Abstract
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.
Highlights
Ataxia-telangiectasia Mutated (ATM), mutated in the human disorder ataxia-telangiectasia, is rapidly activated by DNA double strand breaks
In A-T cells, faulty DNA damage recognition is accompanied by defective cell cycle checkpoint activation, which is likely to contribute to the genetic instability and cancer susceptibility in this syndrome [10, 11]
The extent of ATM activation exceeds that observed in irradiated samples, suggesting that ATP is titrating out all the ATM activity
Summary
A-T, ataxia-telangiectasia; ATM, ataxia-telangiectasia mutated; ATR, ataxia-telangiectasia and Rad3-related; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; BLM, defective in Bloom’s Syndrome; Nbs, defective in Nijmegen breakage syndrome; GST , glutathione S-transferase; Gy, gray; PI3-kinase, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; AMP-PNP, 5Ј-adenylylimidodiphosphate. The gene defective in A-T, ATM (A-T mutated), encodes a protein that is a member of the phosphatidylinositol 3-kinase (PI3-kinase) family [12] This group includes the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), A-T and rad3-related protein (ATR), and proteins in different organisms responsible for DNA damage recognition and cell cycle control [13]. Inositol hexakisphosphate stimulates DNAPK-dependent non-homologous DNA end joining in vitro, and this appears to be achieved by specific interaction with Ku70/ Ku80 [35] Another member of the PI3-kinase family mTOR is altered in its activity by small molecules, acting as a sensor of ATP concentration [36]. To investigate the activation of ATM and the possible involvement of autophosphorylation in this process, we have established an in vitro system involving immunoprecipitation of ATM and activation by ATP
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
