Expression and Characterization of GSK-3 Mutants and Their Effect on β-Catenin Phosphorylation in Intact Cells

• Published in: The Journal of biological chemistry 2002-06, Vol.277 (26), p.23330
• Main Authors: Thilo Hagen, Elena Di Daniel, Ainsley A. Culbert, Alastair D. Reith
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M201364200

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates β-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of β-catenin by GSK-3 does not require priming in vitro , i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3β. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321–1327). Here we have investigated whether phosphorylation of Ser 33 , Ser 37 , and Thr 41 in β-catenin requires priming through prior phosphorylation at Ser 45 in intact cells. We have shown that the Arg 96 mutant does not induce β-catenin degradation but instead stabilizes β-catenin, indicating that it is unable to phosphorylate β-catenin in intact cells. Furthermore, if Ser 45 in β-catenin is mutated to Ala, β-catenin is markedly stabilized, and phosphorylation of Ser 33 , Ser 37 , and Thr 41 in β-catenin by wild type GSK-3β is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro , is still able to phosphorylate β-catenin in intact cells although it has reduced activity. Mutation of Tyr 216 to Phe markedly reduces the ability of GSK-3β to phosphorylate and down-regulate β-catenin. In conclusion, we have found that the Arg 96 mutant has a dominant-negative effect on GSK-3β-dependent phosphorylation of β-catenin and that targeting of β-catenin for degradation requires prior priming through phosphorylation of Ser 45 .