Phosphorylation of FANCA on S1449 Is Important for Fanconi Anemia (FA) Pathway Function.

Previous work in our lab and others has shown that the Fanconi anemia proteins, FANCG and FANCA, are phosphoproteins. FANCG is phosphorylated at mitosis, and these phosphorylations are required for proper exit from chromatin at mitosis. FANCG is also phosphorylated after DNA damage, with the phosphorylation site required for wild-type sensitivity to DNA damaging agents. FANCA is also phosphorylated after DNA damage and localized to chromatin, but the site and significance of this phosphorylation were previously unknown. Mass spectrometry of FANCA revealed one phosphopeptide with phosphorylation on serine 1449. Site-directed mutagenesis of this residue to alanine (S1449A) abolished a slower mobility form of FANCA seen after MMC treatment. Furthermore, the S1449A mutant failed to completely correct the MMC hypersensitivity of FA-A mutant cells. S1449A mutant cells displayed lower than wild-type levels of FANCD2 monoubiquitination following DNA damage, and an increased number of gross chromosomal aberrations were seen in metaphase spreads from S1449A mutant cells when compared to wild type cells. Using a GFP reporter substrate to measure homologous recombination, cells expressing the S1449A FANCA failed to completely correct the homologous recombination defect seen in FA cells. Taken together, cells expressing FANCA S1449A display a variety of FA-associated phenotypes, suggesting that the phosphorylation of S1449 is a functionally significant event. The DNA damage response in human cells is, in large part, coordinated by phosphorylation events initiated by apical kinases ATM and ATR. S1449 is found in a consensus ATM site, therefore studies are underway to determine if ATM or ATR is the kinase responsible for FANCA phosphorylation at S1449. Phosphorylation is a crucial process in transducing the DNA damage response, and phosphorylation of FA proteins appears critical to both localization and function of the proteins. Understanding how phosphorylation marks are placed on FANCA will give insight into the role of FANCA in the DNA damage response.