Abstract
β
-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3
β
(GSK-3
β
)-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-3
β
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-3
β
. 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.