Interactive Fly, Drosophila

GSK-3, APC and Axin

The adenomatous polyposis coli (APC: see Drosophila APC-like) protein binds to the cellular adhesion molecule ß-catenin, a mammalian homolog of Armadillo. Overexpression of APC blocks cell cycle progression. When ß-catenin is present in excess, APC binds to another component of the Wingless pathway, glycogen synthase kinase 3ß, a mammalian homolog of Shaggy/Zeste white 3. APC is a good substrate for GSK3ß in vitro, and the phosphorylation sites map to the central region of APC. Binding of ß-catenin to this region is dependent on phosphorylation of GSK3ß (Rubinfeld, 1996). APC also binds to DLG, the human homolog of the Drosophila Discs Large tumor suppressor protein. This interaction requires the C-terminal region of APC and the PDZ domain repeat region of DLG. APC colocalizes with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function (Matsumine, 1996). Perhaps the PDZ domain of Dishevelled (involved directly in Wingless signaling in Drosophila) interacts with APC and thereby provides a link between DSH and the APC-GSK3-ß-catenin complex (Perrimon, 1996).

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. Mouse Fused is not to be confused with Drosophila fused, which mediates the Hedgehog signal to activate decapentaplegic. The product of the Fused locus, Axin (see Drosophila Axin), displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Axin blocks the stimulation of the Wnt signaling pathway, regulating an early step in axis formation downstream of GSK-3, the mammalian homolog of Drosophila Shaggy. The Axin sequence homologous to the RGS domain is found between amino acids 213 and 338. A C-terminal 51 amino acid segment is 40% identical to a conserved sequence near the N terminus of Drosophila Dsh and its vertebrate homologs. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Based on its ability to block ectopic axis formation in Xenopus embryos by Xwnt8, Dishevelled, or dominant negative GSK-3, Axin appears to negatively regulate signaling through the Wnt pathway, either at the level of GSK-3 or further downstream. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in both mammals and amphibians (Zeng, 1997).

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. A GSK-3 beta-interacting protein has been isolated from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and Dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, this protein has been called rAxin (rat Axin). rAxin interacts directly with, and is phosphorylated by, GSK-3beta. rAxin also interacts directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta is distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promotes GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin (Ikeda, 1998).

Using a yeast two-hybrid method, a novel protein has been identfied that interacts with glycogen synthase kinase 3beta (GSK-3beta) and has 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway. This protein has been termed Axil, for Axin like. Like Axin, Axil ventralizes Xenopus embryos and inhibits Xwnt8-induced Xenopus axis duplication. Axil is phosphorylated by GSK-3beta. Axil binds not only to GSK-3beta but also to beta-catenin; the GSK-3beta-binding site of Axil is distinct from the beta-catenin-binding site. Axil enhances GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating the GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation (Yamamoto, 1998).

Axin antagonizes the developmental effects of Wnt in vertebrates. Axin simultaneously binds two components of the Wnt pathway: beta-catenin and its negative regulator, glycogen synthase kinase-3beta. In mammalian cells, Axin inhibits Wnt-1 stimulation of beta-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks beta-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin, by forming a complex with beta-catenin and glycogen synthase kinase-3beta, can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations (Sakanaka, 1998).

Control of stability of beta-catenin is central in the Wnt signaling pathway. The protein Conductin is found to form a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induces beta-catenin degradation, although conductin mutants are deficient in complex formation stabilize beta-catenin. Fragments of APC that contain a conductin-binding domain also block beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfers with wnt-induced axis formation (Behrens, 1998).

The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. The effects of exogenous APC expression were examined on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC causes a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction is due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lack this activity. Mutational analysis reveals that the central region of the APC protein, which is typically deleted or severely truncated in tumors, is responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin (Munemitsu, 1995).

Regulation of cell adhesion and cell signaling by beta-catenin occurs through a mechanism likely involving the targeted degradation of the protein. Deletional analysis was used to generate a beta-catenin refractory to rapid turnover These deletants were used to examine beta-catenin's effects on complexes containing either cadherin or the adenomatous polyposis coli (APC) protein. The amino-terminal deletion of beta-catenin results in a protein with increased stability, which acts in a dominant fashion with respect to wild-type beta-catenin. Constitutive expression in AtT20 cells of a beta-catenin lacking 89 N-terminal amino acids (deltaN89beta-catenin) results in severely reduced levels of the more labile wild-type beta-catenin. The mutant beta-catenin is expressed at endogenous levels but displaces the vast majority of wild-type beta-catenin associated with N-cadherin. The deltaN89beta-catenin accumulates on the APC protein to a level 10-times that of wild-type beta-catenin; it also recruits a kinase into the APC complex. The kinase is highly active toward APC in vitro and promotes a sodium dodecyl sulfate gel band shift that is also evident for endogenous APC from cells expressing the mutant beta-catenin. Unlike wild-type beta-catenin, which partitions solely as part of a high-molecular-weight complex, the deltaN89 mutant protein also fractionates as a stable monomer, indicating that it has escaped the requirement to associate with other proteins. That similar N-terminal mutants of beta-catenin have been implicated in cellular transformation suggests that their abnormal association with APC may, in part, be responsible for this phenotype (Munemitsu, 1996).

APC is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of Armadillo, a component of the Wingless signaling pathway in Drosophila development. When beta-catenin is present in excess, APC binds to another component of the Wingless pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila Shaggy/Zeste white 3. APC is a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region is dependent on phosphorylation by GSK3 beta (Rubinfeld, 1996).

The mutation cluster region in the APC gene defines a region of approximately 660 bp, in which the vast majority of its somatic mutations are found. These mutations disrupt the polypeptide chain, typically eliminating five of the seven repeated sequences of 20 amino acids (aa), each in the central region of the APC protein. To examine the relationship between loss of this structure and loss of function, APC deletion mutants were constructed that progressively truncate the protein across the mutation cluster region. The mutants were tested for their association with beta-catenin and their ability to down-regulate it in SW480 cells. The binding of beta-catenin to APC fragments requires the inclusion of only a single 20-aa repeat sequence, whereas down-regulation requires the presence of at least three of these repeat sequences; those including the second repeat exhibit the highest activity. The mutation of three conserved serine residues in the second repeat greatly reduce the activity of an otherwise highly active APC fragment. Thus, the repeated 20-aa sequence is directly implicated in beta-catenin turnover. The elimination of at least five of these seven repeats due to somatic mutations suggests that loss of beta-catenin regulation by APC is selected for during tumor progression (Rubinfeld, 1997).

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. The results are reported of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. These results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. A region of Axin was identified that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, it was demonstrated that the C-terminal 100 amino acids of Axin can bind to this same region (Hsu, 1999).

Axin has been identified as a regulator of vertebrate embryonic axis induction that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicate that Axin functions downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin; subsequent studies have shown that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). The roles of different Axin domains were examined in the effects on axis formation and beta-catenin levels. The regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences are required for targeting to either of these sites, whereas COOH-terminal sequences increase localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) reveals strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity (Fagotto, 1999).

The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bind one another. Furthermore, Dvl-1 forms a homo-oligomer. Axin also forms a homo-oligomer, and its DIX domain is necessary. The N-terminal region of Dvl-1, including its DIX domain, bind to Axin directly. Dvl-1 inhibits Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 is required for this inhibitory activity. Expression of Dvl-1 in L cells induces the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolishes this activity. Although expression of Axin in SW480 cells causes the degradation of beta-catenin and reduces the cell growth rate, expression of an Axin mutant that lacks the DIX domain does not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin (Kishida, 1999).

Axin promotes the phosphorylation of beta-catenin by GSK-3beta, leading to beta-catenin degradation. Wnt signals interfere with beta-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of beta-catenin complexes containing TCF transcription factors. Little is known about how GSK-3beta-mediated beta-catenin turnover is regulated in response to Wnt signals. An exploration was carried out of the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that functions upstream of GSK-3beta. Expression of Dvl-2 activates TCF-dependent transcription. This is blocked by co-expression of GSK-3beta or Axin. Expression of a 59 amino acid GSK-3beta-binding region from Axin strongly activates transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevents GSK-3beta binding also generates a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localize within expressing cells. When Dvl-2 localization is altered using a C-terminal CAAX motif, Axin is also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggests that Dvl-association determinants within Axin are contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3beta (Smalley, 1999).

Wnt proteins transduce their signals through Dishevelled (Dvl) proteins to inhibit glycogen synthase kinase 3beta (GSK), leading to the accumulation of cytosolic beta-catenin and activation of TCF/LEF-1 transcription factors. To understand the mechanism by which Dvl acts through GSK to regulate LEF-1, the roles of Axin and Frat1 (a novel proto-oncogene) in Wnt-mediated activation of LEF-1 were examined in mammalian cells. Dvl interacts with Axin and with Frat1, both of which interact with GSK. Similarly, the Frat1 homolog GBP binds Xenopus Dishevelled in an interaction that requires GSK. Dvl, Axin and GSK can form a ternary complex bridged by Axin, and Frat1 can be recruited into this complex, probably by Dvl. The observation that the Dvl-binding domain of either Frat1 or Axin is able to inhibit Wnt-1-induced LEF-1 activation suggests that the interactions between Dvl and Axin and between Dvl and Frat may be important for this signaling pathway. Furthermore, Wnt-1 appeared to promote the disintegration of the Frat1-Dvl-GSK-Axin complex, resulting in the dissociation of GSK from Axin. Thus, formation of the quaternary complex may be an important step in Wnt signaling, by which Dvl recruits Frat1, leading to Frat1-mediated dissociation of GSK from Axin (Li, 1999).

Proviral tagging in tumor-prone transgenic mice has been used to identify collaborating oncogenes and genes contributing to tumor progression. This approach has yielded a series of oncogenes that could be assigned to different complementation groups in transformation: the myc, Pim, Bmi1, and Frat1 complementation groups. Frat1 is involved in tumor progression and appears to function in the Wnt signaling pathway. Overexpression of Fratl confers a growth advantage to transplanted tumor cells in vivo and to cells grown in vitro at high density. Frat1 might exert its activity by impairing the kinase activity of Gsk3beta, which is involved in the degradation of beta-catenin (Berns, 1999).

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it has not been clear whether and how Wnt signaling regulates the beta-catenin complex. Axin has been shown to be dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery (Willert, 1999).

A model is presented for the role of Axin in Wnt signal transduction. In an unstimulated cell, GSK-3beta is active and phosphorylates Axin, which in turn, recruits beta-catenin into the Axin/GSK-3beta complex. By virtue of its proximity to GSK-3beta, beta-catenin is then phosphorylated. Phosphorylated beta-catenin is then targeted for degradation. Upon transduction of the Wnt signal through the Frizzled (Fz) receptors to Dishevelled, GSK-3beta kinase activity is inhibited so that PP2A dephosphorylates Axin. Unphosphorylated Axin, in turn, no longer recruits beta-catenin to the complex. Failure of beta-catenin to associate with the Axin/GSK-3beta complex prevents its phosphorylation by GSK-3beta so that it can accumulate to high levels in the cytoplasm and nucleus and activate transcription in concert with the Tcf/Lef-1 family of transcription factors. GSK-3beta also phosphorylates APC, which may facilitate beta-catenin recruitment into the complex; however, this event has not been shown to be regulated by Wnt signaling (Willert, 1999).

An implication of these results is that the primary target for GSK-3beta phosphorylation is Axin. Previous models have argued that GSK-3beta directly phosphorylates beta-catenin and thereby targets it for degradation. However, GSK-3beta does not bind directly to beta-catenin, and efficient in vitro phosphorylation of beta-catenin by GSK-3beta requires the presence of Axin, which binds both proteins. In contrast, efficient Axin phosphorylation by GSK-3beta does not require additional proteins. Thus, in a Wnt-stimulated cell, beta-catenin fails to be phosphorylated by GSK-3beta because it is not recruited into the Axin/GSK-3beta complex. It should also be noted that phosphorylation of APC by GSK-3beta increases beta-catenin binding to APC; however, in contrast to Axin, phosphorylation of APC has not been shown to be regulated by Wnt signaling (Willert, 1999 and references).

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, GSK-3 binding protein (GBP or FRAT1 ), which is required for axis formation in Xenopus, binds and inhibits GSK-3. It has been shown that GBP inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, a dominant-negative GSK-3 mu- tant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating GSK-3's catalytic activity. 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 a 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 (Farr, 2000).

The coordinated regulation of GSK-3 activity by both positive and negative regulators is critical for a wide range of downstream effects, from insulin regulation to developmental processes. The experiments presented here advance the understanding of GSK-3 regulation and suggest the following model for Xgsk-3 regulation in the fertilized Xenopus egg. On the ventral side, beta-catenin levels are kept low by Xgsk-3-dependent phosphorylation in a complex including Axin and APC. Phosphorylation of Axin and APC by Xgsk-3 may be required to assemble this complex. On the dorsal side, GBP inhibits phosphorylation of beta-catenin either by displacing Xgsk-3 from the Axin/APC/Xgsk-3 complex or by prebinding GSK-3 and preventing its association with the Axin-APC complex. GBP binding to Xgsk-3 may block access of protein substrates to the active site. GBP might also prevent Xgsk-3 from phosphorylating Axin and APC, and thereby further inhibit complex formation. The dominant-negative Xgsk-3 acts in a manner analogous to GBP since it either displaces wild-type Xgsk-3 from the complex, or binds endogenous Xgsk-3 and prevents its binding to Axin, thus, inhibiting beta-catenin degradation. The complete chain of molecular events linking fertilization and the dorsal enrichment of beta-catenin has yet to be determined, but this study brings closer an understanding of the interactions of key players. It will ultimately be very interesting to elucidate the relative dorso-ventral abundance of each of these proteins in the Xenopus oocyte and fertilized embryo. However, it may not be just the relative abundance of each of these players that specifies the endogenous axis. Alternatively, posttranslational modifications and/or the presence of yet unidentified factors could affect dorso-ventral differences in known players. GBP is a candidate for the dorsal determinant, and the reagents to examine the possible dorsal localization or dorsal modification of GBP are being developed. Alternatively, GBP may be ubiquitous, but be activated or recruited to function by another factor that becomes dorsally localized in response to sperm entry. The determinant might, for example, bind to the Axin/APC/Xgsk-3 complex and alter its conformation in such a way as to allow GBP to bind Xgsk-3 and remove it from the complex. In this regard, the observation that Dishevelled interacts with GBP and Axin and is dorsally enriched in the early Xenopus embryo is very suggestive of such a mechanism. Alternatively, GBP might function together with Dishevelled to prevent the initial formation of complexes between Axin and GSK-3 in the dorsal cortical cytoplasm, rather than functioning to disrupt complexes once they have formed (Farr, 2000).

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating ß-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization. The microtubule binding domain is deleted in the transforming mutations of APC; however, the direct effect of APC protein on microtubules has never been examined. Binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3ß. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation (Zumbrunn, 2001).

Zebrafish embryos homozygous for the masterblind (mbl) mutation exhibit a striking phenotype in which the eyes and telencephalon are reduced or absent and diencephalic fates expand to the front of the brain. mbl minus embryos carry an amino-acid change at a conserved site in the Wnt pathway scaffolding protein, Axin1. The amino-acid substitution present in the mbl allele abolishes the binding of Axin to Gsk3 and affects Tcf-dependent transcription. Therefore, Gsk3 activity may be decreased in mbl minus embryos and in support of this possibility, overexpression of either wild-type Axin1 or Gsk3ß can restore eye and telencephalic fates to mbl minus embryos. These data reveal a crucial role for Axin1-dependent inhibition of the Wnt pathway in the early regional subdivision of the anterior neural plate into telencephalic, diencephalic, and eye-forming territories (Heisenberg, 2001).

This study reveals a novel role for Axin1 in the regional subdivision of prospective forebrain territories. It is proposed that Wnt signaling must be suppressed to allow the development of telencephalic and optic fates and that if this fails to occur, prospective forebrain cells adopt a more caudal, diencephalic identity. It is intriguing that headless/tcf3 mutant zebrafish embryos, in which the canonical Wnt pathway is also likely to be overactivated, show expansion of midbrain (or midbrain/hindbrain boundary) fates at the expense of forebrain fates. Similarly, graded overexpression of Wnt8 can lead to expansion of midbrain-specific gene expression into prospective forebrain territories. These observations raise the possibility that thresholds of Wnt activity may specify different posterior to anterior fates within the neural plate in a manner analogous to that proposed for graded Bmp activity in the allocation of fates along the dorsoventral axis of the neural plate (Heisenberg, 2001).

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. A tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of GSK-3ß. These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). These results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway (F.-Q. Zhou, 2004).

Crystal structure of Glycogen synthase kinase 3ß

Glycogen synthase kinase 3ß (GSK3ß) plays a key role in insulin and Wnt signaling, phosphorylating downstream targets by default, and becoming inhibited following the extracellular signaling event. The crystal structure of human GSK3ß shows a catalytically active conformation in the absence of activation-segment phosphorylation, with the sulphonate of a buffer molecule bridging the activation-segment and N-terminal domain in the same way as does the phosphate group of the activation-segment phospho-Ser/Thr in other kinases. The location of this oxyanion binding site in the substrate binding cleft indicates direct coupling of P+4 phosphate-primed substrate binding and catalytic activation, explains the ability of GSK3ß to processively hyperphosphorylate substrates with Ser/Thr pentad-repeats, and suggests a mechanism for autoinhibition in which the phosphorylated N terminus binds as a competitive pseudosubstrate with phospho-Ser 9 occupying the P+4 site (Dajani, 2001).

Unlike many protein kinases involved in signal transduction, GSK3ß is active in the absence of the external signal and becomes inhibited in response to signals from the membrane-bound receptor. In insulin signaling, GSK3ß phosphorylates (and thereby inhibits) glycogen synthase (GS), the translation initiation factor eIF2B, and the C/EBPalpha transcription factor. Insulin binding to the extracellular domains of the receptor activates a phosphorylation cascade, resulting in inhibitory phosphorylation of GSK3ß by PKB/Akt with consequent hypophosphorylation and activation of the downstream targets. The regulation of GSK3 by insulin has been shown to be mediated by protein kinase B (PKB). Upon insulin stimulation, threonine 308 (Thr-308) and serine 473 (Ser-473) residues of PKB are phosphorylated and PKB is activated. Subsequently, both GSK3 isotypes (GSK3alpha and GSK3ß) in mammalian cells are phosphorylated on a serine residue at the N terminus (serine 21 of GSK3alpha and serine 9 of GSK3ß), which leads to a decrease in GSK3 activity. Although this has usually been detected as a 50%-70% drop, it is apparently sufficient to relieve the inhibition of GS and allow cells to complete glycogen synthesis. In Wnt signaling, GSK3ß phosphorylates ß-catenin, promoting its ubiquitination and subsequent destruction so that ß-catenin levels are minimized. Binding of Wnt glycopeptide to the Frizzled receptor inhibits GSK3ß via Dishevelled, allowing increased ß-catenin levels with consequent up-regulation of proliferative genes such as c-Myc, which are regulated by ß-catenin-dependent Tcf/Lef transcription factors (Dajani, 2001 and references therein).

Despite their common GSK3ß component, the Wnt and insulin signaling pathways are insulated from each other by incorporation of Wnt-signaling GSK3ß into a multiprotein complex involving Axin/Conductin, and the adenomatous polyposis coli-associated protein (APC). The Axin-APC complex also binds ß-catenin, which is efficiently phosphorylated by GSK3ß in the presence of the Axin-APC scaffold, but not in its absence. The Axin-APC complex may serve a dual role of enhancing access of GSK3ß to ß-catenin, while restricting its access to non-Wnt substrates. The requirement of an active Axin-APC-GSK3ß complex for phosphorylation and consequent degradation of ß-catenin confers a tumor suppression role on this complex. Defects in APC that prevent the formation of the complex are associated with the most common form of hereditary colorectal cancer, and somatic loss of functional APC is a frequent occurrence in spontaneous colorectal tumors (Dajani, 2001 and references therein).

Phosphorylation of glycogen synthase (GS) by GSK3ß is unusual in requiring a prior phosphorylation by another protein kinase. Thus, GSK3ß only phosphorylates GS once Ser 656 of GS has been phosphorylated by casein kinase II (CK2). With GS thus 'primed,' GSK3ß will sequentially phosphorylate Ser 652, Ser 648, Ser 644, and Ser 640 in a carboxy-terminal to amino-terminal direction, with each subsequent phosphorylation dependent on prior phosphorylation of the P+4 position. A similar process of sequentially primed phosphorylation is suggested by the periodicity of the phosphorylation sites (Ser 45, Thr 41, Ser 37, Ser 33, and Ser 29) in the main Wnt target ß-catenin. The kinase providing the priming phosphorylation at Ser 45 is unknown. Efficient phosphorylation of Wnt targets is different from phosphorylation of insulin targets in requiring formation of a ß-catenin-APC-Axin-GSK3ß complex, and is sensitive to inhibition by GBP/FRAT proteins and fragments thereof. A requirement for priming phosphorylation of substrates has also been reported for casein kinase I (CK1), but here the priming position is located three residues to the N-terminal side of the target Ser/Thr. The crystal structure of human GSK3ß, expressed in insect cells, at 2.8 Å, has been determined. The structure is devoid of activation segment phosphorylation, but is nonetheless in an activated conformation stabilized by the sulphonate group of a buffer molecule bound in the substrate binding cleft. Analysis of the structure and comparison with other protein kinase structures suggests a mechanism of catalytic activation coupled to binding of phosphorylated substrates, serendipitously mimicked by the bound buffer. This explains the requirement of GSK3ß for phosphorylation 'priming' of substrates and its ability to processively hyperphosphorylate substrates such as glycogen synthase and ß-catenin, and suggests a mechanism for phosphorylation-dependent autoinhibition of GSK3ß following phosphorylation by PKB/Akt (Dajani, 2001).

Table of contents

shaggy: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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