RESUMO
BRCA1 plays an important role in mechanisms of response to double-strand breaks, participating in genome surveillance, DNA repair, and cell cycle checkpoint arrests. Here, we identify a constitutive BRCA1-c-Abl complex and provide evidence for a direct interaction between the PXXP motif in the C terminus of BRCA1 and the SH3 domain of c-Abl. Following exposure to ionizing radiation (IR), the BRCA1-c-Abl complex is disrupted in an ATM-dependent manner, which correlates temporally with ATM-dependent phosphorylation of BRCA1 and ATM-dependent enhancement of the tyrosine kinase activity of c-Abl. The BRCA1-c-Abl interaction is affected by radiation-induced modification to both BRCA1 and c-Abl. We show that the C terminus of BRCA1 is phosphorylated by c-Abl in vitro. In vivo, BRCA1 is phosphorylated at tyrosine residues in an ATM-dependent, radiation-dependent manner. Tyrosine phosphorylation of BRCA1, however, is not required for the disruption of the BRCA1-c-Abl complex. BRCA1-mutated cells exhibit constitutively high c-Abl kinase activity that is not further increased on exposure to IR. We suggest a model in which BRCA1 acts in concert with ATM to regulate c-Abl tyrosine kinase activity.
Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA1/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Sítios de Ligação , Proteínas de Ciclo Celular , Células Cultivadas , Proteínas de Ligação a DNA , Humanos , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/efeitos da radiação , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/efeitos da radiação , Radiação Ionizante , Proteínas Supressoras de TumorRESUMO
BRCA1 is a central component of the DNA damage response mechanism and defects in BRCA1 confer sensitivity to a broad range of DNA damaging agents. BRCA1 is required for homologous recombination and DNA damage-induced S and G(2)/M phase arrest. We show here that BRCA1 is required for ATM- and ATR-dependent phosphorylation of p53, c-Jun, Nbs1 and Chk2 following exposure to ionizing or ultraviolet radiation, respectively, and is also required for ATM phosphorylation of CtIP. In contrast, DNA damage-induced phosphorylation of the histone variant H2AX is independent of BRCA1. We also show that the presence of BRCA1 is dispensable for DNA damage-induced phosphorylation of Rad9, Hus1 and Rad17, and for the relocalization of Rad9 and Hus1. We propose that BRCA1 facilitates the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle checkpoint arrest and apoptosis, but that BRCA1 is dispensable for the phosphorylation of DNA-associated ATM and ATR substrates.
Assuntos
Proteína BRCA1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Proteína BRCA1/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA , Ativação Enzimática , Genes BRCA1 , Histonas/metabolismo , Humanos , Técnicas In Vitro , Modelos Biológicos , Mutação , Fosforilação , Proteínas de Schizosaccharomyces pombe , Transdução de Sinais , Proteínas Supressoras de Tumor , Raios UltravioletaRESUMO
BRCA1, a breast and ovarian tumor suppressor, colocalizes with markers of the inactive X chromosome (Xi) on Xi in female somatic cells and associates with XIST RNA, as detected by chromatin immunoprecipitation. Breast and ovarian carcinoma cells lacking BRCA1 show evidence of defects in Xi chromatin structure. Reconstitution of BRCA1-deficient cells with wt BRCA1 led to the appearance of focal XIST RNA staining without altering XIST abundance. Inhibiting BRCA1 synthesis in a suitable reporter line led to increased expression of an otherwise silenced Xi-located GFP transgene. These observations suggest that loss of BRCA1 in female cells may lead to Xi perturbation and destabilization of its silenced state.