Abstract A04: Structural and functional caracterization of BARD1/CDK13 interaction

Abstract

Listen

Translate article

Abstract DNA damage response (DDR) is crucial for cell survivor through genomic integrity maintenance. Dysfunctions in proteins implicated in DDR prompt cell to diseases, such as cancer. BRCA1 is a central protein involved in DDR and, once damaged, plays a pivotal role in cancer development. BRCA1 structure comprises two major domains, a RING finger domain and two tandem BRCT domains (tBRCT). This structure is shared by BRCA1 major interaction partner: BARD1. The tBRCT domain is commonly found in DDR-associated proteins. To study interactions in DDR, our group charted the tBRCT interactome for 7 different proteins, using three different strategies: tandem affinity purification followed by mass spectrometry (TAP-MS), yeast two hybrid (Y2H) screening, and literature curation. CDK13 was identified as a putative BARD1 tBRCT interactor by both TAP-MS and Y2H strategies. CDK13 is described as involved in several processes, such as chromatin remodeling, transcription regulation, and splicing coordination; however, its biological role remains unclear. To confirm BARD1/CDK13 interaction, whole cell extracts obtained from MCF-7 cells were used in coimmunoprecipitation assays using anti-CDK13 and anti-BARD1. Constructs encoding the HA-tagged regions 1-706aa (N-terminal), 706-982aa (kinase domain), and 1006-1452aa (C-terminal) of CDK13 were ectopically coexpressed with Flag-tagged full-length BARD1 or GFP-tagged BARD1 tBRCT in HEK293Tcells; whole cellular extracts were used in coimmunoprecipitation assays. BARD1 and CDK13 interaction was confirmed both in ectopic and constitutive expression approaches. CDK13 silencing was performed using lentiviral constructs encoding five different short hairpin RNAs (shRNA); MCF-7 cells were transduced and submitted to puromycin selection; extracts were used to evaluate CDK13 silencing by immunoblotting. Our data suggest that BARD1 tBRCT and the CDK13 N-terminal region, as well as the kinase domain, are critical for the formation of BARD1/CDK13 complex. MCF-7 cells lacking CDK13 expression will be used to investigate the kinase role during DDR. Citation Format: Vanessa C. Fernandes, Thales C. Nepomuceno, Renato S. Carvalho, Guilherme Suarez-Kurtz, Alvaro N. Monteiro, Marcelo A. Carvalho. Structural and functional caracterization of BARD1/CDK13 interaction [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A04.