Induction of phosphorylation on BRCA1 during the cell cycle and after DNA damage.
BRCA1, the familial breast cancer susceptibility gene product, is a 220-kDA phosphorylated protein. BRCA1 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 BRCA1 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 BRCA1 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 BRCA1 resulted in the conversion of the slower-migrating form to the faster-migrating form. Although these results suggested that BRCA1 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 (H2O2) also promoted BRCA1 hyperphosphorylation. These same stimuli also eliminated the punctate nuclear staining pattern normally observed for BRCA1 in control cells. These results indicate that BRCA1 undergoes cyclic hyperphosphorylation during the cell cycle; however, this modification, as well as changes in BRCA1 nuclear staining, also occurs in response to DNA damage.
Thomas
J E
JE
Department of Cancer Research, Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana 46285, USA. Thomas_James_E@lilly.com
Smith
M
M
Tonkinson
J L
JL
Rubinfeld
B
B
Polakis
P
P
D016590:Aphidicolin / Q000494:pharmacology; D019313:BRCA1 Protein / Q000378:metabolism*; D001769:Blood; D001943:Breast Neoplasms; D002453:Cell Cycle / Q000502:physiology*; D016213:Cyclins / Q000096:biosynthesis; D004249:DNA Damage / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000096:biosynthesis; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays
D019313:BRCA1 Protein; D016213:Cyclins; D004791:Enzyme Inhibitors; D019384:Nucleic Acid Synthesis Inhibitors; D016160:Retinoblastoma Protein; D019319:Okadaic Acid; D016590:Aphidicolin; D006861:Hydrogen Peroxide; D010749:Phosphoprotein Phosphatases