CDK-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity

Fanconi anemia (FA) is a rare genetic disease characterized by increased risk for bone marrow failure and cancer. The FA proteins function together to repair damaged DNA. A central step in the activation of the FA pathway is the monoubiquitination of the FANCD2 and FANCI proteins under conditions of cellular stress and during S-phase of the cell cycle. The regulatory mechanisms governing S-phase monoubiquitination, in particular, are poorly understood. In this study, we have identified a CDK regulatory phospho-site (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by LC-MS/MS and by immunoblotting with a S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during S-phase. Furthermore, FA-D2 (FANCD2-/-) patient cells expressing S592 mutants display reduced proliferation under conditions of replication stress and increased mitotic aberrations, including micronuclei and multinucleated cells. Our findings describe a novel cell cycle-specific regulatory mechanism for the FANCD2 protein that promotes mitotic fidelity. Author SummaryFanconi anemia (FA) is a rare genetic disease characterized by high risk for bone marrow failure and cancer. FA has strong genetic and biochemical links to hereditary breast and ovarian cancer. The FA proteins function to repair DNA damage and to maintain genome stability. The FANCD2 protein functions at a critical stage of the FA pathway and its posttranslational modification is defective in >90% of FA patients. However, the function, and regulation of FANCD2, particularly under unperturbed cellular conditions, remains remarkably poorly characterized. In this study, we describe a novel mechanism of regulation of the FANCD2 protein during S-phase of the cell cycle. CDK-mediated phosphorylation of FANCD2 on S592 promotes the ubiquitination of FANCD2 during S-phase. Disruption of this phospho-regulatory mechanism results in compromised mitotic fidelity and an increase in mitotic chromosome instability.