title: Autophosphorylation and ATM activation: additional sites add to the complexity.
abstract: The recognition and signaling of DNA double strand breaks involves the participation of multiple proteins, including the protein kinase ATM (mutated in ataxia-telangiectasia). ATM kinase is activated in the vicinity of the break and is recruited to the break site by the Mre11-Rad50-Nbs1 complex, where it is fully activated. In human cells, the activation process involves autophosphorylation on three sites (Ser(367), Ser(1893), and Ser(1981)) and acetylation on Lys(3016). We now describe the identification of a new ATM phosphorylation site, Thr(P)(1885) and an additional autophosphorylation site, Ser(P)(2996), that is highly DNA damage-inducible. We also confirm that human and murine ATM share five identical phosphorylation sites. We targeted the ATM phosphorylation sites, Ser(367) and Ser(2996), for further study by generating phosphospecific antibodies against these sites and demonstrated that phosphorylation of both was rapidly induced by radiation. These phosphorylations were abolished by a specific inhibitor of ATM and were dependent on ATM and the Mre11-Rad50-Nbs1 complex. As found for Ser(P)(1981), ATM phosphorylated at Ser(367) and Ser(2996) localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser(367) and Ser(2996) was functionally important because mutant forms of ATM were defective in correcting the S phase checkpoint defect and restoring radioresistance in ataxia-telangiectasia cells. These data provide further support for the importance of autophosphorylation in the activation and function of ATM in vivo.
authors.affiliation: Radiation Biology and Oncology, Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia. sergei.kozlov@qimr.edu.au
authors.forename: Sergei V
authors.initials: SV
authors.lastname: Kozlov
authors.forename: Mark E
authors.initials: ME
authors.lastname: Graham
authors.forename: Burkhard
authors.initials: B
authors.lastname: Jakob
authors.forename: Frank
authors.initials: F
authors.lastname: Tobias
authors.forename: Amanda W
authors.initials: AW
authors.lastname: Kijas
authors.forename: Marcel
authors.initials: M
authors.lastname: Tanuji
authors.forename: Philip
authors.initials: P
authors.lastname: Chen
authors.forename: Phillip J
authors.initials: PJ
authors.lastname: Robinson
authors.forename: Gisela
authors.initials: G
authors.lastname: Taucher-Scholz
authors.forename: Keiji
authors.initials: K
authors.lastname: Suzuki
authors.forename: Sairai
authors.initials: S
authors.lastname: So
authors.forename: David
authors.initials: D
authors.lastname: Chen
authors.forename: Martin F
authors.initials: MF
authors.lastname: Lavin
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D001260:Ataxia Telangiectasia / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002461:Cell Line, Transformed; D053903:DNA Breaks, Double-Stranded; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D000076228:MRE11 Homologue Protein; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011839:Radiation, Ionizing; D016196:S Phase / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D046912:Multiprotein Complexes; C112156:NBN protein, human; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes