FANCD2 Purification and Identification of a Functionally Important Phosphorylation Event at Serine 331.

Fanconi anemia (FA) is an inherited hematological disorder characterized by birth defects, bone marrow failure, and cancer susceptibility. FA cells are characterized by spontaneous chromosomal instability and hypersensitivity to cross linking reagents such as mitomycin C (MMC). Cloned FA genes cooperate in a signaling pathway activated by DNA damage during replication, and monoubiquitination of FANCD2 is the critical downstream event. The check point kinase ATR is required for the efficient monoubiquitination of FANCD2. Inducible ATR-dependent phosphorylation of threonine 691 and serine 717 are reported to enhance the monoubiquitination of FANCD2 and resistance to MMC. In the present study, we have devised means to purify the FANCD2 protein into multiple subcomplexes. In doing so, we have identified at least 3 distinct subcomplexes: a pure multimeric precursor form comprised of FANCD2 polypeptides, a 1 MD form that is chromatin bound, and a 2 MD form that is in the nucleosol. Electron microscopic analysis of the precursor form reveals homogenous appearing complexes. Mass spectroscopic analysis of the 1MD FANCD2 complex has identified the FANCI protein, among others, which confirms recent reports of FANCD2-FANCI interaction. Further mass spectroscopic analysis of FANCD2 has identified a novel phosphorylation site on FANCD2, serine 331, which is further confirmed by immunoblot using phosphospecific antibody against phospho-serine 331. This phosphorylation site is conserved in other species, including mouse, rat, xenopus, and tetraodon nigroviridis. Single amino acid mutation by alanine at this site results in a complete loss of monoubquitination of FANCD2 and resistance to MMC, suggesting that the phosphorylation of S331 is a functionally significant event. Moreover, this site fits the consensus sequence for a CHK1 substrate, which in turn is a substrate of target for CHK1, a substrate of ATR, which is also required for monoubiquitination of FANCD2. These findings further support the correlation between phosphorylation and monoubiquitination of FANCD2 in the DNA damage response with the intervention of CHK1 as a mediator of ATR and suggest dynamic regulation of FANCD2 subcomplexes within the FA pathway.