Phosphorylation and ubiquitination of oncogenic mutants of beta-catenin containing substitutions at Asp32.
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.
Al-Fageeh
Mohamed
M
Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, OR 97331-6512, USA.
Li
Qingjie
Q
Dashwood
W Mohaiza
WM
Myzak
Melinda C
MC
Dashwood
Roderick H
RH
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites; D003110:Colonic Neoplasms / Q000235:genetics*; D003598:Cytoskeletal Proteins / Q000235:genetics* / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D010766:Phosphorylation; D051381:Rats; D012694:Serine; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D051176:beta Catenin
C495268:Ctnnb1 protein, rat; D003598:Cytoskeletal Proteins; D015534:Trans-Activators; D025801:Ubiquitin; D051176:beta Catenin; D001224:Aspartic Acid; D012694:Serine; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D038362:Glycogen Synthase Kinase 3