ATM-dependent phosphorylation of human Rad9 is required for ionizing radiation-induced checkpoint activation

ATM (ataxia-telangiectasia-mutated) is a Ser/Thr kinase involved in cell cy cle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schi zosaccharomyces pombe Rad9 protein that plays a critical role in cell cycle checkpoint control. To examine the potential signaling pathway linking ATM and hRad9, we investigated the modification of hRad9 in response to DNA da mage, Here we show that hRad9 protein is constitutively phosphorylated in u ndamaged cells and undergoes hyperphosphorylation upon treatment with ioniz ing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interesti ngly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser( 272) Of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad 9 is phosphorylated on Ser(272) in response to IR in vivo, and this modific ation is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant p rotein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S check point activation and increased cellular sensitivity to IR. Together, our re sults suggest that the ATM-mediated phosphorylation of hRad9 is required fo r IR-induced checkpoint activation.