Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance

The Fanconi anemia (FA) pathway is a DNA damage-activated signaling pathway which regulates cellular resistance to DNA cross-linking agents. Cloned FA genes and proteins cooperate in this pathway, and monoubiquitination of FANCD2 is a critical downstream event. The cell cycle checkpoint kinase ATR is required for the efficient monoubiquitination of FANCD2, while another checkpoint kinase, ATM, directly phosphorylates FANCD2 and controls the ionizing radiation (IR)-inducible intra-S-phase checkpoint. In the present study, we identify two novel DNA damage-inducible phosphorylation sites on FANCD2, threonine 691 and serine 717. ATR phosphorylates FANCD2 on these two sites, thereby promoting FANCD2 monoubiquitination and enhancing cellular resistance to DNA cross-linking agents. Phosphorylation of the sites is required for establishment of the intra-S-phase checkpoint response. IR-inducible phosphorylation of threonine 691 and serine 717 is also dependent on ATM and is more strongly impaired when both ATM and ATR are knocked down. Threonine 691 is phosphorylated during normal S-phase progression in an ATM-dependent manner. These findings further support the functional connection of ATM/ATR kinases and FANCD2 in the DNA damage response and support a role for the FA pathway in the coordination of the S phase of the cell cycle.