β-Catenin transduces cytosolic signals to the nucleus in the Wnt pathway. The Wnt ligand stabilizes cytosolic β-catenin protein, preventing its phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3). Serine-33 and -37 of β-catenin are GSK3 phosphorylation sites that serve as recognition sites for the β-TRCP-ubiquitin ligase complex, which ultimately triggers β-catenin degradation. Mutations at those two sites, as well as in Ser-45, stabilize β-catenin. Recently, casein kinase Iε (CKIε) has been shown to be a positive regulator of the Wnt pathway. Its action mechanism, however, remains unknown. Here I show that Ser-45 is phosphorylated not by GSK3 but by CKIε. Axin, a scaffold protein that binds CKIε and β-catenin, enhances this CKIε-mediated phosphorylation. Overexpression of CKIε in cells increases the amount of β-catenin phosphorylated at Ser-45. Ser-45 phosphorylated β-catenin is a better substrate for GSK3, which suggests that CKIε and GSK3 may co-operate in destabilizing β-catenin. In spite of the fact that CKIε was found as a positive regulator of the Wnt pathway, mutational analysis suggests that mutation of Ser-45 regulates β-catenin stability by inhibiting the ability of GSK3 to phosphorylate Ser-33 and -37, thereby disrupting the interaction between β-catenin, β-TRCP and Axin. I propose that phosphorylation of Ser-45 by CKIε plays an important role in regulating β-catenin stability.
