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 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...

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.

the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca2"-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP'yS-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25. smg p25A/rab3A p25 is a member of the rab subfamily of the ras p21-related small GTP-binding protein superfamily (20, 27, 52; for reviews, see references 5, 8, and 50). Accumulating evidence indicates that the rab family is involved in the regulation of intracellular vesicle traffic such as exocytosis and endocytosis (36, 40; for reviews, see references 4, 49, and 50). rab3A p25 is abundant in the presynapse and specifically enriched in the synaptic plasma membrane and the synaptic vesicle near the active zone (6, 11, 13, 14, 28, 30-34, 44, 45). Available evidence suggests that rab3A p25 is implicated in neurotransmitter release from the presynapse. rab3A p25 has GDP-bound inactive and GTP-bound active forms which are interconvertible by GDP/GTP exchange and GTPase reactions. The GDP/GTP exchange reaction is regulated by GDP/GTP exchange proteins. We have previously purified an inhibitory GDP/GTP exchange protein for rab3A p25 named smg p25 GDI (26,47). smg p25 GDI has another action, i.e., to regulate the translocation of rab3A p25 between the cytoplasm and the membrane (3). Moreover, we have shown that smg p25 GDI is also active on rabll p24, a member of the rab family, and SEC4 p24, a yeast member of the rab family (46, 54). In collaboration with our group, Zerial's group has recently found that smg p25 GDI is active on almost all of the rab family members, including the rablA, -2, -3A, - 4B, -5, -7, -8, -9, -10, and -11 proteins (55). Therefore, we have renamed smg p25 GDI as rab GDI.On the basis of the available evidence, we have proposed a possible mode of action of rab3A p25: the GDP-bound form of rab3A p25 complexed with rab GDI stays in the synaptic cytoplasm. Once the GDP-bound form of rab3A * Corresponding author. p25 dissociates from rab GDI, the GDP-bound form of rab3A p25 is converted to the GTP-bound form. The GTPbound form of rab3A p25 binds to the target protein on the synaptic vesicle. The synaptic vesicle containing the GTPbound form of rab3A p25 is translocated to the synaptic plasma membrane. Targeting of the synaptic ve...

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.

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The Wnt signaling pathway is conserved in various species from worms to mammals, and plays important roles in cellular proliferation, differentiation, and migration. Wnt stabilizes cytoplasmic beta-catenin and then the accumulated beta-catenin is translocated into the nucleus, where it activates the transcriptional factor T-cell factor (Tcf)/lymphoid enhancer factor (Lef), and thereby stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Tight regulation of this response involves post-translational modifications of the components of the Wnt signaling pathway. Phosphorylation, ubiquitination, and sumoylation have been shown to affect the half-life of beta-catenin and the transcriptional activity of Tcf/Lef. The precise spatio-temporal patterns of these multiple modifications determine the driving force of various cellular responses.