Abstract 1117: Characterization of BAT1 (UAP56) interaction with BARD1

Abstract

Abstract BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid screen as a binding partner of BRCA1. The functional heterodimer BRCA1-BARD1 is required for several of the cellular and tumour-suppressor functions of BRCA1. Both proteins interact through the N-terminal RING domain to form a heterodimeric E3 ubiquitin ligase that constitutes the major catalytic activity of the BRCA1-BARD1 complex. BARD1 is also associated to p53-mediated apoptosis, in a BRCA1-independent manner. The carboxy terminus of BRCA1 is highly acidic and contains two tandem BRCA1 C-terminal (BRCT) domains, which are characteristic of members of a large superfamily of proteins involved in DNA repair and cell cycle checkpoint control. These domains are protein-protein interacting regions that mediate the association with a number of other proteins with a role in DNA replication, DNA damage repair (DDR) pathway, transcription, cell cycle control, and ubiquitination. BARD1 also contains two tandem BRCT repeats at its C-terminus, a region shown to bind serine-phosphorylated peptides in vitro. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a yeast two hybrid (Y2H) screening using BARD1 BRCT tandem domain (aa 554-777) to screen a human testis cDNA library. We identified, among other hits, a cDNA coding for residues 308-418 of BAT1 (UAP56, C-terminal region [OMIM 142560]). UAP56 is a member of DEAD box family of ATP-dependent RNA helicases. Several studies indicate that UAP56 protein is not only essential in pre-mRNA splicing but is also important in mRNA nuclear export and cytoplasmic mRNA localization. We confirmed BAT1-BARD1 interaction by conducting co-IP analysis of ectopic expressed BARD1 and BAT1 in HEK293T cells. We are using a Y2H system approach to determine the minimal region of interaction between BARD1 and BAT1; in parallel, we are also checking for the presence of a multicomponent complex, investigating the presence of BRCA1. In order to assess the biological significance of the interaction between BARD1 and BAT1 we will determine whether this interaction is important for the activation of, or recovery from, the G2/M cell cycle checkpoint after IR and also investigate its putative involvement in p53-dependent apoptosis pathway. 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 1117.