Cell Cycle Differences in DNA Damage-induced BRCA1 Phosphorylation Affect Its Subcellular Localization

Phosphorylation of BRCA1 tumor suppressor protein is regulated during the cell cycle and in response to DNA damage. Several Ser/Thr kinases have been implicated in BRCA1 phosphorylation, including ATM/ATR, cdk2, and hChk2 kinases. In this study, phospho-Ser-specific antibodies recognizing Ser-988, -1423, -1497, and -1524 residues of BRCA1 were employed to study BRCA1 phosphorylation during the S and G2/M phases under conditions of DNA damage. We observed that IR (ionizing radiation) treatment induced phosphorylation of Ser-988/Ser-1524 during the S phase and of Ser-988/Ser-1423 during the G2/M phase. UV treatment induced phosphorylation of Ser-988 during the S phase and of Ser-1423 during the G2/M phase. Phosphorylation of serines 1423 and -1524 was not induced in HCC1937 breast cancer cells, which contain mutant BRCA1 protein. Confocal microscopy revealed that unphosphorylated BRCA1 localizes on chromosomes from metaphase through telophase, whereas Ser-988-phosphorylated BRCA1 resides in the inner chromosomal structure, centrosome, and the cleavage furrow during prophase through telophase. We also found that Ser-988-phosphorylated BRCA1 relocalizes to the perinuclear region when cells are subjected to IR or UV radiation in the S phase. These results reinforce a model wherein phosphorylation of specific residues of BRCA1 after DNA damage affects its localization and function.