Shosei Kishida

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

Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization 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 β-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 β-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of β-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3β (GSK-3β) and β-catenin and that it promotes GSK-3β-dependent phosphorylation of β-catenin, these results suggest that Axin, APC, GSK-3β, and β-catenin make a tetrameric complex, resulting in the regulation of the stabilization of β-catenin.

PHOSPHORYLATION OF AXIN, A WNT SIGNAL NEGATIVE REGULATOR, BY GLYCOGEN SYNTHASE KINASE-3BETA REGULATES ITS STABILITY

Axin forms a complex with glycogen synthase kinase-3β (GSK-3β) and β-catenin and promotes GSK-3β-dependent phosphorylation of β-catenin, thereby stimulating the degradation of β-catenin. Because GSK-3β also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3β 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-3β were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3β, inhibited the phosphorylation of Axin by GSK-3β in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated β-catenin in L cells and expression of Dvl-1ΔPDZ, 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.

Small G protein Ral and its downstream molecules regulate endocytosis of EGF and insulin receptors.

The involvement of Ral and its downstream molecules in receptor‐mediated endocytosis was examined. Expression of either RalG23V or RalS28N, which are known to be constitutively active and dominantnegative forms, respectively, in A431 cells blocked internalization of epidermal growth factor (EGF). Stable expression of RalG23V or RalS28N in CHO‐IR cells also inhibited internalization of insulin. Internalization of EGF and insulin was not affected by full‐length 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 receptor‐mediated endocytosis.

Axil, a Member of the Axin Family, Interacts with Both Glycogen Synthase Kinase 3β and β-Catenin and Inhibits Axis Formation of Xenopus Embryos

ABSTRACT Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3β (GSK-3β). 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-3β. Axil bound not only to GSK-3β but also to β-catenin, and the GSK-3β-binding site of Axil was distinct from the β-catenin-binding site. Furthermore, Axil enhanced GSK-3β-dependent phosphorylation of β-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3β-dependent phosphorylation of β-catenin, thereby inhibiting axis formation.

Regulation of Wnt signaling by protein-protein interaction and post-translational modifications.

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 β-catenin and then the accumulated β-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 β-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.

Identification and characterization of a novel protein interacting with Ral-binding protein 1, a putative effector protein of Ral.

Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and exhibits a GTPase activating activity for Rac and CDC42. To clarify the function of RalBP1, we isolated a novel protein that interacts with RalBP1 by yeast two-hybrid screening and designated it POB1 (partner of RalBP1). POB1 consists of 521 amino acids, shares a homology with Eps15, which has been identified as an epidermal growth factor (EGF) receptor substrate, and has two proline-rich motifs. The POB1 mRNA was expressed in cerebrum, cerebellum, lung, kidney, and testis. POB1 interacted with RalBP1 in COS cells and the C-terminal region of POB1 was responsible for this interaction. The binding domain of RalBP1 to POB1 was distinct from its binding domain to Ral. Ral and POB1 simultaneously interacted with RalBP1 in COS cells. The binding of POB1 to RalBP1 did not affect the GTPase activating activity of RalBP1. Furthermore, POB1 bound to Grb2 but not to Nck or Crk. POB1 was tyrosine-phosphorylated in COS cells upon stimulation with EGF and made a complex with EGF receptor. These results suggest that RalBP1 makes a complex with POB1 and that this complex may provide a link between tyrosine kinase, Src homology 3 (SH3)-containing protein, and Ral.

Wnt-3a and Dvl induce neurite retraction by activating Rho-associated kinase.

ABSTRACT Dvl is a key protein that transmits the Wnt signal to the canonical β-catenin pathway and the noncanonical planar cell polarity (PCP) pathway. We studied the roles of Rho-associated kinase (Rho-kinase), which is activated by Dvl in the PCP pathway of mammalian cells. The expression of Dvl-1, Wnt-1, or Wnt-3a activated Rho-kinase in COS cells, and this activation was inhibited by the Rho-binding domain of Rho-kinase. The expression of Dvl-1 in PC12 cells activated Rho and inhibited nerve growth factor (NGF)-induced neurite outgrowth. This inhibition was reversed by a Rho-kinase inhibitor but not by a c-Jun N-terminal kinase inhibitor. Dvl-1 also inhibited serum starvation-dependent neurite outgrowth of N1E-115 cells, and expression of the Rho-binding domain of Rho-kinase reversed this inhibitory activity of Dvl-1. Dvl-1 mutants that did not activate Rho-kinase did not inhibit the neurite outgrowth of N1E-115 cells. Furthermore, the purified Wnt-3a protein activated Rho-kinase and inhibited the NGF-dependent neurite outgrowth of PC12 cells. Wnt-3a-dependent neurite retraction was also prevented by a Rho-kinase inhibitor and a Dvl-1 mutant that suppresses Wnt-3a-dependent activation of Rho-kinase. These results suggest that Wnt-3a and Dvl regulate neurite formation through Rho-kinase and that PC12 and N1E-115 cells are useful for analyzing the PCP pathway.

Complex Formation of Adenomatous Polyposis Coli Gene Product and Axin Facilitates Glycogen Synthase Kinase-3β-dependent Phosphorylation of β-Catenin and Down-regulates β-Catenin

Adenomatous polyposis coli gene product (APC) functions as a tumor suppressor and its mutations in familial adenomatous polyposis and colorectal cancers lead to the accumulation of cytoplasmic β-catenin. The molecular mechanism by which APC regulates the stability of β-catenin was investigated. The central region of APC, APC-(1211–2075), has the β-catenin- and Axin-binding sites and down-regulates β-catenin. Glycogen synthase kinase-3β (GSK-3β) phosphorylated β-catenin slightly in the presence of either APC-(1211–2075) or AxinΔ β -catenin, in which the β-catenin-binding site is deleted, and greatly in the presence of both proteins. The enhancement of the GSK-3β-dependent phosphorylation of β-catenin was eliminated by the APC-binding site of Axin. Axin down-regulated β-catenin in SW480 cells, but not AxinΔ β -catenin. In L cells where APC is intact, AxinΔ β -catenin inhibited Wnt-dependent accumulation of β-catenin but not Axin-(298–832)Δ β -catenin in which the APC- and β-catenin-binding sites are deleted. These results indicate that the complex formation of APC and Axin enhances the phosphorylation of β-catenin by GSK-3β, leading to the down-regulation of β-catenin.

Axin prevents Wnt-3a-induced accumulation of β-catenin

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