Cdk1 Protein-mediated Phosphorylation of Receptor-associated Protein 80 (RAP80) Serine 677 Modulates DNA Damage-induced G2/M Checkpoint and Cell Survival

Abstract

Post-translational phosphorylation plays critical roles in the assembly of signaling and repair proteins in the DNA damage response pathway. RAP80, a component of the BRCA1-A complex, is crucial in cell cycle checkpoint activation and DNA damage repair. However, its molecular mechanism is unclear. In this study, we identified Cdk1 as a new RAP80-binding protein and demonstrated that the Cdk1-cyclin B1 complex phosphorylates RAP80 at Ser-677 using an in vitro kinase assay and a phosphopeptide-specific antibody against phospho-Ser-677 of RAP80. RAP80 Ser-677 phosphorylation occurred in the M phase of the cell cycle when Cdk1 was in an active state. In addition, ionizing radiation (IR) induced RAP80 phosphorylation at Ser-677. Mutation of Ser-677 to alanine sensitized cells to IR and functioned in G2/M checkpoint control. These results suggest that post-translational phosphorylation of RAP80 by the Cdk1-cyclin B1 complex is important for RAP80 functional sensitivity to IR and G2/M checkpoint control.Background: RAP80, a component of the BRCA1-A complex, is crucial in the cell cycle checkpoint and DNA damage repair.Results: RAP80 phosphorylation by Cdk1 is important for sensitivity to ionizing radiation and G2/M checkpoint control.Conclusion: Cdk1-mediated RAP80 phosphorylation is important for the DNA damage response.Significance: The findings provide new implications for the interplay of the DNA damage signaling pathway and RAP80 phosphorylation. Post-translational phosphorylation plays critical roles in the assembly of signaling and repair proteins in the DNA damage response pathway. RAP80, a component of the BRCA1-A complex, is crucial in cell cycle checkpoint activation and DNA damage repair. However, its molecular mechanism is unclear. In this study, we identified Cdk1 as a new RAP80-binding protein and demonstrated that the Cdk1-cyclin B1 complex phosphorylates RAP80 at Ser-677 using an in vitro kinase assay and a phosphopeptide-specific antibody against phospho-Ser-677 of RAP80. RAP80 Ser-677 phosphorylation occurred in the M phase of the cell cycle when Cdk1 was in an active state. In addition, ionizing radiation (IR) induced RAP80 phosphorylation at Ser-677. Mutation of Ser-677 to alanine sensitized cells to IR and functioned in G2/M checkpoint control. These results suggest that post-translational phosphorylation of RAP80 by the Cdk1-cyclin B1 complex is important for RAP80 functional sensitivity to IR and G2/M checkpoint control. Background: RAP80, a component of the BRCA1-A complex, is crucial in the cell cycle checkpoint and DNA damage repair. Results: RAP80 phosphorylation by Cdk1 is important for sensitivity to ionizing radiation and G2/M checkpoint control. Conclusion: Cdk1-mediated RAP80 phosphorylation is important for the DNA damage response. Significance: The findings provide new implications for the interplay of the DNA damage signaling pathway and RAP80 phosphorylation.