Abstract 3925: RNF138, a novel ubiquitin E3 ligase is overexpressed in colon cancer cell lines and interacts with RAD51D

Abstract

Abstract The RAD51 protein family provides core components of the Homologous Recombination (HR) complex, which is critical for repairing DNA double-strand breaks and maintaining genome stability. The purpose of this study was to identify proteins that regulate HR processes through post-translational modification of the RAD51 paralogs. A yeast two-hybrid screen was performed using the RAD51 paralogs RAD51C, RAD51D, and XRCC2 as bait against a normalized universal mouse cDNA library. Fifty-eight primary candidates were isolated, and five are predicted to function in the ubiquitination pathway. Within this group, Ring Finger Protein 138 (RNF138) is of particular interest since RNF138 is a novel E3-ubiquitin ligase having both an N-terminal RING finger domain and a putative C-terminal ubiquitin interaction motif. Presence of both domains indicates RNF138 induces the transfer of ubiquitin to target substrates and also recognizes ubiquitin moieties on other proteins. Expression of RNF138 is increased by 12-fold in the HCC38 breast cancer cell line and 4-fold in SW-48, RKO, and Wi-DR colon cancer cell lines compared to non-tumorigenic controls. Yeast two-hybrid results indicate that RNF138 interaction is unique to RAD51D, as RNF138 failed to directly interact with RAD51C and XRCC2. Further analysis using N- and C-terminal truncated RAD51D constructs revealed that the RAD51D core domain rather than the N-terminal linker region that governs the interaction between RAD51D and XRCC2 mediates the association with RNF138. Moreover, K113A and K113R mutations of the highly conserved RAD51D Walker A ATP binding motif abolished RAD51D-RNF138 association, suggesting that ATP binding and hydrolysis may regulate the interaction. As further demonstration of this association, Myc-RNF138 co-immunoprecipitated with HA-RAD51D following transient transfections. Future studies will determine whether RNF138 is directly required for HR repair and if the mechanism specifically involves RAD51D ubiquitination. Understanding the regulation of HR proteins by ubiquitination processes will provide important insights into cancer progression and guide the identification of new therapeutic strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3925.