Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.
The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces β-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify β-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in β-catenin, and characterize their breast cancer tissue expression. β-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-β-catenin and His-Rad6B deletion mutants identified amino acids 131-181 and 50-116, respectively, as necessary for their interaction. Ubiquitination assays using β-catenin deletion mutants mapped Rad6B-induced ubiquitination within β-catenin 181-422 encompassing Armadillo repeats 2-7. Lysine to arginine mutations within repeats 5-7 identified K394 as the major Rad6B ubiquitination site in vitro and in vivo, and confirmed by Rad6B ubiquitination of a β-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-β-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-β-catenin, K394R mutation caused ~50% drop in TOP/Flash activity in Wnt-silent MCF-7 cells. Consistent with these data, expression of Rad6B, itself a β-catenin/TCF transcriptional target, was also reduced in K394R-β-catenin transfected cells. Steady-state K394R-β-catenin levels are decreased compared to wild type-β-catenin. The decreased expression is not due to proteasomal degradation as treatment with MG132 failed to rescue its levels. Lymph node-positive breast carcinomas express higher levels of Rad6 protein and Rad6 activity, and K63-linked ubiquitinated β-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of β-catenin ubiquitination that may be important for the stability and activity of β-catenin in breast cancer.
Molecular Biology & Genetics Program, Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA.
Brigitte
B
Gerard
Matthew A
MA
Sanders
Daniel W
DW
Visscher
Larry
L
Tait
Malathy P V
MP
Shekhar
D051186:Armadillo Domain Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D005260:Female; D020868:Gene Silencing; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009361:Neoplasm Invasiveness; D011485:Protein Binding; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation / Q000235:genetics; D051176:beta Catenin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D051186:Armadillo Domain Proteins; C495270:CTNNB1 protein, human; D025801:Ubiquitin; D051176:beta Catenin; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008239:Lysine