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.
Department of Radiation Oncology, University of Virginia Health System, Charlottesville 22908, USA.
Chang Y
CY
Guo
David L
DL
Brautigan
James M
JM
Larner
D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000528:radiation effects; D004268:DNA-Binding Proteins; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism / Q000528:radiation effects*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism / Q000528:radiation effects*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000502:physiology* / Q000528:radiation effects; D011839:Radiation, Ionizing; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1