Monitoring ATM kinase activity in living cells.
DNA double strand breaks (DSB) in mammalian cells result in the activation of the ATM protein kinase. This leads to phosphorylation of numerous downstream transducer and effector proteins that coordinate a cellular response including DNA repair, cell cycle arrest or apoptosis. We have developed a reporter protein that allows the measurement of ATM kinase activity in single living cells. This CFP-YFP FRET-based biosensor uses an ATM phosphorylation site and an FHA phosphospecific binding domain to produce a phosphorylation-induced change in conformation, which alters the FRET efficiency between CFP and YFP. We show that the reporter provides a measurable output in response to DSBs and is specific for ATM over ATR or DNA-PK. We expect the description of the spatiotemporal dynamics of ATM activity in living cells that this reporter provides will be helpful in providing a more detailed understanding of the DNA damage response.
Johnson
Sam A
SA
Molecular and Cell Biology Laboratory, Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
You
Zhongsheng
Z
Hunter
Tony
T
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001426:Bacterial Proteins / Q000378:metabolism*; D015374:Biosensing Techniques; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D010446:Peptide Fragments; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; C485182:Cyan Fluorescent Protein; D004268:DNA-Binding Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D025521:Tumor Suppressor Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases