Abstract 79: The role of BRCA1 and SUMO in ubiquitination of cyclinb1
Authors: Shabana Shabbeer, Alexandra Pietraszkiewicz, Lauren Mahon, Yaron Galanty, Ronit I. Yarden
Journal: Cancer Research (2012)

Abstract BRCA1 is a breast and ovarian tumor susceptibility gene. The gene encodes a protein that is a key player and an integral component of the signaling cascades leading to cell cycle checkpoint arrest following genetic insults induced by replication stress and/or DNA damage. In the absence of BRCA1 expression and following such insults, cells fail to activate the replication checkpoint or the DNA damage checkpoint and continue to progress into mitosis. Thus, loss of BRCA1 expression results in accumulation of genomic instability leading to cancer development. BRCA1 is an E3 ubiquitin ligase when it is bound to its partner protein, BARD1. While a number of BRCA1 substrates have been identified, none of them provide a clear mechanistic understanding of how BRCA1 enzymatic activity relates to its multiple functions in checkpoint activation and tumor suppression. Here we show that BRCA1 E3 ligase when activated by genetic insults, mediates ubiquitination of the cell cycle protein cyclin B1 (CycB) and signals it for proteasomal degradation to prevent mitotic entry. Previously it has been shown that high CycB expression is associated with poor survival in breast cancer, and this overexpression is more frequent among patient carriers of BRCA1 or BRCA2 mutations. We recently identified a consensus SUMOylation site in CycB. Given that BRCA1 E3 ubiquitin ligase activity is induced by SUMOylation, we wished to determine if SUMOylation is a necessary step in the BRCA1-mediated ubiquitination of CycB. By immunoprecipitation assays we found that endogenous CycB binds to both the SUMO1 and SUMO2 proteins in U2OS cells stably expressing GFP-SUMO1 or GFP-SUMO2. Basal levels of CycB SUMOylation with either SUMO1 or SUMO2 are low. Following exposure to ionizing radiation (IR), SUMOylation of CycB with SUMO1 increases in a sigmoidal dose and time-dependent manner while, SUMOylation with SUMO2 follows a different kinetics. Ubiquitination of CycB increases following SUMOylation. When exposed to proteosomal inhibitor alone, both SUMOylated and ubiquitinated CycB accumulates in the cells. The association between SUMO1/2 and CycB was confirmed in vitro by using bacterial pull-down assays. In addition, we identified CycB in a BRCA1 and in a SUMO2 immunocomplex purified from U2OS cells, suggesting an association of these proteins. By immunoprecipitations and FRET, using wild type and Lysine-mutants of CycB we will identify whether the N-terminal ψKXE consensus SUMOylation site within CycB is the site for these interactions in basal and IR-treated cells. Studies are currently underway to elucidate the significance of BRCA1 SUMOylation for CycB ubiquitination and SUMOylation. In summary, we present data to show that SUMOylation could be the bridge for BRCA1's ubiquitin ligase activity toward CycB in order to regulate the G2-M transition, cell cycle checkpoint arrest and thus, maintenance of genomic stability. 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 79. doi:1538-7445.AM2012-79