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 cycle checkpoints and DNA repair. Human Rad9 (hRad9) is the homologue of Schizosaccharomyces 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 damage. Here we show that hRad9 protein is constitutively phosphorylated in undamaged cells and undergoes hyperphosphorylation upon treatment with ionizing radiation (IR), ultraviolet light (UV), and hydroxyurea (HU). Interestingly, hyperphosphorylation of hRad9 induced by IR is dependent on ATM. Ser(272) of hRad9 is phosphorylated directly by ATM in vitro. Furthermore, hRad9 is phosphorylated on Ser(272) in response to IR in vivo, and this modification is delayed in ATM-deficient cells. Expression of hRad9 S272A mutant protein in human lung fibroblast VA13 cells disturbs IR-induced G(1)/S checkpoint activation and increased cellular sensitivity to IR. Together, our results suggest that the ATM-mediated phosphorylation of hRad9 is required for IR-induced checkpoint activation.
Department of Molecular Medicine/Institute of Biotechnology, The University of Texas Health Science Center, San Antonio, Texas 78245-3207, USA.
M J
MJ
Chen
Y T
YT
Lin
H B
HB
Lieberman
G
G
Chen
E Y
EY
Lee
D001260:Ataxia Telangiectasia / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology / Q000528:radiation effects*; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000528:radiation effects*; D004268:DNA-Binding Proteins; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008168:Lung; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011839:Radiation, Ionizing; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D025521:Tumor Suppressor Proteins; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D025521:Tumor Suppressor Proteins; C069930:rad9 protein; D010768:Phosphoserine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea