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-3β-interacting protein from a rat brain cDNA library using a yeast twohybrid 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-3β-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-3β. rAxin also interacted directly with the armadillo repeats of β-catenin. The binding site of rAxin for GSK-3β was distinct from the β-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3β-dependent phosphorylation of β-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3β and β-catenin and mediating the signal from GSK-3β to β-catenin.

The regulators of G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, made a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to ␤-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats bound to the RGS domain of Axin. Axin and ␤-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of ␤-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3␤ (GSK-3␤) and ␤-catenin and that it promotes GSK-3␤-dependent phosphorylation of ␤-catenin, these results suggest that Axin, APC, GSK-3␤, and ␤-catenin make a tetrameric complex, resulting in the regulation of the stabilization of ␤-catenin.Axin, which is a product of the mouse Fused locus, has been identified as a negative regulator of the Wnt signaling pathway (1). Fused is a mutation that causes dominant skeletal and neurological defects and recessive lethal embryonic defects including neuroectodermal abnormalities (2-4). Because dorsal injection of wild type Axin in Xenopus embryos blocks axis formation and coinjection of Axin inhibits Wnt8-, Dsh-, and kinase-negative GSK-3␤ 1 -induced axis duplication (1), Axin could exert its effects on axis formation by inhibiting the Wnt signaling pathway. However, the molecular mechanism by which Axin regulates axis formation has not been shown. We have recently identified rat Axin (rAxin) as a GSK-3␤-interacting protein (5). rAxin is phosphorylated by GSK-3␤, directly binds to not only GSK-3␤ but also ␤-catenin, and promotes GSK-3␤-dependent phosphorylation of ␤-catenin (5). Because the phosphorylation of ␤-catenin by GSK-3␤ is essential for the down-regulation of ␤-catenin (6, 7), our results suggest that rAxin may induce the degradation of ␤-catenin. These actions of rAxin are consistent with the observation that Axin inhibits dorsal axis formation in Xenopus embryos, because the accumulation of ␤-catenin induces the axis duplication (8).It has been shown that besides the phosphorylation by GSK-3␤, the down-regulation of ␤-catenin requires APC, which is a tumor suppressor linked to FAP and to the initiation of sporadic human colorectal cancer (9). The middle portion of APC contains three successive 15-amino acid (aa) repeats followed by seven related but distinct 20-aa repeats. Both types of repeats are able to bind independently to ␤-catenin (10 -12). In FAP and colorectal cancers, most patients carry APC mutations that result in the expression of truncated proteins (9). Almost all mutant proteins lack the C-terminal half including most of the 20-aa repeats but retain the 15-aa repeats. Colorectal carcinoma cells with mutant APC contain large amounts of monom...

The involvement of Ral and its downstream molecules in receptor-mediated endocytosis was examined. Expression of either Ral G23V or Ral S28N , which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of Ral G23V or Ral S28N in CHO-IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by fulllength RalBP1 which is an effector protein of Ral, but was inhibited by its C-terminal region which binds directly to Ral and POB1. POB1 is a binding protein of RalBP1 and has the Eps15 homology (EH) domain. Deletion mutants of POB1 inhibited internalization of EGF and insulin. However, internalization of transferrin was unaffected by Ral, RalBP1, POB1 and their mutants. Epsin and Eps15 have been reported to be involved in the regulation of endocytosis of the receptors for EGF and transferrin. The EH domain of POB1 bound directly to Epsin and Eps15. Taken together with the observation that EGF and insulin activate Ral, these results suggest that Ral, RalBP1 and POB1 transmit the signal from the receptors to Epsin and Eps15, thereby regulating ligand-dependent receptormediated endocytosis.

Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3␤ (GSK-3␤). This protein had 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway.We designated this protein Axil for Axin like. Like Axin, Axil ventralized Xenopus embryos and inhibited Xwnt8-induced Xenopus axis duplication. Axil was phosphorylated by GSK-3␤. Axil bound not only to GSK-3␤ but also to ␤-catenin, and the GSK-3␤-binding site of Axil was distinct from the ␤-catenin-binding site. Furthermore, Axil enhanced GSK-3␤-dependent phosphorylation of ␤-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3␤-dependent phosphorylation of ␤-catenin, thereby inhibiting axis formation.Axin, which is a product of the mouse Fused locus, has been identified as a negative regulator of the Wnt signaling pathway (45). Fused is a mutation that causes dominant skeletal and neurological defects and recessive lethal embryonic defects including neuroectodermal abnormalities (36). Two spontaneous alleles of Fused, called Kinky (Fu Ki ) and Knobbly (Fu Kb ), and a transgenic insertional allele, Fu Tg1 , carry axis duplications and are lethal between 8 and 10 days postcoitus, suggesting that the Fused locus plays a role in the determination of the embryonic axis (9, 14, 33). The cDNA of this locus has been sequenced, and the Fused gene has been renamed Axin. Dorsal injection of wild-type Axin in Xenopus embryos blocks axis formation, and coinjection of Axin inhibits Wnt8-, dishevelled (Dsh)-, and kinase-negative glycogen synthase kinase 3␤ (GSK-3␤)-induced axis duplication (45). These results suggest that Axin exerts its effects on axis formation by inhibiting the signal transduction in the Wnt signaling pathway. However, the molecular mechanism by which Axin regulates axis formation is not known.Wnt and Wg signal many key developmental decisions, regulating anterior-posterior and dorsal-ventral patterns in both vertebrates and flies (22,30,31). In vertebrates, the Wnt signaling pathway consists of an intracellular cascade that includes frizzled, Dsh, GSK-3␤, and ␤-catenin (5). The Wnts are a family of secreted polypeptides, whose receptors are believed to be members of the frizzled family (3). It has been suggested that Dsh acts downstream of frizzled (22,30). GSK-3␤ is a constitutively active protein kinase and antagonizes downstream elements of the Wnt signaling pathway through changes in the ␤-catenin level (10). Wnt inactivates GSK-3␤ activity through Dsh, although by which mechanism is not known (6). In the presence of Wnt, there is a decrease in the phosphorylation of ␤-catenin and an increase in its stability, and ␤-catenin translocates to the nucleus (44). This translocation involves the association of ␤-catenin with the transcriptional enhancers of lymphocyte enhancer binding factor/T cell factor (LEF/ TCF) family (2, 24). ␤-Catenin has a consensus sequence of a phosphorylation site for GSK-3␤, and elimination of this possib...

When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3b (GSK-3b), b-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel ®ltration column chromatography. In this fraction, GSK-3b, b-catenin, and APC were co-precipitated with Axin. Although bcatenin was detected in the high molecular weight fraction in L cells on gel ®ltration column chromatography, addition of conditioned medium expressing Wnt3a to the cells increased b-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of b-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to b-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3b, b-catenin, and APC, resulting in the stimulation of the degradation of bcatenin and that Wnt-3a induces the dissociation of bcatenin from the Axin complex and accumulates bcatenin.