title: Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation.
abstract: In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.
authors.affiliation: Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
authors.forename: Magtouf
authors.initials: M
authors.lastname: Gatei
authors.forename: Katie
authors.initials: K
authors.lastname: Sloper
authors.forename: Claus
authors.initials: C
authors.lastname: Sorensen
authors.forename: Randi
authors.initials: R
authors.lastname: Syljuäsen
authors.forename: Jacob
authors.initials: J
authors.lastname: Falck
authors.forename: Karen
authors.initials: K
authors.lastname: Hobson
authors.forename: Kienan
authors.initials: K
authors.lastname: Savage
authors.forename: Jiri
authors.initials: J
authors.lastname: Lukas
authors.forename: Bin-Bing
authors.initials: BB
authors.lastname: Zhou
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Kum Kum
authors.initials: KK
authors.lastname: Khanna
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D004261:DNA Replication / Q000528:radiation effects; D004268:DNA-Binding Proteins; D016195:G2 Phase / Q000528:radiation effects; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D010766:Phosphorylation / Q000528:radiation effects; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011839:Radiation, Ionizing; D016196:S Phase / Q000528:radiation effects; D012694:Serine / Q000378:metabolism; D025521:Tumor Suppressor Proteins
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D012694:Serine; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases