Abstract 3927: Characterization of galectin 3 as a putative BRCA1/BARD1 ubiquitin E3 ligase substrate

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 tumor-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 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 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 galectin 3 C-terminal region, residues 170-250 (OMIM 153619). Galectin 3 is a galactoside binding protein involved in several cellular processes including apoptosis control and RNA splicing. Moreover there are evidences that galectin 3 variants are correlated with tumor development and progression in breast and thyroid cancer. We confirmed galectin 3/BARD1 interaction by conducting co-immuneprecipitation analysis of ectopically expressed BARD1 and galectin-3 in HEK293T cells. The interaction between endogenous galectin 3 and BARD1 were validated in HeLa nuclear extracts. Using the same approach we co-immuneprecipitated BRCA1 and galectin-3, suggesting the presence of BRCA1, BARD1 and galectin-3 in the same complex. Interestingly the galectin 3 observed in this complex corresponds to a mono-ubiquitinated form, corroborating the idea that galectin 3 is a putative natural target of BRCA1-BARD1 heterodimer E3 ubiquitin ligase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3927. doi:10.1158/1538-7445.AM2011-3927