Abstract 2132: Characterization of galectin 3 as a novel BARD1 interaction partner

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL BARD1 (BRCA1-associated ring domain 1) was originally identified in a yeast-two-hybrid (Y2H) 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. In order to identify putative interaction-proteins with BARD1 C-terminal region we performed a 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 galectin 3 C-terminal region, residues 170-250 (OMIM [153619][1]). 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 conducting co-immunoprecipitation 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, and by confocal microscopy. Using ectopically expressed galectin 3 fragments we determined the interaction region (aa 100-215). Moreover, galectin 3 fragment comprising aminoacids 100-165 is able to disrupt the endogenous interaction with BARD1. Co-immunoprecipitation assays suggested 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. This ubiquitination reaction occurs mainly in the nucleus but is not BRCA1 dependent. The role of galectin 3 in DNA damage pathway was investigated using galectin 3 silenced HeLa cells exposed to increasing doses of ionizing radiation. These cells exhibited a slightly increase in radioresistance, but no effect was observed in cell cycle control. In addition the phosphorylation of histone H2AX seems to be delayed after damage in cells lacking galectin 3. Other DNA damage agents, such as carboplatin and etoposide did not affected differently the cell viability of these cells. We are now evaluating the protein interaction network of galectin 3 using a tandem affinity purification approach. 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 2132. doi:1538-7445.AM2012-2132 [1]: /lookup/external-ref?link_type=OMIM&access_num=153619&atom=%2Fcanres%2F72%2F8_Supplement%2F2132.atom