title: Two serine phosphorylation sites in the C-terminus of Rad9 are critical for 9-1-1 binding to TopBP1 and activation of the DNA damage checkpoint response in HeLa cells.
abstract: A heteromeric proliferating cell nuclear antigen-like ring complex 9-1-1 is comprised of Rad9, Hus1 and Rad1. When assembled, 9-1-1 binds to TopBP1 and activates the ATR-Chk1 checkpoint pathway. This binding in vitro depends on the phosphorylation of Ser-341 and Ser-387 in Rad9 and is reduced to 70% and 20% by an alanine substitution for Ser-341 (S341A) and Ser-387 (S387A), respectively, and to background level by their simultaneous substitution (2A). Here, we show the importance of phosphorylation of these two serine residues in vivo. siRNA-mediated knockdown of Rad9 in HeLa cells impaired UV-induced phosphorylation of checkpoint kinase, Chk1, and conferred hypersensitivity to UV irradiation and to methyl methane sulfonate or hydroxyurea treatments. Either siRNA-resistant wild-type Rad9 (Rad9R(r)) or Rad9R(r) harboring the S341A substitution restored the phosphorylation of Chk1 and damage sensitivity, whereas Rad9R(r) harboring S387A or 2A did not. However, high expression of S387A restored Chk1 phosphorylation and partially suppressed the hypersensitivity. Thus, the affinity of Rad9 to TopBP1 correlates with the activation of the cellular DNA damage response and survival after DNA damage in HeLa cells, and phosphorylation of Ser-341 and Ser-387 of Rad9 is critical for full activation of the checkpoint response to DNA damage.
authors.affiliation: Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ueda
authors.forename: Yukimasa
authors.initials: Y
authors.lastname: Takeishi
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohashi
authors.forename: Toshiki
authors.initials: T
authors.lastname: Tsurimoto
mesh_terms: D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D034622:RNA Interference; D012694:Serine / Q000378:metabolism*; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; C069930:rad9 protein; D012694:Serine; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D006918:Hydroxyurea