Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3beta and beta-catenin and inhibits axis formation of Xenopus embryos.
Journal: 1998/May - Molecular and Cellular Biology
ISSN: 0270-7306
PUBMED: 9566905
Abstract:
Using a yeast two-hybrid method, we identified a novel protein which interacts with glycogen synthase kinase 3beta (GSK-3beta). 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-3beta. Axil bound not only to GSK-3beta but also to beta-catenin, and the GSK-3beta-binding site of Axil was distinct from the beta-catenin-binding site. Furthermore, Axil enhanced GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation.
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Mol Cell Biol 18(5): 2867-2875

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

Department of Biochemistry, Hiroshima University School of Medicine, Minami-ku, Hiroshima 734-8551, and Department of Life Science (Biology), University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan
Corresponding author. Mailing address: Department of Biochemistry, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: 81-82-257-5130. Fax: 81-82-257-5134. E-mail: pj.ca.u-amihsorih.dem.iacm@ihcukika.
Received 1997 Nov 17; Revisions requested 1997 Dec 19; Accepted 1998 Feb 13.

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.

Abstract

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 (FuKi) and Knobbly (FuKb), and a transgenic insertional allele, FuTg1, 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 3β (GSK-3β)-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-3β, and β-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-3β is a constitutively active protein kinase and antagonizes downstream elements of the Wnt signaling pathway through changes in the β-catenin level (10). Wnt inactivates GSK-3β activity through Dsh, although by which mechanism is not known (6). In the presence of Wnt, there is a decrease in the phosphorylation of β-catenin and an increase in its stability, and β-catenin translocates to the nucleus (44). This translocation involves the association of β-catenin with the transcriptional enhancers of lymphocyte enhancer binding factor/T cell factor (LEF/TCF) family (2, 24). β-Catenin has a consensus sequence of a phosphorylation site for GSK-3β, and elimination of this possible phosphorylation site stabilizes β-catenin (22, 26, 44). It has been recently shown that the ubiquitination-proteasome pathway is involved in the degradation of β-catenin and that mutations in the GSK-3β consensus phosphorylation site of β-catenin prevent ubiquitination (1). It is well known that adenomatous polyposis coli gene product (APC) is required for the degradation of β-catenin, although the role of APC is not well understood (35). Furthermore, it has been shown that GSK-3β phosphorylates APC and that the phosphorylation enhances the binding of APC to β-catenin (37). Thus, it appears that GSK-3β is a key mediator in the Wnt signaling pathway to regulate β-catenin turnover and that the phosphorylation of β-catenin by GSK-3β is essential for this process. However, it is not clear how GSK-3β regulates the degradation of β-catenin since GSK-3β does not significantly phosphorylate β-catenin by the use of the mammalian purified proteins.

To obtain insights into the action of GSK-3β on the degradation of β-catenin and the specification of cell fate, we have tried to find a GSK-3β-interacting protein by using a yeast two-hybrid method. We isolated a protein which interacts with GSK-3β and found that this protein has 44% identity with Axin (45). We designated this protein Axil (Axin like). We show here that Axil inhibits Xwnt8-induced axis formation of Xenopus embryos like Axin. Further, we demonstrate that Axil makes a complex with both GSK-3β and β-catenin and that it promotes GSK-3β-dependent phosphorylation of β-catenin. These results suggest that Axil negatively regulates the Wnt signaling pathway by interacting with GSK-3β and β-catenin and by mediating the signal from GSK-3β to β-catenin, resulting in the regulation of axis formation.

ACKNOWLEDGMENTS

We are grateful to Y. Takai, K. Tanaka, A. Nagafuchi, S. Tsukita, S. Nagata, J. R. Woodgett, Y. Hata, F. Tamanoi, C. W. Turck, Q. Hu, and M. Nakata for donating plasmids, cDNA libraries, GSK peptide 1, and antibodies. We thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.

This work was supported by a grant-in-aid for scientific research (B) from the Ministry of Education, Science, and Culture, Japan (1996, 1997), and by grants from the Yamanouchi Foundation for Research on Metabolic Disorders (1996, 1997), the Fukuyama Transporting Shibuya Longevity and Health Foundation (1996), the Tsuchiya Foundation (1996), and the Kato Memorial Bioscience Foundation (1997).

ACKNOWLEDGMENTS

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