Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells

J Biol Chem. 2002 Jun 28;277(26):23330-5. doi: 10.1074/jbc.M201364200. Epub 2002 Apr 19.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / chemistry
  • Calcium-Calmodulin-Dependent Protein Kinases / physiology*
  • Cell Line
  • Cytoskeletal Proteins / metabolism*
  • Glycogen Synthase Kinase 3
  • Glycogen Synthase Kinases
  • Humans
  • Mutation
  • Phosphorylation
  • Structure-Activity Relationship
  • Trans-Activators*
  • beta Catenin

Substances

  • CTNNB1 protein, human
  • Cytoskeletal Proteins
  • Trans-Activators
  • beta Catenin
  • Glycogen Synthase Kinases
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Glycogen Synthase Kinase 3