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

Mohamed Al-Fageeh, Qingjie Li, W. Mohaiza Dashwood, Melinda C. Myzak, Roderick H. Dashwood

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-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-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-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 β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-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 β-catenins, as well as on WT β-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 β-catenin.

Original languageEnglish (US)
Pages (from-to)4839-4846
Number of pages8
JournalOncogene
Volume23
Issue number28
DOIs
StatePublished - Jun 17 2004

Keywords

  • APC
  • CTNNB1
  • Colorectal cancer
  • Ctnnb1
  • Human β-catenin gene
  • Rat β-catenin gene
  • TCF/LEF
  • Wnt signaling
  • β-catenin

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cancer Research

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