Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.

Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). Here, we examine the role of different Axin domains in the effects on axis formation and beta-catenin levels. We find that 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 were required for targeting to either of these sites, whereas COOH-terminal sequences increased localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) revealed strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity.     

Structural basis of the Axin-adenomatous polyposis coli interaction

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.     

Regulation of Glycogen Synthase Kinase 3β and Downstream Wnt Signaling by Axin

Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3beta (GSK-3beta) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3beta in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3beta in vivo, suggesting that these domains are critical for proper regulation of GSK-3beta activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3beta activity. Based on these data, we propose that the axin complex may directly regulate GSK-3beta enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3beta can mimic the effect of lithium in developing Xenopus embryos.     

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.     

Differential Role of Axin RGS Domain Function in Wnt Signaling during Anteroposterior Patterning and Maternal Axis Formation

Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin.

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. We have isolated here a GSK-3beta-interacting protein 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, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted 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.     

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.     

Dorsal downregulation of GSK3beta by a non-Wnt-like mechanism is an early molecular consequence of cortical rotation in early Xenopus embryos

Cortical rotation and concomitant dorsal translocation of cytoplasmic determinants are the earliest events known to be necessary for dorsoventral patterning in Xenopus embryos. The earliest known molecular target is beta-catenin, which is essential for dorsal development and becomes dorsally enriched shortly after cortical rotation. In mammalian cells cytoplasmic accumulation of beta-catenin follows reduction of the specific activity of glycogen synthase kinase 3-beta (GSK3beta). In Xenopus embryos, exogenous GSK3beta) suppresses dorsal development as predicted and GSK3beta dominant negative (kinase dead) mutants cause ectopic axis formation. However, endogenous GSK3beta regulation is poorly characterized. Here we demonstrate two modes of GSK3beta regulation in Xenopus. Endogenous mechanisms cause depletion of GSK3beta protein on the dorsal side of the embryo. The timing, location and magnitude of the depletion correspond to those of endogenous beta-catenin accumulation. UV and D(2)O treatments that abolish and enhance dorsal character of the embryo, respectively, correspondingly abolish and enhance GSK3beta depletion. A candidate regulator of GSK3beta, GSK3-binding protein (GBP), known to be essential for axis formation, also induces depletion of GSK3beta. Depletion of GSK3beta is a previously undescribed mode of regulation of this signal transducer. The other mode of regulation is observed in response to Wnt and dishevelled expression. Neither Wnt nor dishevelled causes depletion but instead they reduce GSK3beta-specific activity. Thus, Wnt/Dsh and GBP appear to effect two biochemically distinct modes of GSK3beta regulation.     

A novel beta-catenin-binding protein inhibits beta-catenin-dependent Tcf activation and axis formation

beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.     

Inhibition of Wnt signaling pathway by a novel axin-binding protein

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.     

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.     

Inhibition of the Wnt signaling pathway by Idax, a novel Dvl-binding protein

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as Dvl in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta-catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta-catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.     

Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-catenin by the proteasome. Siah-1, the human homolog of Drosophila seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.     

GSKIP is homologous to the Axin GSK3beta interaction domain and functions as a negative regulator of GSK3beta

Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.