Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation.

Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.

[1]  Yoichi Taya,et al.  ATM associates with and phosphorylates p53: mapping the region of interaction , 1999, Nature Genetics.

[2]  C. Canman,et al.  The role of ATM in DNA damage responses and cancer , 1998, Oncogene.

[3]  A. Blasina,et al.  A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase , 1999, Current Biology.

[4]  D. Livingston,et al.  ATM phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response , 2000, Nature.

[5]  S. Elledge,et al.  Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. , 1999, Science.

[6]  T. Hunter,et al.  Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. , 1991, Methods in enzymology.

[7]  B. Price,et al.  Caffeine inhibits the checkpoint kinase ATM , 1999, Current Biology.

[8]  S. Carr,et al.  Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway , 1999, Oncogene.

[9]  Y. Shiloh,et al.  Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart. , 1997, Annual review of genetics.

[10]  H. Piwnica-Worms,et al.  A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  M. Gatei,et al.  ATM-dependent phosphorylation of nibrin in response to radiation exposure , 2000, Nature Genetics.

[12]  T. Halazonetis,et al.  Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53. , 2000, Genes & development.

[13]  E. Stavridi,et al.  Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  K. Isselbacher,et al.  Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome. , 1999, Science.

[15]  Y. Shiloh,et al.  Rapid ATM-dependent phosphorylation of MDM2 precedes p53 accumulation in response to DNA damage. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. T. Kim,et al.  Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members* , 1999, The Journal of Biological Chemistry.

[17]  Y Taya,et al.  Enhanced phosphorylation of p53 by ATM in response to DNA damage. , 1998, Science.

[18]  Y Taya,et al.  The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. , 2000, Genes & development.

[19]  Bo Xu,et al.  ATM phosphorylates p95/nbs1 in an S-phase checkpoint pathway , 2000, Nature.

[20]  Jong-Soo Lee,et al.  hCds1-mediated phosphorylation of BRCA1 regulates the DNA damage response , 2000, Nature.

[21]  K. Khanna,et al.  Caffeine Abolishes the Mammalian G2/M DNA Damage Checkpoint by Inhibiting Ataxia-Telangiectasia-mutated Kinase Activity* , 2000, The Journal of Biological Chemistry.

[22]  S. Elledge,et al.  Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. , 1998, Science.

[23]  Y. Shiloh,et al.  Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products , 2000, Nature.

[24]  S. Elledge,et al.  DNA damage-induced activation of p53 by the checkpoint kinase Chk2. , 2000, Science.

[25]  B. Vogelstein,et al.  A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia , 1992, Cell.

[26]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.

[27]  Y. Shiloh,et al.  ATM: A mediator of multiple responses to genotoxic stress , 1999, Oncogene.

[28]  P. Leder,et al.  Loss of atm radiosensitizes multiple p53 null tissues. , 1998, Cancer research.