Induction of Phosphorylation on BRCAI during the Cell Cycle and after DNA Damage

BRCAI, the familial breast cancer susceptibility gene product, is a 220-kDa phosphorylated protein. BRCAI immunoprecipitated from MCF7 cells blocked in G1-S phase or progressing through S-phase of the cell cycle migrated more slowly through SDS polyacrylamide gels than BRCAI from cells maintained in serum-supplemented media, serum-free media for 24 h, or delayed in G2-M phase by treatment with colchicine. Restoration of BRCAI to the faster-migrating form, which occurred on release of cells from the G1-S-phase block, was prevented by the phosphatase inhibitor okadaic acid. Phosphatase treatment of immunoprecipitated BRCAI resulted in the conversion of the slower-migrating form to the faster-migrating form. Although these results suggested that BRCAI was preferentially hyperphosphorylated near the G1-S-phase boundary of the cell cycle, exposure of cells to DNA-damaging agents including UV light or treatment with hydrogen peroxide (H2O) also promoted BRCAI hyperphosphorylation. These same stimuli also eliminated the punctate nuclear staining pattern normally observed for BRCA1 in control cells. These results indicate that BRCAI undergoes cyclic hyperphosphorylation during the cell cycle; however, this modification, as well as changes in BRCAI nuclear staining, also occurs in response to DNA damage