␦-Catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby ␦-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates ␦-catenin and thus affects its stability. Initially, we found that the level of ␦-catenin was greater and the half-life of ␦-catenin was longer in GSK-3
fibroblasts than those in GSK-3؉/؉ fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3␣ and -3 kinase dead constructs, consistently showed that the levels of endogenous ␦-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3␣ and -3 interact with and phosphorylate ␦-catenin. The phosphorylation of ⌬C207-␦-catenin (lacking 207 C-terminal residues) and T1078A ␦-catenin by GSK-3 was noticeably reduced compared with that of wild type ␦-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr 1078 residue of ␦-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased ␦-catenin levels and caused an accumulation of ubiquitinated ␦-catenin. It was also found that GSK-3 triggers the ubiquitination of ␦-catenin. These results suggest that GSK-3 interacts with and phosphorylates ␦-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.
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