Identification of a Domain of Axin That Binds to the Serine/Threonine Protein Phosphatase 2A and a Self-binding Domain

Abstract

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and ␤-catenin. Axin has been shown to have distinct binding sites for GSK-3 and ␤-catenin and to promote the phosphorylation of ␤-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through ␤-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of ␤-catenin. 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 binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin⅐APC⅐GSK-3⅐␤-catenin complex, where it could modulate the effect of GSK-3 on ␤-catenin or other proteins in the complex. We also identified a region of Axin 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.

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 used human erythrocyte PP2A that consists of the C and A subunits since it is the major phosphoprotein phosphatase in human erythrocytes (Usui et al, 1988). Yeast two hybrid experiments have shown that the C subunit of PP2A but not the A subunit interacts with the C-terminal region of mouse Axin, which corresponds to rAxin-(508-713) (Hsu et al, 1999). Our results demonstrate that PP2A binds to not only the Cterminal region of rAxin (rAxin-(508-832)) but also its central region of rAxin-(298-506).…”

Controlled levels of canonical Wnt signaling are required for neural crest migration

“…Under unstimulated conditions cytoplasmic level of ß-catenin is kept low through the dual phosphorylation (Liu et al, 2002) by glycogen synthase kinase 3β (GSK-3β) (Hart et al, 1999;Yost et al, 1996) and casein kinase 1α (CK1α) (Price, 2006;Amit et al, 2002), which together with the adenomatous polyposis coli tumor suppressor protein (APC) (Hart et al, 1999;Ha et al, 2004), protein phosphatase 2A (PP2A) (Hsu et al, 1999;Seeling et al, 1999;Ratcliffe et al, 2000;Yamamoto et al, 2001) and Axin (Hart et al, 1999;Ikeda et al, 1998;Dajani et al, 2003) form a destruction complex (Stamos and Weis, 2013).…”

β-Catenin destruction complex: insights and questions from a structural perspective

“…The C subunit has been shown to bind Axin (Hsu et al, 1999;Ratcliffe et al, 2000) and APC was shown to bind two B subunits, B56a and B56d (Seeling et al, 1999). The role of PP2A, however, remains unclear.…”