Autophosphorylation and ATM activation: Additional sites add to the complexity

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 (Ser³⁶⁷, Ser¹⁸⁹³, and Ser¹⁹⁸¹) and acetylation on Lys³⁰¹⁶. 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 Ser²⁹⁹⁶, 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 Ser³⁶⁷ and Ser²⁹⁹⁶ localized to sites of DNA damage induced by radiation, but ATM recruitment was not dependent on phosphorylation at these sites. Phosphorylation at Ser³⁶⁷ and Ser²⁹⁹⁶ 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.