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.

Wnt-5a is a representative ligand that activates a B-cateninindependent pathway in the Wnt signaling. Although abnormal activation of B-catenin-dependent pathway is often observed in human cancer, the relationship between Bcatenin-independent pathway and tumorigenesis is not clear. We sought to clarify how Wnt-5a is involved in aggressiveness of gastric cancer. Abnormal expression of Wnt-5a was observed in 71 of 237 gastric cancer cases by means of immunohistochemistry. The positivity of Wnt-5a expression was correlated with advanced stages and poor prognosis of gastric cancer. Wnt-5a had the abilities to stimulate cell migration and invasion in gastric cancer cells. Wnt-5a activated focal adhesion kinase and small GTP-binding protein Rac, both of which are known to play a role in cell migration. Cell migration, membrane ruffling, and turnover of paxillin were suppressed in Wnt-5a knockdown cells. Furthermore, anti-Wnt-5a antibody suppressed gastric cancer cell migration. These results suggest that Wnt-5a stimulates cell migration by regulating focal adhesion complexes and that Wnt-5a is not only a prognostic factor but also a good therapeutic target for gastric cancer.

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 bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did 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.

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...

beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the b-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrindependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and b-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of b-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the b-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalizationdependent and -independent mechanisms.

Axin forms a complex with glycogen synthase kinase-3␤ (GSK-3␤) and ␤-catenin and promotes GSK-3␤-dependent phosphorylation of ␤-catenin, thereby stimulating the degradation of ␤-catenin. Because GSK-3␤ also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3␤ inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3␤ were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3␤, inhibited the phosphorylation of Axin by GSK-3␤ in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated ␤-catenin in L cells and expression of Dvl-1 ⌬PDZ , in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, we found that Wnt3a acts in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively.