Ionizing radiation activates nuclear protein phosphatase-1 by ATM-dependent dephosphorylation.

Ionizing radiation (IR) is known to activate multiple signaling pathways, resulting in diverse stress responses including apoptosis, cell cycle arrest, and gene induction. IR-activated cell cycle checkpoints are regulated by Ser/Thr phosphorylation, so we tested to see if protein phosphatases were targets of an IR-activated damage-sensing pathway. Jurkat cells were subjected to IR or sham radiation followed by brief (32)P metabolic labeling. Nuclear extracts were subjected to microcystin affinity chromatography to recover phosphatases, and the proteins were analyzed by two-dimensional gel electrophoresis. Protein sequencing revealed that the microcystin-bound proteins with the greatest reduction in (32)P intensity following IR were the alpha and delta isoforms of protein phosphatase 1 (PP1). Both of these PP1 isoforms contain an Arg-Pro-Ile/Val-Thr-Pro-Pro-Arg sequence near the C terminus, a known site of phosphorylation by Cdc/Cdk kinases, and phosphorylation attenuates phosphatase activity. In wild-type Jurkat cells or ataxia telangiectasia (AT) cells that are stably transfected with full-length ATM kinase, IR resulted in net dephosphorylation of this site in PP1 and produced activation of PP1. However, in AT cells that are deficient in ATM, IR failed to induce dephosphorylation or activation of PP1. IR-induced PP1 activation in the nucleus may be a critical component in an ATM-mediated pathway controlling checkpoint activation.

ATM-dependent dissociation of B55 regulatory subunit from nuclear PP2A in response to ionizing radiation.

Ionizing radiation (IR) is known to activate multiple cell cycle checkpoints that are thought to enhance the ability of cells to respond to DNA damage. Protein phosphatase 2A (PP2A) has been implicated in IR-induced activation of checkpoints; therefore, Jurkat cells were exposed to an activating dose of IR or sham treatment as control, and nuclear extracts were analyzed for PP2A by Mono Q anion exchange chromatography and microcystin affinity chromatography. PP2A exists in eukaryotic cells both as a heterodimer consisting of a 65-kDa scaffolding subunit (A) plus a 36-kDa catalytic subunit (C) and as ABC heterotrimers, containing one of a variety of regulatory (B) subunits. Here we show that IR produces a transient and reversible reduction in the amount of nuclear AB55C heterotrimer without affecting the AB'C heterotrimer or AC heterodimer. In ataxia telangiectasia-mutated (ATM)-deficient cells the amount of nuclear PP2A heterotrimer relative to heterodimer was not reduced by radiation, but the radiation response was restored by transfection of these cells with plasmids encoding ATM. Wortmannin, an inhibitor of kinases such as phosphatidylinositol 3-kinase, also prevented the IR-induced reduction in nuclear PP2A heterotrimer. The changes in nuclear PP2A occurred without a noticeable difference in the carboxyl-terminal methylation of the C subunit, which is known to influence association with B subunits. We conclude a novel ATM-dependent mechanism is regulating association of B55 subunits with nuclear PP2A in response to IR.