin-stimulated tyrosine phosphorylation of Cbl is an essential co-event in this process. Cbl is recruited to the insulin receptor by the adapter protein CAP (Cblassociated protein). The phosphorylated Cbl is translocated to lipid rafts. Blocking this step is shown to completely inhibit the stimulation of GLUT4 translocation by insulin. Phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G activates TC10, a small GTP-binding protein. The activation of TC10 is shown to be essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI 3-K to fully stimulate GLUT4 translocation in response to insulin.
Elevated cell surface levels of GLUT4 facilitate enhanced glucose uptake from the circulation and storage in adipose and muscle tissue. Hence, defects in insulin stimulated GLUT4 translocation are important in insulin resistance. Consequently GLUT4 has become a major pharmacological target for the treatment of diabetes mellitus and insulin resistance. In addition to an impairment in insulin stimulated recruitment of GLUT4 transporter from its intracellular storage compartment to the cell surface, insulin resistance observed in obesity and type II diabetes is characterized by many other defects, including decreases in the number of insulin receptors and their tyrosine kinase activity, the concentration and phosphorylation of IRS-1 and -2, and PI 3-K activity. These abnormalities result in a variety of metabolic defects, including hyperglycemia, hyperlipidemia, and hyperinsulinemia, which contribute to the development of metabolic syndrome X and increase the risk of cardiovascular disorders and
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premature death. Adipose tissue plays a vital role in the development of insulin resistance and associated abnormalities. A higher circulating level of free fatty acids (FFA), as seen in obesity and type II diabetes, is considered to be an important contributor to insulin resistance. Elevated FFA levels cause a reduction in insulin-stimulated IRS-1 phosphorylation, IRS-1-associated PI 3-K activity, and increased hepatic glucose production via gluconeogenesis. Higher levels of FFA shift substrate preference from glucose to FFA in the
muscle tissue oxidation, further contributing to hyperglycemia. Long-term exposure of pancreatic -cells to FFAs diminishes their insulin secretory response to glucose. Adipose tissue also secretes a variety of hormones (adipokines) that regulate various cellular processes, including energy expenditure. A higher expression of TNF- in adipose tissue of obese subjects has been linked to insulin resistance. TNF- is known to impair insulin signaling through IRS-1 serine phosphorylation and through reduced expression
Nitric Oxide
Obesity
Elevated Levels of Free Fatty Acids
Angiotensin-2
Vasoconstriction
Reduced b-Cell Function
Type II Diabetes
Insulin Resistance
Hyperinsulinemia
Glucose Intolerance Elevated Triglycerides Low HDL-C High LDL-C
Uncoupled Oxidative Phosphorylation
Elevated Levels of Free Fatty Acids
Hyperglycemia Protein Glycation Advanced End-product Glycation
Oxidative Stress Thrombosis Lipid Peroxidation Membrane Damage DNA & Protein Damage
Tissue Damage
Microvessel Damage
Obesity and type II diabetes - causative factors in the development of metabolic syndrome X.
of IRS-1 and GLUT4. Deficiency of leptin, another hormone of adipose origin, is also linked with insulin resistance in db/db and ob/ob mice. Leptin replacement improves glycemic control and reduces circulating lipid levels. Resistin, a hormone of adipose origin, is found at much higher levels in animal models of diabetes and obesity, and treatments with insulin sensitizing agents, such as thiazolidinediones (TZD) is shown to reduce circulating levels of resistin. TZDs are also shown to increase the expression of adiponectin, an insulin-sensitizing factor in adipose tissue, which reduces serum FFAs by promoting their flux into adipose tissue.
TZDs belong to a new class of insulin sensitizers that are used for the treatment of type II diabetes. They act as direct, high-affinity ligands of peroxisome proliferator-activated receptor (PPAR) - an adipocyte-specific nuclear hormone receptor. Although PPAR is expressed in most organs, the level of PPAR mRNA is about 50-fold higher in adipose tissue. When compared to some natural ligands, such as 15- deoxy-D- 12, 14-prostaglandin J2, TZDs exhibit much higher affinity for PPAR (EC50 = 20-400 nM). In the cell, PPAR forms a heterodimer with the retinoid X receptor (RXR). Without TZD binding the heterodimer is associated with a co-repressor
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Hypertension Inflammation
complex that includes a histone deacetylase, which keeps DNA in a transcriptionally repressed state. Upon TZD binding to PPAR, the co-repressor complex dissociates and a co-activator complex containing histone acetylase associates. This promotes binding of the PPAR-RXR complex to PPAR response elements (PPRE) in target genes resulting in modification of the transcription of these genes. PPREs are commonly found in genes involved in lipid metabolism and energy balance, including those encoding lipoprotein lipase, adipocyte fatty acid binding protein, fatty acyl-CoA synthase, glucokinase, and the glucose transporter GLUT4. TZDs may also have cardiovascular benefits in type II diabetes subjects who exhibit metabolic syndrome X, characterized by clustering of atherosclerotic cardiovascular disease risk factors, including insulin resistance, obesity, hypertension, and dyslipidemia/hyperlipidemia. The characteristic features of metabolic syndrome X emerge from interactions between molecular pathways of glucose and lipid metabolism and blood pressure control. Insulin is known to promote the activity of lipoprotein lipase, which participates in converting VLDL into LDL. A few clinical studies have shown that TZDs raise HDL levels, reduce triglyceride levels, and improve endothelium-mediated vasodilation. TZD-induced activation of PPAR triggers signaling from adipocytes to skeletal muscle, which ameliorates insulin resistance. This may be linked to a significant reduction of FFA levels by TZDs. It is widely recognized that cardiovascular complications observed in type
II diabetes develop through inflammatory and procoagulant pathways with increased oxidative stress as a major etiologic mechanism. In addition to their insulin-sensitizing effects, TZDs also exhibit antioxidant, anti-inflammatory, and anti-procoagulant properties. These important links have increased our understanding of the relationship between hyperglycemia, insulin resistance, obesity, and the onset of cardiovascular disease.
References: Huang, S., and Czech M.P. 2007. Cell Metab. 5, 237. Kanzaki, M. 2006. Endocr. J. 53, 267. Reynolds, K., and Goldberg, R.B. 2006. Treat. Endocrinol. 5, 25. Jessen, N., and Goodyear, L.J. 2005. J. Appl. Physiol. 99, 330. Lehrke, M., and Lazar, M.A. 2005. Cell 123, 993. Liberman, Z., and Eldar-Finkelman, H. 2005. J. Biol. Chem. 280, 4422. Luo, M., et al. 2005. Endocrinology 146, 4410. Kim, S.H., and Reaven, G.M. 2004. Diab. Vasc. Dis. Res. 1, 68. Boden G. 2003. Exp. Clin. Endocrinol. Diabetes 111, 121. Haber, E.P. et al. 2003. J. Cell Physiol. 194, 1. Greene, M.W., et al. 2003. J. Biol. Chem. 278, 8199. Albrektsen, T., et al. 2002. Diabetes 51, 1042. Arner, P. 2002. Diab. Metab. Res. Rev. 18, S5. Hauner, H. 2002. Diab. Metab. Res. Rev. 18, S10. Jiang, G., and Zhang, B.B. 2002. Front. Biosci. 7, 903. Stumvoll, M., and Haring, H.U. 2002. Ann. Med. 34, 217. Bevan, P. 2001. J. Cell Sci. 114, 1429. Chiang, S-H., et al. 2001. Nature 410, 944 Ozes, O.N., et al. 2001. Proc. Natl. Acad. Sci. USA 98, 4640 Saltiel, A.R., and Kahn, C.R. 2001. Nature 414, 799. Tsuruzoe, K., et al. 2001. Mol. Cell Biol. 21, 26 Contreres, J.O., et al. 1998. J. Biol. Chem. 273, 22007. Martin, L.B., et al. 1998. J. Biol. Chem. 273, 1444. White, M.F., and Yenush, L. 1998. Curr. Top. Microbiol. Immunol. 228, 179. Withers, D.J., et al. 1998. Nature 391, 900. Hubbard, S.R. 1997. EMBO J. 16, 5572. Mohan, C., et al. 1989. Curr. Top. Cell. Regul. 30, 105. Rosen, O.M., et al. 1983. Proc. Natl. Acad. Sci. USA 80, 3237.
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Chapter Thirty two
Alzheimers Disease: The Role of -Amyloid Peptides and Oxidative Stress
Alzheimers disease (AD), the principal cause of senile dementia, is characterized by regional neuronal degeneration, synaptic loss, and the presence of neurofibrillary tangles (NFTs) and senile plaques. NFTs are aggregates of hyperphosphorylated
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microtubular Tau protein, whereas the senile plaques are complex extracellular lesions composed of a -amyloid (A)containing core that is surrounded by activated microglia, fibrillary astrocytes, and dystrophic neurites.
A decade of research has established that reactive oxygen species (ROS) contribute extensively to the neuronal damage in AD. Oxidative damage is probably one of the early markers of neuronal dysfunction in AD. With advancing age there is increased production of ROS and diminished capacity to protect against ROS, leading to an increased oxidizing cellular environment. A strong correlation exists between the extent of free radical generation by A and neurotoxicity. In addition to its direct neurotoxic effects, A may also fragment into free radical peptides (containing 25-35 amino acids) that act as potent initiators of lipid peroxidation. Deposition of A is an early event in the pathogenesis of AD that precedes the formation of Tau-positive paired helical filaments (PHFs) in NFTs. AD is also characterized by a progressive deposition of the A peptide in senile plaques. In normal healthy individuals, A peptides are present only in small quantities as soluble monomers that circulate in the cerebrospinal fluid and blood. In AD patients, however, their levels increase significantly and they begin to accumulate as insoluble, fibrillar plaques. The A in senile plaques vary in length from 40 to 43 amino acids, however,
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A142 occurs more frequently and forms fibrillar aggregates far more readily than the A140 peptide. A peptides originate from the proteolytic cleavage of the amyloid precursor protein (APP). The -amyloid gene, located on chromosome 21, encodes the transmembrane APP. APP occurs in three common isoforms, APP695, APP751, and APP770. The APP695 is expressed exclusively in neurons, whereas APP751 and APP770 are present in both neural and non-neural cells. The primary structure of APP contains a small signal sequence, a large extramembranous N-terminal region, a single transmembrane domain, and cytoplasmic C-terminal tail. Processing of APP in vivo occurs by two major pathways. Cleavage of APP at the N-terminus of the A region by -secretase and at the C-terminus by -secretases represents the amyloidogenic pathway for processing of APP. The -secretase cleaves APP between residues Met671 and Asp672 and yields sAPP and C99. The -secretase has also been identified as an aspartyl protease (BACE or Asp-2) of unusual nature. It has a C-terminal transmembrane domain and two active site motifs located in the luminal domain. Newly synthesized BACE contains a propeptide domain,
which is cleaved at residue Glu46 to produce the mature enzyme. The active site of BACE and the -secretase cleavage site of APP are in precise topological orientation for endoproteinases. Succeeding the -secretase cleavage, a second cleavage occurs at the C-terminus of A peptide that releases A from C99. This cleavage occurs in the vicinity of residue 712 of the C-terminus. The -secretase can cleave the C-terminal region at either Val711 or Ile713 to produce the shorter A peptide (A1-40) or the longer A peptide (A1-42). The predominant form of A found in the cerebrospinal fluid is the shorter A1-40 peptide. Despite its lower rate of synthesis, A142 is the peptide that is initially deposited within the extracellular plaques of AD patients. In addition, A142 is shown to aggregate at a much lower concentration than the A140 form. APP can also be processed by -secretase, which cleaves within the A domain between Lys687 and Leu688 and produces a large soluble -APP domain and the C-terminal fragment containing P3 (C83). The latter can then be cleaved by -secretase at residue 711 or 713 to release the P3 fragment. This pathway does not yield A peptide. Hence, shunting APP towards the -secretase pathway
Oxidative stress - a contributory factor in the pathogenesis of Alzheimers disease
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may have a beneficial effect in lowering A peptide levels. It is reported that -secretase shares many of its properties with the secretase that cleaves angiotensin-converting enzyme and is believed to be a zinc metalloproteinase of the ADAMs family. Muscarinic agonists (M1 and M3) and some PKC activators are reported to enhance -secretase activity and are under consideration for their therapeutic value as AD treatment tools. Neuronal toxicity to A occurs via several different mechanisms, of which free radical induced damage appears to the most prominent one. A can activate inflammatory pathways by enhancing the microglial secretion of inflammatory cytokines, such as IL-1 and IL-6. Additionally, it can trigger the production of ROS, nitrogen intermediates, and TNF- from microglia. A also increases the accumulation of H2O2 in a Cu2+/Zn2+-dependent manner, which can lead to free radical-induced lipid peroxidation and cell death. In vitro, A142 is shown to induce apoptosis in cultured cortical neurons, possibly through alterations of cellular calcium homeostasis. Interaction between A and ApoE3 or E4 is considered to be an important determinant of amyloidosis. ApoE3 is shown to inhibit A aggregation in vitro by decreasing A multimers, whereas ApoE4 is reported to accelerate the rate of amyloid fibril formation (A1-42 > A1-40). Another set of proteins, known as presenilins (PS1 and PS2), is also reported to play an important role in APP processing. They are tightly linked to -secretase mediated cleavage. Mutations in presenilin, PS1 and PS2, genes are reported to enhance amyloid deposition. PS1 has been suggested to have either an inherent -secretase activity or act as a co-factor for -secretase. Studies of Li et al. (2000) have indicated that the active site of -secretase is shared between the N- and C-terminal fragments of presenilin. Cells obtained from PS1/PS2 double knockout mice do not show any -secretase activity. Presenilins are also involved in the regulation of Notch signaling that is important in framing cell destiny during embryogenesis, hematopoiesis, and neural stem cell differentiation. PS1 also plays an important role in the formation of the axial skeleton and in neurogenesis and survival of progenitor cells and neurons in specific brain regions.
In AD patients all mutations in APP are shown to increase A142 production. Most cases of familial AD are reported to result from mutations in one of the three genes, APP, PS1 and PS2. Any mutation in these genes results in elevated levels of A peptide. The mutation in APP gene, located on chromosome 21, accounts for about 2% of all cases of familial AD (FAD) and approximately 5 - 20% of early-onset FAD. A substitution of Glu to Gln at codon 693 of APP is termed Dutch mutation, which is responsible for hereditary cerebral hemorrhage with amyloidosis (Dutch type). Here amyloid deposits containing the A peptide are found in cerebral vessel walls with diffuse plaques in the brain parenchyma. Another mutation known as Flemish mutation occurs at codon 692 and where Ala is replaced by Gly. It causes an intermediate phenotype between congophilic angiopathy and AD. A well-studied mutation, Swedish mutation, results from the replacement of Lys and Met by Asn and Leu at codons 670 and 671, respectively. The Swedish mutation does not lie within A peptide region but lies in the proximity of the secretase cleavage sites and produces mainly the soluble A140 peptide. Fibroblast cell lines transfected with the Swedish mutation are shown to produce elevated levels of the soluble form of A peptide. Over 40 different mutations have been reported in PS1, which account for about 30 to 50% of all presenile FAD cases. The PS2 gene mutations are rather rare and account for less than 2% of all early-onset FAD. Mutations in both PS1 and PS2 are associated with an increased production of the A1-42 peptide, the more amyloidogenic form of A. It has been suggested that mutant PS1 proteins alter the proteolytic processing of APP at the C-terminus of A and favor the deposition of A142 peptide. The localization of Tau protein in the AD brain is markedly abnormal and may contribute to neuronal dysfunction. In AD, normal soluble cytoskeletal elements, such as Tau and neurofilaments are transformed into insoluble PHFs. This is linked to the post-translational change in Tau, primarily the hyperphosphorylation of Tau by a number of protein kinases. Phosphorylation is intimately tied to oxidative stress via the MAP kinase pathway and through activation of NF-B. Pyramidal neurons of the hippocampus undergoing degeneration are reported to show higher levels
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of free carbonyls, lipid peroxide adduction, and nitrotyrosine. Tau is a neuronal protein located mostly in the axon and, to a lesser extent in cell bodies, but is almost absent from dendrites. In vitro, Tau is a substrate for a multitude of protein kinases including CaM kinase II, casein kinase II, PKA, ERK2, and GSK-3. Cyclin-dependent kinase 5 (Cdk5) in conjunction with its neuron-specific activator p35 (cdk5/p35) is another protein kinase implicated in Tau hyperphosphorylation. Proteolytic cleavage of the regulatory unit p35 by calpain produces p25 that accumulates in the AD brain. This cleavage is reported to be induced by A in cortical neurons. Another protein kinase that has been implicated in the phosphorylation of Tau in Alzheimers disease is MARK (microtubule regulating kinase), which preferentially phosphorylates KXGS motifs in the microtubule affinity binding domains. MARK predominantly phosphorylates Tau on Ser262, although Ser293, Ser324, and Ser356 are also phosphorylated. In fact it has been suggested that phosphorylation of Ser262 in AD may be a primary event contributing to Tau dysfunction and, eventually, PHF and NFT formation. PHF-Tau is reported to be at least partially phosphorylated at 19 sites, of which 9 sites have a Ser/Thr-Pro motif. A significant consequence of Tau hyperphosphorylation in AD is a reduction in its ability to bind microtubules and promote microtubule assembly. Hyperphosphorylated Tau may contribute to a destabilized microtubule network, impaired axonal transport, and ultimately in NFT formation and neuronal death. Phosphorylation of Tau at only a few sites within the microtubule binding regions (Ser262, Ser356, and to a lesser extent Ser293 and Ser324) is sufficient to diminish its ability to bind microtubules. Another interesting fact to note is that hyperphosphorylated Tau is far more resistant to degradation by calpain, a calcium-activated protease. This may be the result of Tau self-association that occurs throughout microtubule binding domains, thereby reducing the accessibility of these sites to calpain. Selfassociation of Tau is potentiated in an oxidizing
environment. Another significant outcome of increased Tau aggregation in an oxidizing environment is glycosylation, the nonenzymatic addition of a reducing sugar to a protein. This often occurs on a lysine residue and may result in the formation of Schiff bases. PHF Tau that is both glycated and hyperphosphorylated shows a greater reduction in microtubule-binding capacity compared to soluble Tau from AD brain that is hyperphosphorylated, but not glycated. Oxidative cross-linking also makes proteins more resistant to proteolytic removal by inhibiting the activity of proteasomes. Therefore, oxidative cross-linking may be a significant contributor to the accumulation of ubiquitin conjugates in NFTs. Higher levels of ubiquitin have been reported in several neurodegenerative diseases. An important question that arises is whether reducing oxidative stress has any therapeutic value in minimizing the pathogenesis of AD. Agents that inhibit free radical formation have been shown to reduce the incidence and progression of AD. Addition of antioxidants, such as propyl gallate, vitamin E, and spin traps, such as N-tert-butyl-phenylnitrone, reduces neurotoxicity in cultured cells exposed to A. Vitamin E has been reported to promote hippocampal neuronal survival in vitro and restore hypofunctioning cholinergic neurons in rats. Another approach to minimize neuronal damage appears to be reducing or preventing the release of A from APP. The characterization of the secretases during the past few years has provided significant advancement in therapeutic strategies that may lead to limiting the build up of A peptides in the brain and eliminating or delaying the pathological effects of AD. Inhibiting the activity of - or -secretase is therapeutically attractive because clinical intervention at this step affects the early events that lead to plaque formation and neuronal death. Small molecules that inhibit - and/or - secretase would therefore be expected to decrease production of A and retard the progression of AD. Major focus in AD research is to identify more genetic and environmental factors responsible for A build-up in nerve cells.
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SIGNAL TRANSDUCTION

A short overview of its role in health and disease

intracellular signals by using a complex network of communication pathways,

A Word to Our Colleagues in Life Science Research

Calcium Signaling ....................................................................................................115

Signal Transduction: A Brief Introduction

| Signal Transduction: A Brief Introduction

A typical signaling cascade

G-Proteins (GTP Binding Proteins):

Cell Adhesion Molecules:

| Signal Transduction: A Brief Introduction

Cell Cycle: A Brief Overview

2 | Cell Cycle: A Brief Overview

| Cell Cycle: A Brief Overview

Phosphoinositide 3-Kinase (PI 3-K): A Multifunctional Signaling Molecule

| Phosphoinsositide 3- Kinase (PI 3-K): A Multifunctional Signaling Molecule

PI 3-Kinase Signaling - An Overview

| Phosphoinsositide 3- Kinase (PI 3-K): A Multifunctional Signaling Molecule

Akt Signaling in Cell Death and Cell Survival

IKK PTE PTEN PTEN TEN T Activated Akt C Caspase-9

IkB p50 p65 p p50 p65 N NF-kB

Apoptosis Caspase-9 Survival

PDK1 Other h kinases

Bad Apoptosis Bad

Proteolytic d degradation Glycogen synthase activation

C Cell cycle arrest

p21 p21 C Cytoplasmic retention

Cell cycle arrest

Multiple roles of activated Akt in cell survival and death

4 | Akt Signaling in Cell Death and Cell Survival

Receptor and Non-Receptor Protein Tyrosine Kinases

5 | Receptor and Non-Receptor Protein Tyrosine Kinases

MAP Kinase Signaling: Synergistic Response to Upstream Signals

Map Kinase signaling cascade

6 | MAP Kinase Signaling: Synergistic Response to Upstream Signals

Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential

Tissue and Cell Specific Distribution of Protein Kinase C Isozymes

+ = present; - = absent; blank = unknown

7 | Protein Kinase C Isozymes: Molecular Switches with Tumorigenic Potential

Protein Kinase C signaling

Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease

Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease

Shh Wnt Fzl LRP G Ptc Smo Ptc

| Glycogen Synthase Kinase-3: Its Signaling Role in Development and Disease

GSK-3,Wnt and Shh signaling

Organizational and Functional Diversity of Protein Phosphatases

Major Protein Phosphatases Involved in Signal Transduction

9 | Organizational and Functional Diversity of Protein Phosphatases

9 | Organizational and Functional Diversity of Protein Phosphatases

Acetylation and Methylation: Epigenetic Modulators

and has anti-inflammatory activity in peripheral blood mononuclear cells.

10 | Acetylation and Methylation: Epigenetic Modulators

10 | Acetylation and Methylation: Epigenetic Modulators

Nuclear Import - Export: A Nuclear Revolving Door

| Nuclear Import - Export: A Nuclear Revolving Door

An Overview of nuclear protein import and export

11 | Nuclear Import - Export: A Nuclear Revolving Door

Poly (ADP-ribosylation): PARP in DNA Repair and Apoptosis

PARP in DNA Repair and Apoptosis

Oxidative stress (O2, NO, OH)

Oxidative stress (O2, NO, OH)

Oxidative stress, DNA damage, and PARP activation

12 | Poly(ADP-ribosylation): PARP in DNA Repair and Apoptosis