| Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that plays a central role in controlling the cellular response to DNA double-strand breaks caused by ionizing radiation (IR). IR causes an increase in ATM protein kinase activity and induces autophosphorylation of ATM on serine 1981, however the precise mechanisms that regulate ATM activation are not fully understood. Here, I describe the purification and characterization of human ATM from nuclear HeLa cell extracts. Microgram quantities of highly stable, kinase-active ATM were prepared. Purified ATM was phosphorylated on serine 1981 and was active towards a variety of known ATM substrates. A biochemical characterization of ATM protein kinase activity is presented, including the effects of chemical inhibitors, activators and an analysis of the native molecular weight of ATM by size exclusion chromatography. I also show that the protein phosphatase inhibitor okadaic acid (OA) induces autophosphorylation of ATM on serine 1981 in unirradiated cells at concentrations that inhibit protein phosphatase 2A (PP2A)-like activity in vitro. OA did not induce gamma-H2AX foci, suggesting that it induces ATM autophosphorylation by inactivation of a protein phosphatase rather than by inducing DNA double-strand breaks. In support of this, I show that the scaffolding (A) and catalytic (C) subunits of PP2A interact with ATM in undamaged cells and that immunoprecipitates of ATM from undamaged cells contain PP2A-like protein phosphatase activity. Moreover, I demonstrate that IR induces phosphorylation-dependent dissociation of PP2A from ATM and loss of the associated protein phosphatase activity. A model for the role of PP2A in the activation of ATM is proposed. Finally, I show that protein phosphatase 5 (PP5), which has been implicated in the activation of ATM-dependent pathways after irradiation, is phosphorylated after exposure to IR and other DNA damaging agents. PP5 phosphorylation occurs with similar kinetics to the activation of ATM pathways, although ATM is dispensable for PP5 phosphorylation after IR. Possible roles for PP5 phosphorylation in the response to IR-induced DNA double-strand breaks are discussed. |
