Cyclin-Dependent Kinase-Mediated Phosphorylation of FANCD2 Promotes Mitotic Fidelity

ABSTRACT

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, which occurs upon exposure to DNA-damaging agents and during the 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 cyclin-dependent kinase (CDK) regulatory phosphosite (S592) proximal to the site of FANCD2 monoubiquitination. FANCD2 S592 phosphorylation was detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by immunoblotting with an S592 phospho-specific antibody. Mutation of S592 leads to abrogated monoubiquitination of FANCD2 during the 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.

KEYWORDS:

• Fanconi anemia
• FANCD2
• CDK phosphorylation
• cell cycle
• chromosome stability
• ubiquitination
• cell cycle
• ubiquitin

ACKNOWLEDGMENTS

We thank members of the Howlett, Camberg, and Dutta laboratories for critical discussions. We thank Andrew A. Deans and Winnie Tan at the University of Melbourne for purified proteins and Martin A. Cohn at the University of Oxford for HeLa FANCD2^−/− cells.

N.G.H. designed the research, supervised the experiments, analyzed the data, and wrote the paper. J.A.C.-V. performed the majority of experiments, analyzed the data, and wrote the paper with N.G.H. J.L.B., D.A.V., R.A.B., J.L.G., and B.P. performed experiments and analyzed data. W.T. and A.J.D. contributed vital reagents and advice.

We declare that we have no conflicts of interest.

This work was supported by NIH/NHLBI grant R01HL149907 ( to N.G.H.), an American Society of Hematology Bridge grant (to N.G.H.), Rhode Island IDeA Network of Biomedical Research Excellence (RI-INBRE) grant P20GM103430 from the National Institute of General Medical Science, and Rhode Island Experimental Program to Stimulate Competitive Research (RI-EPSCoR) grant no. 1004057 from the National Science Foundation.