ATR Dependent Phosphorylation of FANCM Is Required for the Fanconi Anemia Pathway.

Fanconi anemia (FA) is a rare, recessive disorder characterized by progressive bone marrow failure, developmental abnormalities, chromosome instability, cellular hypersensitivity to DNA cross-linking agents, and predisposition to cancer, mainly leukemias and squamous cell carcinomas of the head and neck. We have shown that FANCM which is one of the FA core complex proteins is hyperphosphorylated in response to DNA damage suggesting that it may serve as a signal transducer through which the activity of the FA-core complex is regulated. The cell cycle checkpoint kinase, ATR has been shown to act upstream of the FA pathway, however, its substrate within the FA-core complex has not been identified yet. FANCM contains multiple predicted ATR phosphorylation sites suggesting that FANCM could be a direct ATR target. In this study, we examined the roles of ATR in regulating FANCM phosphorylation in response to DNA damage:

1. by kinetics study we found that phosphorylation of FANCM is concurrent with FANCD2 monoubiquitination;

2. siRNA mediated suppression of ATR activity abrogates both phosphorylation of FANCM and monoubiquitination of FANCD2; and

3. ATR knock out HCT116 cells display defective phosphorylation of FANCM as well as defective monoubiquitination of FANCD2 indicating that DNA damage induced phosphorylation of FANCM is ATR dependant.

Furthermore, we used mass spectrometry to identify the in vivo phosphorylation sites of FANCM and found a novel DNA damage-inducible phosphorylation site (S-1045; one of the potential ATR phosphorylation sites) within FANCM protein. Using ATR knock out HCT116 cells and the anti-p-S1045 antibody, we show that phosphorylation of FANCM at S-1045 is ATR dependant. The biological relevance of phosphorylation of FANCM at S1045 in FA pathway will be investigated by functional complementation analysis with non phosphorylatable FANCM mutants in FANCM deficient cells.