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GSK3 at the Edge: Regulation of Developmental Specification and Cell Polarization

Leung Kim1 and Alan R. Kimmel2,*
1

Department of Biological Sciences, Florida International University, Miami, FL 33199, USA and 2Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-8028, USA
Abstract: GSK3 is a multifunctional protein kinase that is pivotal for the regulation of metabolism, the cytoskeleton, and gene expression. Multicellular eukaryotes utilize GSK3 as a molecular switch to specify distinct cell fates, but also to organize these cells spatially within the developing organism. We discuss the central role of GSK3 in control of the Wnt, Hedgehog, cAMP (in Dictyostelium), and other signaling pathways, but also focus on significant new evidence that GSK3 is required to establish cell polarity.

Key Words: Wnt, Frizzled, Hedgehog, Presenilin, PAR, Cell Motility, Chemotaxis, Dictyostelium. THE OVERVIEW As inferred from its original name glycogen synthase kinase 3, GSK3 function was initially associated with metabolic pathways. But, it is now clear that GSK3 activity is required for multicellular development and for a broad range of cellular functions, including stem-cell renewal and differentiation, apoptosis, and even circadian rhythm. Aberrations in GSK3 regulation can promote tumorigenesis, neurodegeneration, diabetes and insulin resistance, among other abnormalities (see [1-5]). Mammalian cells have three closely related forms of GSK3, GSK3, GSK3, and the minor splice variant GSK32. Their catalytic domains are nearly identical and they share many modes of regulation. Two sites for phosphorylation of GSK3 directly affect kinase activity. Phosphorylation at S9 of GSK3 (S21 on GSK3) by Akt, and other protein kinases, is specifically inhibitory. In general, GSK3 requires a priming phosphorylation at position +4 in the downstream position of the optimal consensus sequence (SXXXpS) for substrate recognition. S9 inhibition appears to function as an intramolecular pseudo-substrate, blocking access to the catalytic site [3] of these primed substrates [6]. In the absence of Akt activation during insulin resistance, GSK3 is active and constitutively represses glycogen synthase. In addition, active GSK3 will phosphorylate and inhibit IRS-1 (insulin receptor substrate-1), an essential component of the insulin signaling pathway [7]. Loss of GSK3 repression exacerbates insulin resistance. In contrast to the inhibitory action of Akt at S9, tyrosine phosphorylation at Y216 in the activation loop of GSK3 (Y279 of GSK3 ) enhances kinase activity. While the activation is modest as compared to that usually observed with other kinases [8], an explanation became apparent with the elucidation of the GSK3 structure. In the unphosphorylated protein, the activation loop is already in an open configuration. Tyrosine phosphorylation may serve to reposition Y216 through the interaction with a proximate arginine to increase substrate access and kinase activity [8]. Data from Dictyostelium (c.i.) indicate that additional phosphorylation at Y222 is essential for the full activation of GSK3 [8]. Alteration in subcellular localization of GSK3 is the other major regulatory mode. GSK3 does not have specific subcellular localization motifs, but various extracellular signals can impact the interaction of GSK3 with its target substrates. Other signals influence the levels of GSK3 in the nucleus, where it can phosphorylate a variety of transcription factors. Many of these are inhibited, as
*Address correspondence to this author at the Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892-8028, USA; Tel: (301) 496-3016; Fax: (301) 496-5239; E-mail: ark1@helix.nih.gov 1389-4501/06 $50.00+.00

phosphorylation by GSK3 can promote nuclear export. The most widely studied is NFAT (Nuclear Factor of Activated T-cells), a central regulator of thymic development and immune response, cardiac morphogenesis, and neural survival [9, 10]. Despite the apparent essential role of GSK3 throughout development it came as an initial surprise that GSK3 -deficient mice did not exhibit an embryonic lethality until mid gestation [11]. Most certainly, progression through early embryogenesis was rescued by the endogenous expression of GSK3 in these mice. Nonetheless, lethality is the result of a liver failure that is consistent with a hypersensitivity to TNF due to loss of NF-B activation. The observations defined a previously unappreciated requirement of GSK3 function and a link to apoptosis and other NF-B regulated pathways [11-13]. The GSK3-deficient mice provided an additional view to GSK3 function in vivo. gsk3 +/- mice, which are haploinsufficient for GSK3, mimic the behavioral effects observed upon treatment of control animals with clinical doses of lithium, a GSK3 inhibitor [14]. Lithium has been one of the effective treatments for human bipolar disorder and these results provide strong support that a primary target of action may be GSK3. The variety of cellular and developmental processes that are controlled by GSK3 is indeed too vast to detail. Rather, this review will focus on selected GSK3-dependent pathways that have been well described at the molecular level. Particular attention will be directed toward mechanisms that organize cell fate specification and regulate cell polarity and directed cell movement. Since GSK3 is of such critical import, it is subject to continuous discussion and review; accordingly, emphases here will be limited to the most recent developments. GSK3-LINKED PATHWAYS THAT SPECIFY DEVELOPMENTAL FATES Canonical Wnt Signaling and Complexity Beyond The Wnts are a large family of secreted, lipid-modified, glycoproteins [15] that regulate major developmental and proliferative events in the metazoa [4, 5, 16]. Loss of Wnt signaling in mammals disrupts embryonic axis formation, gastrulation, and CNS (central nervous system), thymic, and limb development. Stem cell proliferation is also regulated by Wnt signaling, and mutations that lead to the constitutive activation of the pathway promote tumorigenesis [1-5, 16, 17]. Wnt signaling is multi-facetted and broadly classified as either canonical or non-canonical and can directly impact transcriptional as well as cytoskeletal processes. Canonical Wnt signaling functions primarily by regulating GSK3 to promote the rapid, cytosolic accumulation of the multifunctional protein -catenin (Fig. 1). In quiescent cells, -catenin is principally found at the cell cortex in association with cytoskeletal
2006 Bentham Science Publishers Ltd.

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components (e.g. E-cadherin), where it functions to orchestrate actin dynamics and regulate cell adhesion, cell-cell interactions, and intercellular communication. Unstimulated cells (Fig. 1A) possess relatively low levels of cytosolic -catenin which, in general, is sequestered to multi-protein scaffolds anchored by Axin and Axin2 (conductin). Within these Axin complexes, -catenin is phosphorylated by CK1 (Casein Kinase I), priming it for additional phosphorylations by GSK3 [18, 19]. Polyphosphorylated -catenin is then subject to proteasomal destruction [20, 21] that is mediated by APC (Adenomatous Polyposis Coli) and TrCP [5]. These Axin scaffolds are, thus, often denoted as destruction complexes, and although Axin is also a kinase substrate when complexed with GSK3, Axin does not appear to be de-stabilized upon phosphorylation by GSK3. In addition to its role as a cytoskeletal component, -catenin can also function as a transcriptional co-factor; complexes of catenin and Lef/Tcf transcription factors serve as nuclear activators [22-24] of gene sets that promote specific developmental pathways or tumorigenesis [5]. Canonical Wnt signaling serves to inhibit catenin phosphorylation and thus promotes the accumulation of an uncomplexed, cytosolic population of -catenin that is available for interaction with and activation of Lef/Tcf family members (Fig. 1B). In the canonical pathway, extracellular Wnt signals are detected at the cell surface by both the seven-transmembrane Frizzled (Fz) [25, 26] and the single-pass, LDL receptor-related proteins (LRP) 5/6 [27-29] that act as co-receptors (Fig. 1B). Data suggest that Wnt treatment disassembles the Axin/GSK3 complexes (see [5]), thus decreasing GSK3 phosphorylation of both -catenin and Axin. While unphosphorylated -catenin is consequently stabilized, unphosphorylated Axin is subject to proteasomal degradation, further reducing Axin/GSK3 complex levels [30-33]. While the precise molecular events that transmit Wnt signals are not well characterized, many additional components are involved (see [5]). Principal to this is Dishevelled (Dvl/Dsh), which functions downstream of Fz/LRP but upstream of GSK3 to mediate -catenin stabilization and may play an essential, but as yet undefined, role in the disruption of the Axin destruction complex [3436]. The structural similarity of Fz to bona fide G protein coupled receptors had led to early speculation that heterotrimeric G protein signaling may participate in Wnt regulation of GSK3. Although there is good evidence that Fz receptors may interact with G proteins for Ca+2 signaling, a non-canonical, non-GSK3 Wnt pathway [37], until recently, definitive evidence [38, 39] for G proteindependent, -catenin signaling had been elusive. Epistasis experiments in Drosophila now provide strong support that G o acts downstream of Fz, but upstream of Dsh during Wnt regulation of GSK3/-catenin signaling [40]; further, activated Go-GTP appears to function independently of receptor activation in this pathway [40]. In addition, we have shown that depletion of Go and Gq in mammalian cells with siRNA will inhibit Wnt-induced stabilization of -catenin [40a] and that direct activation of G proteins with GTPS can promote -catenin accumulation in the absence of Wnt stimulation [40a]. These data are consistent with an intermediary role for G proteins in signal transduction from Fz/LRP to Axin/GSK3. Yet, it is not clear why G protein genes had not been identified previously in various genetic screens for Wnt modifiers. There may be regulatory paths that potentiate -catenin stabilization independently of G -signaling. Potentially, LRP5/6 can transduce a Wnt signal directly to Axin/GSK3 [41-44] by a mechanism that requires interaction with Fz, but that bypasses G activation. Other evidence exists for a mechanistic parallel. Fz receptors are characterized by their extended, extracellular N-terminal, cysteinerich domain (CRD). Although the CRD is the Wnt-binding region of Fz, in vivo data in Drosophila indicate that the CRD is not en-

tirely required for Wnt signaling [45]. The direct binding of Wnt to Fz may not be absolutely essential for signal transduction, but perhaps can be compensated by interaction with other components of the receptor complex. To this extent, disruption of Fz/G -signaling may have a less severe effect on Wnt response during development than would be the loss of an individual Wnt, Fz, or LRP gene member. Finally, it should be emphasized that additional non-canonical Wnt receptor systems (e.g. Ryk, Ror) may function independently of Fz/G -signaling and, thus, also bypass G protein dependency [46-51]. Still, definitive downstream linkages of these signaling pathways have not been established. Hedgehog The Hedgehog (Hh) proteins constitute another family of secreted proteins that regulate a broad variety of growth and developmental processes through the targeted regulation of GSK3 function. Hh signaling was first described in Drosophila but is now understood to be essential throughout vertebrate development. Disruption of component genes in humans leads to neural defects, tumorigenesis, and other severe and complex syndromes (see [52]). In general Hh and Wnt responses occur in non-overlapping cell populations, yet their signaling mechanisms have remarkable parallels. In the absence of Hh protein, the 155 kDa, zinc-finger transcription factor Ci [cubitus interruptus in Drosophila; Gli (from glioblastoma) in vertebrates] is subject to proteolytic cleavage mediated by TrCP/Slimb (supernumerary limbs) in concert with the proteasome pathway (see [52]). The resulting product is an N-terminal Ci75 fragment that retains the zinc-finger domain, but that functions largely as a transcriptional repressor. Hh signaling inhibits Ci processing, thus directing the accumulation of full-length Ci-155 and the transcriptional activation of Hh-response genes. While it had been clear that phosphorylation of Ci by the cAMP-dependent protein kinase (PKA) was essential for proteolytic cleavage in the absence of Hh, mechanistic links were only poorly understood. Now, several recent studies have proven an essential role for GSK3 in Hh signaling and suggest a new model for its antagonistic regulation [53-60]. In unstimulated cells, Ci-155 is associated with a cytoplasmic scaffolding complex consisting of Cos2, Fused (Fu), PKA, GSK3, and CKI (Fig. 2A). Within the complex, PKA phosphorylates Ci155 at several sites [54, 55, 61]; each of these in turn serves as a priming phosphorylation for recognition by both GSK3 and CKI (Fig. 2B). Hyper-phosphorylation of Ci-155 by all 3 kinases is required for the proteolytic processing that generates Ci-75. Mutation of the PKA, the GSK3, or the CKI sites within Ci-155 prevents proteolysis and mimics activation by Hh [54, 55]. In addition, mutation of Cos2 also suppresses Ci-75 production, presumably through disruption of the Cos2-Ci-kinase destruction complex. Two transmembrane proteins, Patched and Smoothened (Smo), function to transduce the Hh signal (Fig. 2A). Patched is a 12transmembrane domain protein and the presumed Hh receptor [52]. Historically, Patched has been denoted Ptc, but recently several workers have adopted Ptch to distinguish it from PTC, the bittertaste receptor of humans. In the absence of an Hh ligand, Ptc/Ptch is suggested to inhibit the function of the 7-transmembrane domain protein Smo (Fig. 2A). Inhibition is still poorly understood mechanistically, but appears to require phosphorylation-dependent events that promote destabilization and, perhaps, localization to intracellular vesicles. Although the phosphorylation sites on Smo are remarkably similar in sequence to those of Ci, phosphorylation of Smo by GSK3 is not required for function [53, 57]. In contrast, Smo proteins that lack either the 3 consensus PKA or 3 consensus CKI sites are not subject to Hh-independent degradation, and are also unable to support signaling in the presence of Hh (v.i.). Hh binding to Ptc/Ptch stimulates Smo hyper-phophorylation and relieves Smo inhibition (Fig. 2C), potentially through stabiliza-

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Fig. (1). GSK3 regulation of -catenin degradation. A. In the absence of a Wnt ligand, -catenin is associated with an Axin scaffold in complex with GSK3. Phosphorylation by GSK3 promotes the proteasomalmediated degradation (X) of -catenin. Transcriptional paths that require -catenin for activation are, thus, inhibited (X). See text for details and additional components. B. Wnt binding to the Fz/LRP co-receptor complex leads to the disassembly of the Axin destruction complex, by a process mediated by Dsh. The precise relocalizations of these various components have not been established. -catenin is no longer associated with or phosphorylated by GSK3. -catenin degradation is attenuated, and accumulated -catenin is now able to associate with Tcf transcription factors to activate gene expression. C. A potential -catenin destruction complex mediated by PS scaffolding and insensitive to Wnt signaling. -catenin is subject to phosphorylation and degradation (X).

tion and recruitment to the cell surface [56, 58-60]. Activated Smo is suggested to bind Cos2 in a manner that disassembles the Cos2Ci-kinase destruction complex. As a consequence, Ci-155 phosphorylation and proteolysis are diminished, production of Ci-75 is attenuated, and Ci-155 accumulates to promote expression of Hhresponse genes. The similarity in phosphorylated sequences shared by Smo and Ci suggests a simple mechanism whereby activated, hyper-phosphorylated Smo may displace and, hence, release Ci-155 from association with the Cos2-kinase complex. Phosphorylation of Smo by both PKA and CKI (but not by GSK3) is required for Hhinduced accumulation of Ci-155. Significant parallels are easy drawn between the Hh and canonical Wnt pathways. Both utilize GSK3 to regulate the stability of a core transcription factor, Ci for Hh and -catenin for Wnt. Under unstimulated conditions, GSK3 is functionally active, directing the phosphorylation and proteolysis of Ci/-catenin. Ligand engagement blocks phosphorylation in both pathways, but in neither is GSK3 activity inhibited, per se; rather, signal transduction serves to disassemble the Cos2 and Axin destruction complexes releasing GSK3 from association with its substrate targets and its dependent priming kinases. One additional link between the Hh and Wnt paths should be noted. The signal-transducing proteins for each, Smo and

Fz, respectively, share high sequence similarity and belong to the same subfamily of 7-transmembrane receptors. Presenilin Alzheimers disease is marked by the elevated accumulation of the 40 and 42 amino acid amyloid (A ) peptides. Mutation of the gene for Presenilin (PS) is a major cause of familial, early-onset Alzheimers disease; the PS/Alzeimers connection is now well established. A peptides are generated by sequential cleavages of the amyloid precursor protein (APP), a single-span transmembrane protein. The first proteolytic cleavage releases the extracellular domain and facilitates subsequent intramembranous proteolysis by -secretase; -secretase activity requires PS, as well as nicastrin, Aph1, and Pen2, but the catalytic activity appears to reside within PS. Dominant mutations in PS can alter the sites for intramembrane cleveage of APP and increase the relative proportion of A 42, the presumed pathogenic product. PS is a multi-transmembrane protein that, in addition to exhibiting proteolytic activity, possesses binding domains for both GSK3 and -catenin. Recent studies have focused on the relationship between GSK3 and -secretase activities and a potential role for PS as a multi-functional scaffold of GSK3.

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Fig. (2). GSK3 regulation of Ci-155 processing. A. In the absence of an Hh ligand, Ci-155 is in a Cos2 scaffold complex with GSK3, PKA, CKI, and Fu. Phosphorylation of Ci-155 by GSK3, PKA, and CKI promotes Ci-155 proteolysis and the production of C-75. (See text for details and additional components.) B Consensus sites for Ci-155 phosphorylation by PKA, GSK3, and CKI. PKA recognizes the sequence RRXS, creating a priming phosphorylation recognized by GSK3 (at position +4 within the consenus site, SXXXpS) and by CKI (at position -3, pSXXS). C. Upon Hh binding to Ptc, Smo becomes activated and promotes disassembly of the Cos2 destruction complex. Ci-155 is no longer associated with or phosphorylated by GSK3. Ci155 degradation is attenuated and now activates expression of the Hh-responsive genes.

A role for GSK3 in APP processing has been confirmed [62]. Briefly, pharmacological inhibition of GSK3 specifically inhibits the processing of APP and the accumulation of A peptides. This is not caused by a global inhibition of -secretase, since GSK3 is not required for the proteolysis of other PS substrates (e.g. Notch, v.i. [62, 63]), and is not mimicked by overexpression of -catenin, a cofactor that may accumulate under conditions of GSK3 inhibition (v.s.). Further, directed siRNA approaches indicate a requirement for GSK3, but not GSK3, in APP proteolysis, underscoring a unique and restricted association of GSK3 to PS function. In addition, although GSK3 is not required for Notch processing, phosphorylation of the intracellular domain of Notch by GSK3 does decrease its stability and suppresses its function (v.i. [63]). Linkage between PS and GSK3/-catenin signaling has been less clear. PS was identified in genetic screens for novel modifiers of the Wnt (Wg) pathway in Drosophila [64, 65] and data suggest that PS acts as a negative regulator of the pathway. PS has also been identified in a multi-protein complex with PKA, GSK3, and catenin in mammalian cells [66]. Here, again it appears to antagonize Wnt signaling (Fig. 1C ). In this context, PS functions analogously to Axin, scaffolding -catenin to GSK3 and a priming

kinase (PKA). This complex would promote the de-stabilization of -catenin, but would be unresponsive to disassembly by Wnt. However, depletion of PS (or PKA) by RNAi did not alter -catenin (armadillo) levels in Drosophila tissue culture cells [67]. PS cleaves a diverse group of single-span transmembrane proteins. Other than APP, Notch is among the most well characterized. PS processing of Notch releases the intracellular C-terminal domain (ICD) that functions as an essential transcriptional activator for embryogenesis, and ps1-/-ps2-/- mice carrying mutations that fully ablate PS function exhibit an embryonic Notch-like lethality. Thus, it has been difficult to analyze an in vivo role for PS in GSK3/catenin regulation in mammals. However, ps1+/-ps2-/- mice that are partially deficient for PS survive and their derived fibroblasts exhibit significantly elevated levels of -catenin as compared with wild-type controls [68]. These data are consistent with a scaffold function for PS that promotes GSK3-mediated degradation of catenin (Fig. 1C). Non-Canonical Wnt Although most developmental control appears to be directed towards the negative regulation of GSK3, two organisms, Cean-

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horabditis elegans and Dictyostelium discoideum, utilize an activating signal to specify cell fate determination (Fig. 3 ). In the nematode C. elegans, endoderm and mesoderm fates derive from an EMS precursor cell, which arises after the second cell division of the embryo. The EMS cell is oriented along an anterior/posterior axis and its subsequent division generates an anterior MS and a posterior E cell that, respectively, define mesoderm and endoderm lineages. These cell fates are regulated, at least in part, by Wnt signal induction from the neighboring P2 cell [69-72]. Two mutations that inhibit E cell differentiation define the pathway. MOM-2 (more mesoderm) is a Wnt homolog produced in P2 cells, and MOM-5 is a Fz receptor expressed in EMS cells (Fig. 3). In addition, a Dsh protein is required for the intracellular transduction of the Wnt signal. However, Wnt signaling through Fz and Dsh does not repress GSK3 function as in the canonical pathway, but requires active GSK3. Thus, disruption of GSK3 in C. elegans does not promote endoderm induction as would occur if GSK3 were inhibited by Wnt, but produces a MOM (more mesoderm) phenotype (Fig. 3) that parallels the loss-of-function mutations in mom-2 (Wnt) and mom-5 (Fz). -catenin and Tcf homologs function downstream of GSK3, but again their genetic interactions differ from that of the canonical Wnt pathway [69-72]. Despite the requirement for active GSK3 during E/MS specification, C. elegans utilizes canonical Wnt pathway members, including PRY-1, a highly diverged but functionally active Axin ortholog, to regulate neuroblast and vulval differentiation [73]. Dictyostelium provides further evidence for an activating pathway for GSK3 during cell fate determination (Fig. 3). There are 2 major cell classes at terminal differentiation of Dictyostelium. These are the spore and stalk cells which derive from the non-

terminally differentiated prespore and prestalk progenitors (see [74]). GSK3 acts as developmental switch to establish these cell fates (Fig. 3 ). In Dictyostelium, GSK3 is not regulated by a Wnt signal (making it extremely non-canonical). Instead, the prestalk/prespore axis is specified by signaling via secreted cAMP that binds and activates a family of 7-transmembrane domain, cell-surface receptors (CARs), which are distantly related to Fz. CAR3, the related tyrosine kinases ZAK1 and ZAK2, and GSK3 are required for prespore cell fate differentiation, but inhibit prestalk differentiation ([8, 17, 74-77] Kim and Kimmel, unpublished). ZAK1/2 are activated by cAMP/CAR3 and, in turn, ZAK1/2 are required to phosphorylate and activate GSK3. The sites phosphorylated by ZAK1 have been mapped to the activation loops of both Dictyostelium GSK3 and mammalian GSK3 and these phospho-tyrosines are required for GSK3 activation in vivo and in vitro [8, 75]. It should be noted that a Src-related tyrosine kinase functions in concert with GSK3 to activate MS differentiation in C. elegans [71]. Although the target of Src has not yet been determined, GSK3 is a potential candidate that may be subject to an activating tyrosine phosphorylation in this pathway (Fig. 3). In contrast to the role of CAR3, Dictyostelium that are deficient for CAR4 exhibit the phenotypic loss of prestalk cell gene expression and expansion of prespore gene expression (Fig. 3 ). car4-nulls also have persistent tyrosine phosphorylation and hyper-activation of GSK3 [8, 17, 74, 75]. Developmental defects of car4 -nulls can be partially rescued by biochemical and genetic reduction of GSK3 activity in vivo, indicating that GSK3 is a downstream negative target of CAR4. The absence of CAR4 has no affect on cAMP/ CAR3 regulated activation of ZAK1, indicating that CAR4 does not

Fig. (3). The role of GSK3 for cell fate determination in C. elegans and Dictyostelium. In contrast to the inactivating signal transmitted to GSK3 in the canonical Wnt pathway (see Fig. 1), C. elegans requires an active GSK3 to regulate EMS fates. See text for details. In Dictyostelium, GSK3 acts as developmental switch to establish the fates of the prespore and prestalk cells, the two major developmental progenitors. Activated GSK3 is required for the prespore pathway, whereas de-activated GSK3 promotes prestalk differentiation. The data are consistent with phosphorylation/de-phosphorylation of GSK3 as the mechanism for activation/de-activation of GSK3. The different levels of tyrosine phosphorylation of GSK3 resulting from the antagonistic actions of the tyrosine kinases ZAK1/2 or the CAR4-PTPase constitute the core of the regulatory machinery on GSK3 activity in the context of cell fate decision regulated by the 7-TM CARs. See text for details.

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inhibit GSK3 by repressing ZAK1, the activating tyrosine kinase of GSK3. Rather, CAR4 activates a specific PTPase. This has been confirmed in biochemical assays using a specific phosphoGSK3 substrate [8]. These data are consistent with phosphorylation/dephosphorylation as a mechanism for activation/de-activation of GSK3. The different levels of tyrosine phosphorylation of GSK3 resulting from the antagonistic actions of the tyrosine kinases ZAK1/2 or the CAR4-PTPase constitute the core of the regulatory machinery on GSK3 activity in the context of cell fate decision by the 7-TM CARs in Dictyostelium (Fig. 3 ). GSK3 AND CELL POLARITY Analysis of P2/EMS interactions in C. elegans provided new perspectives into GSK3 regulation of cell specification. However, Wnt signaling to the EMS cells has complexity beyond the control of transcriptional events [69, 78]. Prior to cell division, the EMS precursor is organized along an anterior/posterior (A/P) axis. Initially however, the mitotic spindle is not aligned with the A/P axis. The spindle must rotate during mitosis to adopt the appropriate A/P orientation. Spindle rotation and re-orientation relies on the same upstream members of the MOM-2/MOM-5 (i.e. Wnt/Fz) signaling pathway that specify E/MS fates (see Figs. 3 and 4). Both pathways require, but diverge at GSK3. Thus, spindle rotation occurs independently of activated gene expression. Other developmental aspects in C. elegans have demonstrated a requirement for GSK3 in organizing cell polarity. Early embryogenesis in C. elegans involves a series of asymmetric cell divisions. par embryos that are defective in partitioning are unable to undergo normal asymmetric divisions, with resulting defects in differentiation. Genetic screens for such abnormalities identified six PAR genes essential to polarize cells for partitioning. Five of these genes are conserved in mammals, where they also regulate cell polarization. Par6 and Par3 are PDZ domain-containing proteins that form a heterotrimer with aPKC (an atypical protein kinase C). Mutations in any of these genes disrupt cell polarity in C . elegans, Drosophila, and vertebrates. The Par6 complex [79, 80] is intracellularly polarized to the anterior pole of the C . elegans zygote, but also to the cell-cell junctions of epithelial cells where it regulates apical/basolateral specificity, to nascent axons on neurites, and in certain cells migrating toward a directional signal. Astrocyte migration involves cellular protrusions that are driven by microtubule elements. In scratch assays that mimic wound healing, primary astrocytes re-orient and migrate to repair the abrasion. Scratch-induced migration causes a re-orientation of the centrosome towards the leading edge and the formation of microtubule protrusions. cdc42 and Rac1, members of the Rho family of small GTPases, also become activated and re-localized to the leading edge of migrating astrocytes. Active, GTP-bound cdc42 will induce cytoskeletal rearrangements through interaction with WASp (Wiscott-Aldrich syndrome protein) and PAK and their reorganization of actin filaments. But, cdc42 has not been generally associated with establishing microtubule polarity. Recent data now connect cdc42 to microtubule assembly through the regulation and function of Par6/PKC and GSK3 (Fig. 4 ). Par6 binding to cdc42 at the leading edge promotes the activation of the associated aPKC leading to the phosphorylation of GSK3 at S9 and its consequent inactivation [80, 81]. Inhibition of cdc42 or of aPKC blocks GSK3 phosphorylation, microtubule orientation, and astrocyte migration. Expression of the nonphosphorylatable, S9A variant of GSK3 is similarly insensitive to scratch induction. Remarkably, expression of a constitutively-active (GTP-locked) cdc42 or a kinase-inactive GSK3 also disrupts cell polarization [80, 81]. The global inhibition of GSK3 does not prevent cell protrusions, but it induces them along the entire cell periphery, emphasizing the significance of localized activation and (GSK3 inhibition) for cell polarization.

Downstream of GSK3, APC is also recruited to the leading edge. In addition to its role as a mediator of -catenin degradation during canonical Wnt signaling (see Fig. 1), APC can associate with the plus-ends of microtubules to regulate their stability and dynamics. Expression of an APC fragment that lacks a microtubule interacting domain blocks centrosome re-orientation in migrating astrocytes [80, 81]. Localized inhibition of GSK3 by Par6/aPKC is also required to establish and maintain neuronal polarity (Fig. 4 [82-87]). Axons and dendrites constitute distinct structural elements that define the polarized neuron. The Par6/aPKC complex localizes to a single tip of the immature neurite and ultimately specifies the axon [83]. An APC fragment, which will interact with microtubules but not with phosphorylated GSK3, acts as a dominant-negative for axon formation. Likewise, expression of the non-phosphorylatable (constitutively-active) S9A GSK3 will also inhibit axon formation, whereas global inhibition of GSK3 promotes multiple axons and transforms pre-existing dendrites into new axons [85]. Semaphorin 3A (Sema 3A), a molecule that suppresses axonal growth, promotes GSK3 de-phosphorylation and its consequent activation at the leading edge of the growing neuron [86]. Studies on another Par protein have provided an additional linkage for GSK3 regulation of cell polarity. Several mammalian epithelial cell culture models will spontaneously polarize when confluent. Apical brush borders form and sort from basolateral markers. Par6/Par3 and cdc42-GTP modulate this process, but another Par protein, Par4, is also essential [88, 89]. The Par-4 ortholog in humans is LKB1, a tumor suppressor linked to Peutz-Jeghers cancer syndrome. LKB1 is activated by two proteins, STRAD and MO25, and the induced expression of STRAD will activate LKB1 and rapidly remodel and polarize individual epithelial cells prior to confluence. Conversely, depletion of LKB1 in these epithelial cells will delay the spontaneous formation of apical/basolateral polarity [88, 89]. LKB1 is itself multifunctional. Like Par6, it can associate with aPKC to promote the phosphorylation and inhibition of GSK3. Although it has been controversial whether enzymatic inhibition of GSK3 may synergize with the Wnt effects on GSK3 (see Fig. 1) to increase -catenin stabilization (see [17]), recent data clearly indicate that LKB1-mediated phosphorylation of GSK will activate catenin dependent transcriptional pathways [90, 91]. Part of the LKB1 effect could be mediated by the Par1 kinase, a substrate of LKB1. Indeed, Par1 is suggested to facilitate Wnt signaling [92]. However, a related study found that LKB1 expression appeared to antagonize the pathway rather than activate it [93]. Perhaps additional complexities explain the apparent contradictions [92]. There are multiple Par1 kinases and multiple functions for the individual Par1 proteins. Activation by LKB1 may redirect Par1 localization or complex formation. It should also be noted that AMPK (AMP protein kinase) is a member of the Par1 family and a downstream target of LKB1. AMPK is also positive regulator of TOR (target of rapamycin), and although new TOR complexes have been identified that regulate the actin cytoskeleton and cell polarity [94], the direct input of LKB1 and AMPK into these TOR processes has not been established. PERSPECTIVES GSK3 participates in multiple pathways that regulate cell fate specification and cell polarization. While these events have been considered distinct, the most recent results suggest an intimate interrelationship between them. It may now be prudent to re-examine several non-canonical Wnt pathways that have been thought to function independently of GSK3 [16]. Planar cell polarity (PCP) establishes wing bristle and ommatidia polarity in Drosophila and convergent movement during vertebrate gastrulation. PCP is regulated through Fz/Dsh, but does not require -catenin stabilization. Mis-expression of GSK3 can also cause polarity defects [95], but

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Fig. (4). GSK3 and cell polarity. The GSK3 regulatory modes for several cell polarization systems are compared and contrasted. See text for details and additional components.

whether this is related to an alteration in PCP, to mis-regulation of Par, or an interaction between the pathways remains to be explored. Astrocyte migration differs widely from another type of directed cell movement, chemotaxis (see [74]). Yet, a linkage to GSK3 regulation is potentially related. When Dictyostelium or neutrophils migrate within a chemoattractant gradient, they preferentially localize PI3K and activated Akt to their leading edges (see [74, 96, 97]). The antagonistic actions at the leading edge and rear of a chemotaxing cell are essential to maintain polarity and directionality and to suppress spurious lateral movement (see [74, 96, 97]). Microtubule assembly is critical to regulate these balanced and localized activities [98, 99]. Disruption of the microtubule network in neutrophils suppresses leading edge formation and cell polarization in response to a chemoattractant signal, yet broadly activates Rho and Rho kinase, regulators that normally specify the rear of chemotaxing cells [98, 99]. While there may be a similar downregulation of GSK3 activity via Akt at the leading edge of neutrophils as in migrating astrocytes (see Fig. 4), a dependency on GSK3 inhibition and microtubule regulation in chemotaxing cells is not proven. Note that the Nterminus of GSK3 in Dictyostelium is unrelated to that of GSK3, and although it is Ser-rich, phosphorylation (or inhibition) by Akt has not been examined. However, one observation in Dictyostelium may further the connection of GSK3 function to cell polarity during chemotaxis. As would be consistent, disruption of GSK3 in Dictyostelium leads to defects at the backs of chemotaxing cells (Kim and Kimmel, unpublished). It is interesting to consider that localized activation of GSK3 by tyrosine phosphorylation may also influence cell polarization by restricting microtubule formation to promote essential posterior functions during chemotaxis. NOTE ADDED IN PROOFS A phosphorylated motif within the intracellular domain of LRP5/6 can function as a docking site for Axin [100]. Data now indicate that GSK3 directs this phosphorylation [101], which induces the subsequent phosphorylation of LRP5/6 by casein kinase 1 and recruits Axin binding [101, 102]. The rapid disruption of Axin/ GSK3 interactions, upon Wnt stimulation [40a], may promote LRP5/6 phosphorylation and potentiate the destabilization of the Axin destruction complex [103].

ACKNOWLEDGEMENTS We are indebted to continuous and insightful discussions with Drs. Xunxian Liu, Yingzi Yang, Jeffrey Rubin, and Pamela Schwartzberg. REFERENCES
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Received: August 11, 2005

Accepted: April 20, 2006