Glycogen Synthase Kinase-3 Mutant Research Articles

Glycogen Synthase Kinase-3 modulates α1A-adrenergic receptor action and regulation

The possibility that glycogen synthase kinase 3 (GSK3) could modulate α1A-adrenergic receptor (α1A-AR) function and regulation was tested employing LNCaP and HEK293 cells transfected to express the enhanced green fluorescent protein-tagged human α1A-AR. Receptor phosphorylation and internalization, intracellular free calcium, α1A-AR-GSK3 colocalization, and coimmunoprecipitation were studied. The effects of the pharmacological GSK3 inhibitor, SB-216763, and the coexpression of a dominant-negative mutant of this kinase, as well as the signaling, desensitization, and internalization of receptors with S229, S258, S352, and S381 substitutions for alanine or aspartate, were also determined. SB-216763 inhibited agonist- and phorbol myristate acetate (PMA)-mediated α1A-AR phosphorylation, reduced oxymetazoline-induced desensitization, and magnified that induced by PMA. Agonists and PMA increased receptor-GSK3 colocalization and coimmunoprecipitation. Expression of a dominant-negative GSK3 mutant reduced agonist- but not PMA-induced receptor internalization. α1A-AR with the GSK3 putative target sites mutated to alanine exhibited reduced phosphorylation and internalization in response to agonists and increased PMA-induced desensitization. Agonist-induced, but not PMA-induced, receptor-β arrestin intracellular colocalization was diminished in cells expressing the GSK3 putative target sites mutated to alanine. Our data indicated that GSK3 exerts a dual action on α1A-AR participating in agonist-mediated desensitization and internalization and avoiding PMA-induced desensitization.

Loss of GSK-3 Causes Abnormal Astrogenesis and Behavior in Mice.

Altered activity of glycogen synthase kinase-3 (GSK-3) is associated with psychiatric diseases and neurodegenerative diseases. GSK-3 is a key regulator in multiple aspects of neuronal differentiation in the brain. However, little is known about the role of GSK-3 in astrocyte development. To examine the role of GSK-3 in astrocytes, we generated a conditional knockout mouse using a glial fibrillary acidic protein (GFAP)-cre driver, in which the GSK-3 alpha and beta genes are deleted in astrocytes. We found that GFAP-cre-mediated GSK-3 deletion led to a larger brain. The number and size of astrocytes were increased in GSK-3 mutant brains. The levels of GFAP and phospho-STAT3, indicators of astrogenesis, were elevated in GSK-3 mutants. Furthermore, we found upregulation of astrocyte regulatory molecules such as phospho-AKT, phospho-S6, and cyclin D in GSK-3 mutant brains. Finally, GSK-3 mutant mice exhibited aberrant anxiety and social behavior. Our results suggest that GSK-3 plays a significant role in astrocyte development and behavioral control in mice.

Regenerative potential of targeting glycogen synthase kinase-3 signaling in neural tissues.

Multiple roles of glycogen synthase kinase-3 (GSK-3) in neural tissues: GSK-3 is a serine/threonine kinase that has two isoforms encoded by two different genes, GSK-3α and GSK-3β, in mammals. GSK-3 has several sites of serine and tyrosine phosphorylation. Its activity is negatively regulated by phosphorylation of serine 21 for GSK-3α and serine 9 for GSK-3β, while it is positively regulated by phosphorylation of tyrosine 279 for GSK-3α and tyrosine 216 for GSK-3β. GSK-3 was initially found to be an important component of glycogen metabolism. However, recent studies have revealed that GSK-3 is a multifunctional kinase in various cell types, including neural cells. GSK-3α and GSK-3β are highly expressed in neural tissues such as the cerebral cortex, the hippocampus, the cerebellum, and the spinal cord. In particular, GSK-3β is elevated in the aged hippocampus, and more abundant than GSK-3α in rodents (Salcedo-Tello et al., 2011). Also, GSK-3β is highly expressed in neurons and astrocytes in the developing brain and spinal cord. In neurons, GSK-3 directly leads to the phosphorylation of several neuronal microtubule-associated proteins (MAPs), especially microtubule plus-end tracking proteins (+TIP), including collapsin response mediator protein-2 (CRMP-2), adenomatous polyposis coli (APC), cytoplasmic linker associated protein (CLASP), MAP1B, MAP2, microtubule actin cross-linking factor 1 (MACF1), and Tau (Kim and Snider, 2011). GSK-3 phosphorylation of primed-MAPs generally decreases their activity and thus leads to a decrease in microtubule stability in neurons. Localized inhibition of GSK-3 activity at the axon terminal is required for axon growth during development and regeneration after injury (Alabed et al., 2010). Meanwhile, phosphorylation by GSK-3 activates some unprimed-substrates such as MAP1B, which stabilizes microtubules for axon extension. This is why global inhibition of GSK-3 at a high degree using pharmacological inhibitors or genetic elimination of both isoforms suppresses axon growth (Kim et al., 2006). GSK-3 is also a master regulator of neural stem cell proliferation and differentiation. Loss of both GSK-3 alleles leads to an increase in neural stem cell and progenitor proliferation (Kim et al., 2009). Similarly, pharmacological inhibition of GSK-3 by SB-216763 maintains pluripotency in neural stem cells. Additionally, GSK-3 plays an important role in astrocyte and oligodendrocyte development. The rate of astrocyte apoptosis is increased by overexpression of a constitutively-active GSK-3β mutant in primary cortical astrocytes. Both the number and size of astrocytes are significantly increased in GSK-3 mutant mice when both GSK-3 isoforms are genetically eliminated in astrocyte progenitors and mature astrocytes using a GFAP-cre driver (Jung et al., 2015). Also, pharmacological inhibition of GSK-3 with lithium and indirubin results in increased numbers of oligodendrocyte progenitors and mature oligodendrocytes. Finally, elevated GSK-3 activity is correlated with neuronal death. For example, overexpression of GSK-3β significantly increases neuronal cell death, and pharmacological inhibition of GSK-3 promotes the survival of several types of neural cells. Therefore, GSK-3 is a major factor in many facets of neural cell regulation, such as neurogenesis, neural stem cell proliferation, neural cell death, neuronal differentiation, and gliogenesis (Kim and Snider, 2011).

Akt-independent GSK3 inactivation downstream of PI3K signaling regulates mammalian axon regeneration

Inactivation of glycogen synthase kinase 3 (GSK3) has been shown to mediate axon growth during development and regeneration. Phosphorylation of GSK3 by the kinase Akt is well known to be the major mechanism by which GSK3 is inactivated. However, whether such regulatory mechanism of GSK3 inactivation is used in neurons to control axon growth has not been directly studied. Here by using GSK3 mutant mice, in which GSK3 is insensitive to Akt-mediated inactivation, we show that sensory axons regenerate normally in vitro and in vivo after peripheral axotomy. We also find that GSK3 in sensory neurons of the mutant mice is still inactivated in response to peripheral axotomy and such inactivation is required for sensory axon regeneration. Lastly, we provide evidence that GSK3 activity is negatively regulated by PI3K signaling in the mutant mice upon peripheral axotomy, and the PI3K–GSK3 pathway is functionally required for sensory axon regeneration. Together, these results indicate that in response to peripheral nerve injury GSK3 inactivation, regulated by an alternative mechanism independent of Akt-mediated phosphorylation, controls sensory axon regeneration.

Inositol hexakisphosphate kinase-1 regulates behavioral responses via GSK3 signaling pathways

Glycogen synthase kinase 3 (GSK3), a prominent enzyme in carbohydrate metabolism, also has a major role in brain function. It is physiologically regulated by the kinase Akt, which phosphorylates GSK3 to inhibit catalytic activity. Inositol hexakisphosphate-1 (IP6K1) generates the inositol pyrophosphate diphosphoinositol pentakisphosphate (IP7), which physiologically inhibits Akt leading to enhanced GSK3 activity. We report that IP6K1 binds and stimulates GSK3 enzymatic activity in a non-catalytic fashion. Physiological relevance is evident in the inhibition of GSK3 activity in the brains of IP6K1-deleted mice. Behavioral alterations of IP6K1 knockout mice resemble those of GSK3 mutants. Accordingly, modulation of IP6K1-GSK3β interaction may exert beneficial effects in psychiatric disorders involving GSK3.

O-31: Expression of glycogen synthase kinase-3 mutants in cells and its effect on glycogen synthase activity

O-31: Expression of glycogen synthase kinase-3 mutants in cells and its effect on glycogen synthase activity

Glycogen Synthase Kinase-3 and Cancer: Good Cop, Bad Cop?

Dogma held that inhibition of the pleiotropic protein kinase glycogen synthase kinase-3 (GSK-3) was procarcinogenic due to its natural repression of beta-catenin. Now, Wang et al. have found the reverse in certain leukemias, possibly paving the way for small-molecule GSK-3 inhibitors as selective anticancer agents.

Glucocorticoid-Induced Degradation of Glycogen Synthase Kinase-3 Protein Is Triggered by Serum- and Glucocorticoid-Induced Protein Kinase and Akt Signaling and Controls β-Catenin Dynamics and Tight Junction Formation in Mammary Epithelial Tumor Cells

Glucocorticoid hormones stimulate adherens junction and tight junction formation in Con8 mammary epithelial tumor cells and induce the production of a stable nonphosphorylated beta-catenin protein localized exclusively to the cell periphery. Glycogen synthase kinase-3 (GSK3) phosphorylation of beta-catenin is known to trigger the degradation of this adherens junction protein, suggesting that steroid-activated cascades may be targeting this protein kinase. We now demonstrate that treatment with the synthetic glucocorticoid dexamethasone induces the ubiquitin-26S proteasome-mediated degradation of GSK3 protein with no change in GSK3 transcript levels. In transfected cells, deletion of the N-terminal nine amino acids or mutation of the serine-9 phosphorylation site on GSK3-beta prevented its glucocorticoid-induced degradation. Expression of stabilized GSK3 mutant proteins ablated the glucocorticoid-induced tight junction sealing and resulted in production of a nonphosphorylated beta-catenin that localizes to both the nucleus and the cell periphery in steroid-treated cells. Serine-9 on GSK3 can be phosphorylated by Sgk (serum- and glucocorticoid-induced protein kinase) and by Akt. Expression of dominant-negative forms of either Sgk- or Akt-inhibited glucocorticoid induced GSK3 ubiquitination and degradation and disrupted the dexamethasone-induced effects on beta-catenin dynamics. Furthermore, the steroid-induced tight junction sealing is attenuated in cells expressing dominant-negative forms of either Sgk or Akt, although the effect of blunting Sgk signaling was significantly greater. Taken together, we have uncovered a new cellular cascade in which Sgk and Akt trigger the glucocorticoid-regulated phosphorylation, ubiquitination, and degradation of GSK3, which alters beta-catenin dynamics, leading to the formation of adherens junctions and tight junction sealing.

Regulation of Nanog Expression by Phosphoinositide 3-Kinase-dependent Signaling in Murine Embryonic Stem Cells

Embryonic stem (ES) cell pluripotency is regulated by a combination of extrinsic and intrinsic factors. Previously we have demonstrated that phosphoinositide 3-kinase (PI3K)-dependent signaling is required for efficient self-renewal of murine ES cells. In the study presented here, we have investigated the downstream molecular mechanisms that contribute to the ability of PI3Ks to regulate pluripotency. We show that inhibition of PI3K activity with either pharmacological or genetic tools results in decreased expression of RNA for the homeodomain transcription factor Nanog and decreased Nanog protein levels. Inhibition of glycogen synthase kinase 3 (GSK-3) activity by PI3Ks plays a key role in regulation of Nanog expression, because blockade of GSK-3 activity effectively reversed the effects of PI3K inhibition on Nanog RNA, and protein expression and self-renewal under these circumstances were restored. Furthermore, GSK-3 mutants mimicked the effects of PI3K or GSK-3 inhibition on Nanog expression. Importantly, expression of an inducible form of Nanog prevented the loss of self-renewal observed upon inhibition of PI3Ks, supporting a functional relationship between PI3Ks and Nanog expression. In addition, expression of a number of putative Nanog target genes was sensitive to PI3K inhibition. Thus, the new evidence provided in this study shows that PI3K-dependent regulation of ES cell self-renewal is mediated, at least in part, by the ability of PI3K signaling to maintain Nanog expression. Regulation of GSK-3 activity by PI3Ks appears to play a key role in this process.

Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

BackgroundThe expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3.ResultsWe found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of β-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level.ConclusionBecause we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth.

Glycogen Synthase Kinase 3-Dependent Phosphorylation of Mdm2 Regulates p53 Abundance

The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21(WAF-1). We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.

Regulation of the Interaction between Glycogen Synthase Kinase 3 and the Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein stabilizes beta-catenin by the novel mechanism of binding to the negative regulator, glycogen synthase kinase 3 (GSK-3), and depleting cytoplasmic GSK-3 levels. The two domains of LANA required for interaction with GSK-3 were further characterized. Evidence for similarity between the C-terminal LANA interaction domain and the axin GSK-3 interaction domain was obtained using GSK-3 and LANA mutants. GSK-3(F291L), which does not interact with axin, also failed to bind to LANA, and a mutation in the axin homology domain of LANA, L1132P, destroyed binding to GSK-3. The N-terminal LANA interaction domain was found to mediate interaction by acting as a substrate for GSK-3. GSK-3(R96A), a priming pocket mutant, did not bind to LANA, suggesting that LANA was a primed GSK-3 substrate. Phosphorylation of endogenous LANA precipitated from primary effusion lymphoma cells was inhibited by the GSK-3 inhibitor LiCl. GST-LANA(1-340) was phosphorylated by GSK-3, and mitogen-activated protein kinase (MAPK) and casein kinase I functioned as priming kinases in vitro. Mutation of consensus GSK-3 sites revealed that sites between LANA amino acids 219 and 268 were important for GSK-3 phosphorylation. Immunoprecipitation assays revealed that loss of GSK-3 phosphorylation of this N-terminal domain correlated with loss of GSK-3 interaction. Although LANA-associated GSK-3 actively phosphorylated LANA, GSK-3 coprecipitated with LANA was unable to phosphorylate an exogenous peptide substrate. LANA sequestration of GSK-3 may explain the ability of KSHV-infected cells to tolerate increased levels of nuclear GSK-3.

Expression and Characterization of GSK-3 Mutants and Their Effect on β-Catenin Phosphorylation in Intact Cells

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).

Identification of the Axin and Frat Binding Region of Glycogen Synthase Kinase-3

Glycogen synthase kinase-3 (GSK-3) is a key component of several signaling pathways including those regulated by Wnt and insulin ligands. Specificity in GSK-3 signaling is thought to involve interactions with scaffold proteins that localize GSK-3 regulators and substrates. This report shows that GSK-3 forms a low affinity homodimer that is disrupted by binding to Axin and Frat. Based on the crystal structure of GSK-3, we have used surface-scanning mutagenesis to identify residues that differentially affect GSK-3 interactions. Mutations that disrupt Frat and Axin cluster at the dimer interface explaining their effect on homodimer formation. Loss of the Axin binding site blocks the ability of dominant negative GSK-3 to cause axis duplication in Xenopus embryos. The Axin binding site is conserved within all GSK-3 proteins, and its loss affects both cell motility and gene expression in the nonmetazoan, Dictyostelium. Surprisingly, we find no genetic interaction between a non-Axin-binding GSK-3 mutant and T-cell factor activity, arguing that Axin interactions alone cannot explain the regulation of T-cell factor-mediated gene expression.

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.

Suppression of Glycogen Synthase Kinase Activity Is Not Sufficient for Leukemia Enhancer Factor-1 Activation

Glycogen synthase kinase-3 (GSK) can be regulated by different signaling pathways including those mediated by protein kinase Akt and Wnt proteins. Wnt proteins are believed to activate a transcription factor leukemia enhancer factor-1 (LEF-1) by inhibiting GSK, and Akt was shown to phosphorylate GSK and inhibit its kinase activity. We investigated the effect of an activated Akt on the accumulation of cytosolic beta-catenin and LEF-1-dependent transcription. Although the activated Akt, mAkt, clearly inhibited the kinase activity of GSK, mAkt alone did not induce accumulation of cytosolic beta-catenin or activate LEF-1-dependent transcription. On the contrary, coexpressed Wnt-1 and Frat activated LEF-1 but did not show significant inhibition of GSK-mediated phosphorylation of a peptide substrate. However, mAkt could act synergistically with Wnt-1 or Frat to activate LEF-1. In addition, the interaction of GSK for Axin appeared to decrease in the presence of mAkt, whereas the interaction for Frat remained unchanged. Consistently, a GSK mutant with substitution of a Phe residue for residue Tyr-216, which showed one-fifth of kinase activity of the wild-type GSK, exhibited a reduced association for Axin than the wild-type GSK. These results suggest that inhibition of GSK kinase activity is not sufficient for activation of LEF-1 but may facilitate the activation by reducing the interaction of GSK for Axin. The additional mechanism for LEF-1 activation may require dissociation of GSK from Axin as Frat facilitates the dissociation of GSK from Axin.

Expression and characterization of glycogen synthase kinase-3 mutants and their effect on glycogen synthase activity in intact cells.

In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.

Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos.

Glycogen synthase kinase 3 (GSK-3) is homologous to the product of the Drosophila gene shaggy (zeste-white 3), which is required for signalling by wingless during Drosophila development. To test whether GSK-3 is also involved in vertebrate pattern formation, its role was investigated during early Xenopus development. It was found that dominant-negative GSK-3 mutants induced dorsal differentiation, whereas wild-type GSK-3 induced ventralization. These results indicate that GSK-3 is required for ventral differentiation, and suggest that dorsal differentiation may involve the suppression of GSK-3 activity by a wingless/wnt-related signal.