Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinasemediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gen e products (Brca1 and Brca2) suppress cancer, at least in part, by particip ating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved i n homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for bo th homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phospho rylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and W; however, S er-1387 is specifically phosphorylated after IR, and Ser-1457 is predominan tly phosphorylated after W. These results suggest that different types of D NA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after W) is largely ataxia telangiectasia mutated (ATM) k inase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at t hese sites, indicating that ATR controls Brca1 phosphorylation in vivo afte r the exposure of cells to UV light. Moreover, ATR associates with Brca1; A TR and Brca1 foci co-localize both in cells synchronized in S phase and aft er exposure of cells to DNA-damaging agents. ATR can itself phosphorylate t he region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530), However, there are additional uncharacte rized ATR phosphorylation site(s) between residues 521 and 757 of Brca1, Ta ken together, our results support a model in which ATM and ATR act in paral lel but somewhat overlapping pathways of DNA damage signaling but respond p rimarily to different types of DNA lesion.