Phosphorylation of Axin, a Wnt signal negative regulator, by glycogen synthase kinase-3β regulates its stability

Axin forms a complex with glycogen synthase kinase-3 beta (GSK-3 beta) and beta-catenin and promotes GSK-3 beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSH-3 beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3 beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3 beta were mutated, exhibited a shorter half-life than wild type Axin, Dvl-1, which was genetically shown to function upstream of GSK-3 beta, inhibited the phosphorylation of Axin by GSK-3 beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of DV1-1(Delta PDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a, These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dv1.