title: Monitoring ATM kinase activity in living cells.
abstract: 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.
authors.affiliation: Molecular and Cell Biology Laboratory, Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Sam A
authors.initials: SA
authors.lastname: Johnson
authors.forename: Zhongsheng
authors.initials: Z
authors.lastname: You
authors.forename: Tony
authors.initials: T
authors.lastname: Hunter
mesh_terms: 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*
chemical_list: 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