title: Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells.
abstract: 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 beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-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-3beta. 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 beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta 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 beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).
authors.affiliation: Systems Research-Kinase Biology Discovery Research, GlaxoSmithKline Pharmaceuticals, Harlow, Essex, CM19 5AD, United Kingdom. t.hagen@ucl.ac.uk
authors.forename: Thilo
authors.initials: T
authors.lastname: Hagen
authors.forename: Elena
authors.initials: E
authors.lastname: Di Daniel
authors.forename: Ainsley A
authors.initials: AA
authors.lastname: Culbert
authors.forename: Alastair D
authors.initials: AD
authors.lastname: Reith
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D013329:Structure-Activity Relationship; D015534:Trans-Activators; D051176:beta Catenin
chemical_list: C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D051176:beta Catenin; D038341:Glycogen Synthase Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3