Clinical and Experimental Pharmacology and Physiology (2012) 39, 674679

doi: 10.1111/j.1440-1681.2011.05599.x

3rd AustraliaChina Biomedical Research Conference (ACBRC2011)

GAPDH: A common enzyme with uncommon functions

Craig Nicholls,* He Li* and Jun-Ping Liu*

*Molecular Signalling Laboratory, Murdoch Childrens Research Institute, Department of Immunology, Monash University, Department of Genetics, University of Melbourne, Melbourne, Victoria, Australia and Institute of Aging Research, Hangzhou Normal University School of Medicine, Hangzhou, China

SUMMARY
1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as an important enzyme for energy metabolism and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm. 2. Recent studies have shown that GAPDH has multiple functions independent of its role in energy metabolism. Although increased GAPDH gene expression and enzymatic function is associated with cell proliferation and tumourigenesis, conditions such as oxidative stress impair GAPDH catalytic activity and lead to cellular aging and apoptosis. 3. The mechanism(s) underlying the effects of GAPDH on cellular proliferation remains unclear, yet much evidence has been accrued that demonstrates a variety of interacting partners for GAPDH, including proteins, various RNA species and telomeric DNA. 4. The present mini review summarizes recent ndings relating to the extraglycolytic functions of GAPDH and highlights the signicant role this enzyme plays in regulating both cell survival and apoptotic death. Key words: GAPDH, nuclear signalling, protein interaction, RNA binding, telomere DNA binding.

until recently, been assigned the rather unglamorous title of a housekeeping gene, with but a few scant reports of possible extraglycolytic functions prior to the mid-1990s. However, recent times have seen the ongoing discovery of new roles for GAPDH in a diverse range of cellular processes, clearly demonstrating that there is more complexity to GAPDH than initially meets the eye. Indeed, far from being a run-of-the-mill protein useful only as a mere reference gene in the study of other, more interesting molecules and processes, this enzyme is emerging as a key component in the function and regulation of many fundamental cellular processes.25 Figure 1 serves as an overview, summarising the variety of roles ascribed to GAPDH. The present mini review summarizes recent ndings pertaining to the extraglycolytic roles of GAPDH, particularly its roles in the nucleus and in the regulation cell proliferation, and serves as a baseline for further investigations of the structure and function of GAPDH at the molecular level.

BINDING OF NUCLEIC ACIDS BY GAPDH

Since the rst report identifying GAPDH as a single-stranded DNA binding protein,6 numerous studies have described a range of nucleic acid binding partners (see Table 1). Various RNA species have been shown to interact with GAPDH both in vitro and in intact cells, with binding typically occurring at AU-rich elements (ARE) in the substrate RNA.7,8 Interestingly, the binding of GAPDH to ARE can have varied, and even opposite, consequences for mRNA stability and gene expression, as discussed below. Colony-stimulating factor 1 (CSF1) is a macrophage cytokine positively implicated in tumour progression.9 The binding of CSF1 mRNA by GAPDH has recently been demonstrated to stabilize the transcript and enhance CSF1 protein levels in ovarian cancer, whereas GAPDH depletion consistently leads to a decrease in CSF1 mRNA and protein levels.10 In contrast with its role in stabilizing CSF1 mRNA, GAPDH binds to and facilitates the degradation of mRNA coding for the potent endothelial vasoconstrictor endothelin (ET)-1.11 The GAPDH-mediated degradation of ET-1 mRNA has been suggested to be caused by unwinding of the ET-1 mRNA and subsequent exposure to ribonucleases.11 Intriguingly, although numerous studies have established that interacting with nucleic acids is a function of the N-terminal NAD+ binding region of GAPDH, only full-length GAPDH can bind to ET-1 RNA probes,11 indicating a requirement for the catalytic domain as well. Consistently, both NAD+ and G3P inhibit GAPDH

INTRODUCTION
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway, catalysing the conversion of glyceraldehyde-3-phosphate (G3P) to 1,3-biphosphoglycerate in the presence of NAD+ and inorganic phosphate.1,2 Commonly used as a loading control in gene expression and protein studies, GAPDH has,

Correspondence: Jun-Ping Liu, Institute of Aging Research, Hangzhou Normal University School of Medicine, Hangzhou, Zhejiang Province 310012, China. Email: junping.liu@mcri.edu.au Presented at the 3rd AustraliaChina Biomedical Research Conference (ACBRC2011) Melbourne, Australia, 2830 April 2011. The papers in these proceedings have been peer reviewed. Received 25 July 2011; revision 29 August 2011; accepted 31 August 2011. 2011 The Authors Clinical and Experimental Pharmacology and Physiology 2011 Blackwell Publishing Asia Pty Ltd

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telomeric DNA in in vitro binding assays, as well as the colocalization of GAPDH with telomeres in intact cells. Distinct from its cytoplasmic form, GAPDH in the nucleus has an increased isoelectric point (pI) at pH 8.38.7 and binds to both single- and doublestranded telomeric DNA with high specicity.12 Overexpression of GAPDH has been shown to protect cells from rapid telomere shortening triggered by treatment with chemotherapeutic drugs that induce the sphingolipid ceramide.12 A subsequent study has conrmed that this direct GAPDHtelomere interaction is required for the protective effect of GAPDH against telomere shortening induced by ceramide and that the GAPDH NAD+ binding region is the site of telomeric DNA binding.13 Although it currently remains unclear whether GAPDH plays a role in regulating telomere maintenance in the absence of ceramide, the nding that depletion of GAPDH by gene silencing results in mild telomere shortening over a short time period12 warrants further investigation. This effect is not due to disrupted glycolysis because inhibition of another glycolytic enzyme does not have this effect. Intriguingly, this telomere-protective role appears inconsistent with that of GAPDH in causing cell apoptotic cell death, which is also dependent on GAPDH migration to the nucleus in response to cellular stress (see below).

GAPDH AS A TRANSCRIPTION FACTOR

GAPDH has also been implicated in the regulation of gene transcription. The Oct-1 coactivator complex OCA-S is required for histone2B synthesis in the S phase and contains GAPDH, which interacts directly with Oct-1.14 The presence of GAPDH is critical for the function of OCA-S and the complex is further stimulated by the GAPDH cofactor NAD+. Further evidence for a role of GAPDH in gene transcription is provided by the increase in transcriptional activation of androgen receptor (AR) targets that accompanies binding of AR by GAPDH, as well as reports of GAPDH interactions with RNA polymerase II in yeast.15,16

Fig. 1 The pleiotropic roles of GAPDH in cellular processes. GAPDH participates in a variety of cellular processes in both the cytoplasmic (GADPHcyt) and nuclear (GADPHnuc) compartments. The primary role of GAPDH is enzymatic conversion of glyceraldehyde-3-phosphate (G3P) in the glycolysis cascade, a required step in the metabolism of glucose. This occurs in the cytoplasm, although GAPDH can shuttle into the nucleus (possibly dependent on post-translational modications; PTM), where it functions in several additional nuclear processes. Cytoplasmic GAPDH can also contribute to apoptosis by triggering release of cytochrome c from the mitochondria. S-Nitrosylation of GAPDH facilitates an interaction with Siah1 and subsequent nuclear localization of the complex. The GAPDHSiah1 complex can then participate in several further reactions, including transfer of the nitrosyl moiety to other proteins, ubiquitination of nuclear proteins or the acetylation and stabilization of p300 CREB binding protein (CBP) and subsequent acetylation of further nuclear protein targets, leading to the induction of apoptosis.

INTERACTIONS BETWEEN GAPDH AND OTHER PROTEINS

Given the multitude of processes that have been shown to involve GAPDH, it comes as no surprise that GAPDH has been found to associate with an array of other proteins. In addition to the interactions discussed already, GAPDH interacts with p22 to aid microtubule organization,17 Rab2 to facilitate endoplasmic reticulum (ER) golgi transport,18 transferrin on macrophage cell surfaces,19 lactate dehydrogenase in an in vitro setting that mimics molecular crowding, although the biological signicance of this remains unclear,20 and the DNA repair protein apurinic apyrimidinic endonuclease (APE1), converting oxidized APE1 to the reduced form to re-establish its endonuclease activity.21 In addition, GAPDH has been shown to interact with promyleocytic leukaemia protein (PML) in an RNAdependent fashion, although the physiological role of this interaction is unknown.22 This variety of interactions reects the range of cellular processes in which GAPDH is involved.

binding to ET-1 mRNA, although catalytic activity is not required because the GAPDH mutant C152S retains the ability to bind.11 This suggests that the mechanism governing the GAPDH interaction with ET-1 mRNA is more complex than that for CSF1 mRNA and may account for the differing effects on transcript stability.

TELOMERES AND GAPDH

In addition to interacting with mRNA, Sundararaj et al.12 have demonstrated that GAPDH is a telomere binding protein, describing an interaction between GAPDH and both single- and double-stranded

GAPDH LINKS GLUCOSE AVAILABILITY TO GROWTH SIGNALLING

Recently, GAPDH was implicated in linking glucose availability to the growth and proliferation-promoting mammalian target of rapamycin

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Table 1 Reports of GAPDH binding nucleic acids Substrate Denatured cellular DNA tRNA Various lymphokine mRNAs HPIV3 Hepatitis A virus RNA Hepatitis B virus RNA Hepatitis C virus RNA IFN-c mRNA Antisense ODN Telomeric DNA CSF1 mRNA AT1 receptor mRNA Telomeric DNA Hepatitis B virus RNA JEV mRNA Binding motif NA Not dened AUUUA; ARE Regulatory RNA subunits 5-UTR; likely AU-rich regions PTRE Poly(U) regions ARE in 3-UTR TAAAT motif TTAGGG, CCCTAA AU-rich region AU-rich region in 3-UTR

C Nicholls et al.

Notes GAPDH binds ssDNA and not dsDNA; NAD inhibits the interaction GAPDH may facilitate tRNA export from the nucleus GAPDH binds ARE in the 3-UTR and may regulate mRNA stability Potential role in HPIV3 lifecycle GAPDH binds a single-stranded RNA region, may impact stability GAPDH may have a role in mRNA export GAPDH binding may inuence viral gene expression GAPDH amino acid residues 143 mediate binding GAPDH facilitates nuclear localization of ODN molecules Protects telomeres from drug-induced rapid shortening GAPDH stabilizes CSF1 RNA in ovarian cancer Inhibits expression of AT1 receptor; mediates H2O2-stimulated upregulation of AT1 receptor GAPDH-dependent protection of telomeres against rapid shortening Inhibits PTRE function; decreases antigen expression JEV infection alters cellular GAPDH distribution

Reference 6 63 7 64 65 66 67 8 68 12 10 69 13 70 71

GGT motifs in the telomeric sequence PTRE Not dened

HPIV3, human parainuenza virus Type 3; IFN-c, interferon-c; ODN, oligodeoxynucleotides; CSF1, colony-stimulating factor 1; JEV, Japanese encephalitis virus; PTRE, post-translational regulatory element; ARE, AU-rich elements; UTR, untranslated region; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.

(mTOR) complex. Under low glucose conditions, GAPDH binds directly to and sequesters the GTPase Rheb, a critical component of mTOR.23 Greater availability of glucose increases glycolytic ux, which elevates levels of the GAPDH substrate G3P. This increases GAPDH catalytic activity and liberates Rheb, restoring mTOR signalling. In this way, GAPDH serves as a sensor of glucose availability and couples this signal to cellular growth and proliferation.

APOPTOSIS AND GAPDH

An association between GAPDH and apoptosis was rst made following experiments that identied increased GAPDH expression in neuronal cells immediately prior to their undergoing programmed cell death.2426 Evidence for a functional role for GAPDH in apoptosis is provided by the complete blockade of cytosine arabinoside-induced apoptosis in GAPDH-depleted neuronal cells.27 Consistently, overexpression of GAPDH in COS-7 cells results in an increase in apoptosis.28 Furthermore, cells undergoing apoptosis exhibit elevated levels of GAPDH in the nucleus.29,30 This role in apoptosis is the most heavily studied non-glycolytic role of GAPDH and has been the subject of several prior reviews.3,5,31,32 Apoptosis triggered by GAPDH appears to be mediated by multiple downstream effector pathways. Nitrosative stress conditions induce an interaction between GAPDH and the ubiquitin ligase Siah1, leading to the nuclear translocation and stabilization of the complex, followed by ubiquitination and degradation of nuclear proteins.33 Even more recently, another GAPDH-binding protein, namely GAPDHs competitor of Siah protein enhances life (GOSPEL), has been found to block nuclear translocation of GAPDH by binding GAPDH and preventing its interaction with Siah1.34 This association may serve to protect cells from undergoing cell death in response to small amounts of oxidative stress. An additional study from this same group has demonstrated that nuclear GAPDH can be

acetylated by p300 CREB binding protein (CBP), which, in turn, stabilizes p300 CBP and enhances the acetylation of additional nuclear targets, facilitating apoptosis.35 Furthermore, the induction of apoptosis by oxidative stress requires the integrity of the GAPDH active site cysteine residue C152.36 GAPDH may also be an effector of p53mediated apoptosis, because p53 depletion inhibits GAPDH expression whereas p53 stabilization conversely upregulates GAPDH.37 Furthermore, GAPDH has been shown to facilitate apoptosis when localized to mitochondria, where it aids in mitochondrial membrane permeablization and the release of pro-apoptotic cytochome c.38 Because G3P suppresses caspase 3 activity preventing caspasedependent proteolysis,39 GAPDH conversion of G3P to 1,3-biphosphoglycerate may allow a caspase 3-dependent apoptosis to occur. However, GAPDH overexpression has also been shown to protect cells from caspase-independent cell death following mitochondrial membrane permeabilization by maintaining ATP levels and upregulating the autophagy facilitator Atg12.40 These ndings suggest that the role of GAPDH in cell death is complex and involves additional factors.

GAPDH AS A PRO-SURVIVAL FACTOR

Although GAPDH can trigger cell death via apoptosis, it can also function as a mediator of cell survival. GAPDH facilitates progression through mitosis by reversing SET-induced cyclin B-cdk1 inhibition through direct interactions with both proteins.41 In addition, cancer cells typically rely heavily on glycolysis for ATP generation, even in the presence of oxygen (the Warburg effect), and frequently upregulate GAPDH to meet their energy requirements.4244 Clearly, these cells expressing elevated GAPDH persist and, indeed, proliferate profusely, despite the demonstrated role for GAPDH as a potent inducer of apoptosis. Therefore, GAPDH can both positively and negatively regulate cell survival and proliferation.

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REGULATION OF GAPDH
Because GAPDH is involved in an array of cellular processes, it could be expected to be subject to considerable regulation. Perhaps surprisingly, GAPDH is encoded by a single gene on human chromosome XII that gives rise to an individual mRNA species with no known splice variants. The translated 335 amino-acid GAPDH protein is highly abundant and although an isoform coded by a separate gene has been identied, its expression is restricted to spermatozoa.45 Under normal cellular conditions GAPDH exists predominantly as a homotetramer, a conformation required for its catalytic activity. Despite the common use of GAPDH as an experimental loading control, gene expression levels can change in response to a variety of stimuli, including oxidative stress and hypoxia, both of which cause an upregulation of GAPDH expression.4649 Many tumours develop a state of hypoxia due to their high energy requirements and disorganized vasculature, which could therefore enhance GAPDH expression and facilitate an increase in the rate of glycolysis. However, the sheer number of different functions ascribed to GAPDH and the requirement for GAPDH to localize to various cellular compartments to exert these effects suggest more complex regulation than mere changes in protein levels. Accordingly, many studies have described extensive post-translational modication of GAPDH, which can alter both the catalytic activity and localization of the protein. The details of these are discussed below. Regulation of GAPDH by phosphorylation GAPDH has been shown to contain phosphotyrosines and is a target of several kinases, including skeletal muscle-specic Ca2+ calmodulin-dependent protein kinase and Akt, with phosphorylation upregulating catalytic activity.5052 In addition, GAPDH is involved in vesicle trafcking and phosphorylation by atypical protein kinase C2 and Src kinases regulate this process.53,54 Furthermore, GAPDH can undergo autophosphorylation in the presence of Mg-ATP, enabling GAPDH itself to exert kinase activity on targets such as the GABAA receptor.55,56 Hence, the phosphorylation status of GAPDH regulates both its glycolytic and non-glycolytic functions. Regulation of GAPDH localization Under normal cellular conditions GAPDH is predominantly found in the cytoplasm. This is due, at least in part, to an atypical chromosome region maintenance-1 (CRM1) binding site at residues 259271 in the C-terminal region, which governs nuclear export of GAPDH.57 The importance of this region for regulating GAPDH localization has been demonstrated by the expression of GAPDH harbouring the mutation K259N in colon adenocarcinoma cells, which abolishes the interaction with CRM1 and leads to nuclear accumulation of the protein.57 This also highlights the fact that GAPDH is capable of shuttling into the nucleus under normal conditions. Nuclear translocation of GAPDH is required for many of the extraglycolytic functions described earlier, including DNA repair, telomere binding and facilitation of apoptosis. However, cells expressing a GAPDH mutant harbouring a nuclear localization signal (NLS) that enforces nuclear accumulation of the protein fail to undergo apoptosis.29,58 This suggests that, in addition to nuclear translocation, modications to GAPDH and or interactions with other proteins are

required for its apoptotic function. One such modication is nitric oxide (NO)-mediated S-nitrosylation of the active site cysteine-152 of GAPDH, which abolishes catalytic activity and facilitates the GAPDHSiah1 interaction and subsequent nuclear translocation.33,59 Even more recent is the discovery that S-nitrosylated GAPDH can mediate the trans-nitrosylation of other nuclear proteins, specically the chromosomal regulators DNA protein kinase, Sirtuin-1 and histone deacetylase 2.60 The importance of S-nitrosylation in regulating the function and activity of proteins is increasingly being recognized and the transfer of nitrosyl groups to particular target proteins by GAPDH provides a mechanism of the specicity of NO signalling.

CONCLUDING REMARKS
Reports describing new functions for GAPDH frequently mention an initial surprise at nding this housekeeping gene involved in processes far removed from glycolysis. Oftentimes, the presence of GAPDH is rst presumed to be an artefact due to its high abundance. However, further research conrms a physiological role and the role of GAPDH is now appreciated to be far more complex than that of a simple glycolytic enzyme. GAPDH has functions across all major compartments of the cell and has key roles in many major cellular systems. Interestingly, GAPDH in lower eukaryotes does not appear to possess any moonlighting functions, with the exception of the interaction with yeast Rpb7, suggesting that these extraglycolytic roles are a recent evolutionary development.15,61 The requirement of GAPDH and it protection from S-nitrosylation upon NO stress in yeast is suggested by the recent nding of glutathione peroxidase 3 binding.62 Although elucidation of the mechanisms by which GAPDH inuences cell proliferation remains incomplete, a key feature is its nuclear translocation in response to stress conditions. In the nucleus, GAPDH is a core constituent of a gene transcriptosome involved in cell division. Binding to nucleic acids, GAPDH is implicated in mediating RNA nuclear export, inuencing the stability of mRNA and the repair of damaged DNA. Of further interest, GAPDH can be found at telomeres, where it binds directly to telomeric DNA. These ndings suggest that, in addition to catalysing energy metabolism, GAPDH can safeguard chromosome stability and protect genome integrity. However, GAPDH is also a critical mediator of the cellular response to oxidative stress and directly facilitates apoptosis. These ndings demonstrate that the pleiotropic functions of GAPDH are important for a range of mammalian biological processes.

ACKNOWLEDGEMENTS
The authors work reported herein was supported by grants from the National Health and Medical Research Council of Australia, Cancer Council Victoria and National Basic Research Program of China (2012CB911200). CN is a recipient of a Monash Postgraduate Scholarship.

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