Identification of a domain of axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain

Axin is a negative regulator of embryonic axis formation in vertebrates, wh ich acts through a Wnt signal transduction pathway involving the serine/thr eonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanati on for the ability of Axin to inhibit signaling through beta-catenin, In ad dition, a more N-terminal region of Axin binds to adenomatous polyposis col i (APC), a tumor suppressor protein that also regulates levels of beta-cate nin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no bindi ng sites for other proteins have previously been identified, We found that Axin can bind to the catalytic subunit of the serine/threonine protein phos phatase 2A through a domain between amino acids 632 and 836. This interacti on was confirmed by in vitro binding studies as well as by co-immunoprecipi tation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3. beta-catenin complex, where it could modulate the effect of GSK-3 on beta-c atenin or other proteins in the complex. We also identified a region of Axi n that may allow it to form dimers or multimers, Through two-hybrid and co- immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.