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Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.

ABSTRACT: The ubiquitin conjugating enzyme Rad6B is overexpressed in breast cancer and induces ?-catenin transcriptional activation and stabilization via K63-linked polyubiquitination. Here we identify ?-catenin and Rad6B interacting regions, identify potential Rad6B ubiquitination sites in ?-catenin, and characterize their breast cancer tissue expression. ?-catenin and Rad6B colocalize in breast carcinoma and coimmunoprecipitate from MDA-MB-231 cells. Pull-down assays using GST-?-catenin and His-Rad6B deletion mutants identified amino acids 131-181 and 50-116, respectively, as necessary for their interaction. Ubiquitination assays using ?-catenin deletion mutants mapped Rad6B-induced ubiquitination within ?-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 ?-catenin peptide encompassing K394. Ubiquitination of wild type- but not K394R-?-catenin was decreased by Rad6B silencing. Compared to wild type-, K312R-, K335R-, K345R-, or K354R-?-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 ?-catenin/TCF transcriptional target, was also reduced in K394R-?-catenin transfected cells. Steady-state K394R-?-catenin levels are decreased compared to wild type-?-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 ?-catenin than reduction mammoplasties. These data suggest that K394 is a novel site of ?-catenin ubiquitination that may be important for the stability and activity of ?-catenin in breast cancer.

SUBMITTER: Gerard B

PROVIDER: S-EPMC3915360 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.

Gerard Brigitte B Sanders Matthew A MA Visscher Daniel W DW Tait Larry L Shekhar Malathy P V MP

Biochimica et biophysica acta 20120615 10

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 de ...[more]

PMID: 22705350

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Project description:BACKGROUND:Aberrant Wnt/?-catenin signaling is associated with breast cancer even though genetic mutations in Wnt signaling components are rare. We have previously demonstrated that Rad6B, an ubiquitin conjugating enzyme, stabilizes ?-catenin via polyubiqutin modifications that render ?-catenin insensitive to proteasomal degradation. Rad6B is a transcriptional target of ?-catenin, creating a positive feedback loop between Rad6B expression and ?-catenin activation. METHODS:To isolate subpopulations expressing high or low Rad6B levels, we transfected MDA-MB-231 or WS-15 human breast cancer cells with ZsGreen fluorescent reporter vector in which the expression of ZsGreen was placed under the control of Rad6B promoter. ZsGreenhigh and ZsGreenlow subpopulations, reflective of high and low Rad6B promoter activity, respectively, were isolated by FACS. To determine the relevance of Wnt signaling in Rad6B-mediated ?-catenin stabilization/activation, the ZsGreenhigh cells were transfected with signaling-defective Wnt coreceptor LRP6?173. Rad6B expression and promoter activity were determined by RT-PCR, Western blot and Rad6B promoter-mediated luciferase assays. ?-catenin levels and transcriptional activity were determined by Western blot and TOP/FOP Flash reporter assays. Tumor formation and morphologies of ZsGreenlow, ZsGreenhigh, and ZsGreenhigh/LRP6?173 cells compared to unsorted vector controls were evaluated in nude mice. Expression of Wnt signaling related genes was profiled using the Wnt signaling pathway RT2 Profiler PCR arrays. RESULTS:ZsGreenhigh subpopulations showed high Rad6B expression and Rad6B promoter activity as compared to ZsGreenlow cells. ZsGreenhigh (high Rad6B expressors) also showed elevated ?-catenin levels and TOP/Flash activity. Inhibiting Wnt signaling in the high Rad6B expressors decreased ZsGreen fluorescence, Rad6B gene expression, ?-catenin levels and TOP/Flash activity. Tumors derived from high Rad6B expressors were predominantly composed of cells with epithelial mesenchymal transition (EMT) phenotype as compared to control tumors that were composed of both cuboidal and EMT-type cells. Tumors derived from low Rad6B expressors lacked EMT phenotype. Inhibition of LRP6 function in the high Rad6B expressors abrogated the EMT phenotype. Gene expression profiling showed upregulation of several Wnt signaling pathway regulators in high Rad6B expressors that were downregulated by interference of Wnt signaling with mutant LRP6 or by Rad6B silencing. CONCLUSIONS:These data reveal a functional link between the canonical Wnt pathway and Rad6B in ?-catenin activation and breast cancer progression.

Dataset Information

Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.

Publications

Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin.

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