title: Quantitative and Dynamic Imaging of ATM Kinase Activity.
abstract: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.
authors.affiliation: Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA. shyamnya@med.umich.edu.|Department of Radiation Oncology, University of Michigan, 109 Zina Pitcher place, AAT-BSRB, Level A, Room # 628, Ann Arbor, MI, 48109 2200, USA. shyamnya@med.umich.edu.
authors.forename: Shyam
authors.initials: S
authors.lastname: Nyati
authors.affiliation: Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Grant
authors.initials: G
authors.lastname: Young
authors.affiliation: Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA.|Department of Radiology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Brian Dale
authors.initials: BD
authors.lastname: Ross
authors.affiliation: Center for Molecular Imaging, University of Michigan, Ann Arbor, MI, 48109, USA.|Department of Radiation Oncology, University of Michigan, Ann Arbor, MI, 48109, USA.
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins / Q000378:metabolism*; D001483:Base Sequence; D064447:Checkpoint Kinase 2 / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D016513:Mice, SCID; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D012694:Serine; D008156:Luciferases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases
keywords: ATM; Bioluminescence; Complementation; In vivo; Kinase activity; Live cell; Molecular imaging; Reporter; Split-luciferase