ATP activates ataxia-telangiectasia mutated (ATM) in vitro. Importance of autophosphorylation.
Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks. The mechanism of activation remains unresolved, and it is uncertain whether autophosphorylation contributes to activation. We describe an in vitro immunoprecipitation system demonstrating activation of ATM kinase from unirradiated extracts by preincubation with ... ATP. Activation is both time- and ATP concentration-dependent, other nucleotides fail to activate ATM, and DNA is not required. ATP activation is specific for ATM since it is not observed with kinase-dead ATM, it requires Mn2+, and it is inhibited by wortmannin. Exposure of activated ATM to phosphatase abrogates activity, and repeat cycles of ATP and phosphatase treatment reveal a requirement for autophosphorylation in the activation process. Phosphopeptide mapping revealed similarities between the patterns of autophosphorylation for irradiated and ATP-treated ATM. Caffeine inhibited ATM kinase activity for substrates but did not interfere with ATM autophosphorylation. ATP failed to activate either A-T and rad3-related protein (ATR) or DNA-dependent protein kinase under these conditions, supporting the specificity for ATM. These data demonstrate that ATP can specifically induce activation of ATM by a mechanism involving autophosphorylation. The relationship of this activation to DNA damage activation remains unclear but represents a useful model for understanding in vivo activation.
Mesh Terms:
Adenosine Triphosphate, Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caffeine, Cell Cycle Proteins, Cell Line, DNA, DNA Damage, DNA-Binding Proteins, Dose-Response Relationship, Drug, Enzyme Activation, Glutathione Transferase, Humans, Manganese, Peptides, Phosphatidylinositol 3-Kinases, Phosphodiesterase Inhibitors, Phosphopeptides, Phosphorylation, Precipitin Tests, Protein Serine-Threonine Kinases, Recombinant Fusion Proteins, Temperature, Time Factors, Trypsin, Tumor Suppressor Proteins
Adenosine Triphosphate, Ataxia Telangiectasia Mutated Proteins, Blotting, Western, Caffeine, Cell Cycle Proteins, Cell Line, DNA, DNA Damage, DNA-Binding Proteins, Dose-Response Relationship, Drug, Enzyme Activation, Glutathione Transferase, Humans, Manganese, Peptides, Phosphatidylinositol 3-Kinases, Phosphodiesterase Inhibitors, Phosphopeptides, Phosphorylation, Precipitin Tests, Protein Serine-Threonine Kinases, Recombinant Fusion Proteins, Temperature, Time Factors, Trypsin, Tumor Suppressor Proteins
J Biol Chem
Date: Mar. 14, 2003
PubMed ID: 12645530
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