Phosphorylation and ubiquitination of oncogenic mutants of beta-catenin containing substitutions at Asp32

Oncogene. 2004 Jun 17;23(28):4839-46. doi: 10.1038/sj.onc.1207634.

Abstract

Beta-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3beta (GSK-3beta)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3beta region of beta-catenin stabilize the protein and activate beta-catenin/TCF/LEF target genes. This study examined three oncogenic beta-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3beta. Compared with wild-type beta-catenin (WT), the beta-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of beta-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the beta-catenin mutants. Specifically, antiphospho-beta-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated beta-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant beta-catenins, as well as on WT beta-catenin. The relative order of ubiquitination was WT>D32G>D32N>D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of beta-catenin.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Aspartic Acid
  • Binding Sites
  • Colonic Neoplasms / genetics*
  • Cytoskeletal Proteins / genetics*
  • Cytoskeletal Proteins / metabolism
  • Glycogen Synthase Kinase 3 / metabolism
  • Glycogen Synthase Kinase 3 beta
  • Phosphorylation
  • Rats
  • Serine
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Ubiquitin / metabolism
  • beta Catenin

Substances

  • Ctnnb1 protein, rat
  • Cytoskeletal Proteins
  • Trans-Activators
  • Ubiquitin
  • beta Catenin
  • Aspartic Acid
  • Serine
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, rat
  • Glycogen Synthase Kinase 3