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

Abstract

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...

The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase

“…The APC gene encodes a protein of 2843 amino acids that are associated with b-catenin, Axin and conductin/Axil (Rubinfeld et al, 1993;Behrens et al, 1998;Ikeda et al, 1998Nakamura et al, 1998;Yamamoto et al, 1998). These proteins are important components of the Wnt/ Wingless signal transduction pathway, which controls embryonic pattern formation (Nusse and Varmus, 1992;Cadigan and Nusse, 1997).…”

GSK-3β-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by β-catenin and protein phosphatase 2A complexed with Axin

“…We have identi®ed rat Axin (rAxin) and its homolog, Axil (for Axin like), as GSK-3b-interacting proteins Yamamoto et al, 1998). Axin is a product of the mouse Fused locus and functions as a negative regulator of the Wnt signaling pathway (Zeng et al, 1997).…”

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

“…We have identi®ed rat Axin (rAxin) and its homolog, Axil (for Axin like), as GSK-3b-interacting proteins Yamamoto et al, 1998). Axil has 44% amino acid identity with rAxin and inhibits Xwnt-8-induced axis formation of Xenopus embryos like Axin .…”