Rad6B is a positive regulator of beta-catenin stabilization.
Mutations in beta-catenin or other Wnt pathway components that cause beta-catenin accumulation occur rarely in breast cancer. However, there is some evidence of beta-catenin protein accumulation in a subset of breast tumors. We have recently shown that Rad6B, an ubiquitin-conjugating enzyme, is a transcriptional target of beta-catenin/TCF. Here, we show that forced Rad6B overexpression in MCF10A breast cells induces beta-catenin accumulation, which despite being ubiquitinated is stable and transcriptionally active. A similar relationship between Rad6B, beta-catenin ubiquitination, and transcriptional activity was found in WS-15 and MDA-MB-231 breast cancer cells, and mouse mammary tumor virus-Wnt-1 mammary tumor-derived cells, implicating Rad6B in physiologic regulation of beta-catenin stability and activity. Ubiquitinated beta-catenin was detectable in chromatin immunoprecipitations performed with beta-catenin antibody in MDA-MB-231 but not MCF10A cells. Rad6B silencing caused suppression of beta-catenin monoubiquitination and polyubiquitination, and transcriptional activity. These effects were accompanied by a reduction in intracellular beta-catenin but with minimal effects on cell membrane-associated beta-catenin. Measurement of beta-catenin protein stability by cycloheximide treatment showed that Rad6B silencing specifically decreases the stability of high molecular beta-catenin with minimal effect upon the 90-kDa nascent form. In vitro ubiquitination assays confirmed that Rad6B mediates beta-catenin polyubiquitination, and ubiquitin chain extensions involve lysine 63 residues that are insensitive to 26S proteasome. These findings, combined with our previous data that Rad6B is a transcriptional target of beta-catenin, reveal a positive regulatory feedback loop between Rad6B and beta-catenin and a novel mechanism of beta-catenin stabilization/activation in breast cancer cells.
Shekhar
Malathy P V
MP
Breast Cancer Program, Karmanos Cancer Institute, Detroit, MI 4820, USA. shekharm@karmanos.org
Gerard
Brigitte
B
Pauley
Robert J
RJ
Williams
Bart O
BO
Tait
Larry
L
D000818:Animals; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D016764:Cell Polarity / Q000502:physiology; D004847:Epithelial Cells; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D034741:RNA, Small Interfering / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D015854:Up-Regulation; D051176:beta Catenin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D034741:RNA, Small Interfering; D025801:Ubiquitin; D051176:beta Catenin; C471546:UBE2B protein, human; D044763:Ubiquitin-Conjugating Enzymes