Abstract 209: Identification of β-catenin lysine 394 as a novel charge button and ubiquitination site by Rad6B
Authors: Gerard Brigitte , Sanders Matthew A. , Shekhar Malathy P. 
Published: August 2012

Clinical evidence suggests that β-catenin accumulation in breast cancer is associated with poor prognosis. Accumulation of β-catenin occurs even though activating mutations are rare. However, the mechanisms responsible for stabilization of β-catenin in breast cancer are not well known. We have previously shown that Rad6B stabilizes β-catenin by polyubiquitin modifications that render β-catenin insensitive to 26S proteasome. Rad6B silencing in breast cancer cells with autocrine Wnt activity inhibits β-catenin polyubiquitination and β-catenin mediated transcriptional activity. In this study, we identify the Rad6B-induced site of β-catenin ubiquitination. To map the lysine(s) of β-catenin that are ubiquitinated by Rad6B, in vitro ubiquitination assays were performed with recombinant Rad6B and extracts of COS7 cells transiently transfected with various lengths of myc-tagged β-catenin. These experiments indicated lysine residues located between amino acids 181-422 of β-catenin as potential targets of Rad6B mediated ubiquitination sites. To identify the Rad6B responsive ubiquitination site(s) in β-catenin, lysines 312, 335, 345, 354 or 394 located in the transcriptional regulatory domain in armadillo repeats (ARM) 5-7 were mutated to arginine as this maintains the charge distribution and minimizes the risk of disturbing the superhelical armadillo domain structure. In vitro ubiquitination assays identified K394 located in ARM 7 as the major site of Rad6B-induced ubiquitination. In vivo ubiquitination assays in Wnt silent MCF-7 breast cancer cells showed impairment of K345R- and K394R-β-catenin ubiquitination. The impact of these mutations on β-catenin mediated reporter transactivation was determined in MCF-7 cells. Whereas K312R, K335R, K345R or K354R mutations caused none to marginal drop in TOP-mediated reporter activation as compared to wild type β-catenin, K394R mutation of β-catenin caused ∼50% drop in TOP/Flash activity. Consistent with the TOP reporter transactivation data, expression of Rad6B, itself a β-catenin/TCF transcriptional target, was reduced in K394R-β-catenin transfected MCF-7 cells. In vitro ubiquitination assays using recombinant wild type or catalytically inert Rad6B and a synthetic 29 amino acid β-catenin peptide encompassing the K394 residue confirmed Rad6B ubiquitination of K394. We observed decreased expression of K394R-β-catenin in vivo. Treatment with the proteasomal inhibitor MG132 failed to rescue its expression, indicating that decreased expression is not due to proteasomal degradation. These data reveal K394 as a novel site of β-catenin ubiquitination and a novel charge button that may be important for maintaining the overall structure and stability of β-catenin.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 209. doi:1538-7445.AM2012-209