131
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_9, © Springer Science+Business Media LLC 2017
Chapter 9
Quantitative and Dynamic Imaging of ATM Kinase Activity
Shyam Nyati, Grant Young, Brian Dale Ross, and Alnawaz Rehemtulla
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.
Key words ATM, Bioluminescence, Complementation, In vivo, Kinase activity, Live cell, Molecular
imaging, Reporter, Split-luciferase
1 Introduction
Protein kinases constitute one of the largest gene families, comprising ~2% of the human genome. It is estimated that approximately 30% of all cellular proteins are phosphorylated on at least
one residue. Thus, protein kinases have key roles in many fundamental processes of cellular signaling in cancer as well as normal
cells. Biochemical methods have been widely used to investigate
whether or not a protein kinase of interest is active. Although biochemical methods are robust in vitro, they generally do not provide information about protein kinase activity in specific subcellular
compartments; nor do they provide information about activity
changes at the single-cell level. We and others have developed
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_9, © Springer Science+Business Media LLC 2017
Chapter 9
Quantitative and Dynamic Imaging of ATM Kinase Activity
Shyam Nyati, Grant Young, Brian Dale Ross, and Alnawaz Rehemtulla
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.
Key words ATM, Bioluminescence, Complementation, In vivo, Kinase activity, Live cell, Molecular
imaging, Reporter, Split-luciferase
1 Introduction
Protein kinases constitute one of the largest gene families, comprising ~2% of the human genome. It is estimated that approximately 30% of all cellular proteins are phosphorylated on at least
one residue. Thus, protein kinases have key roles in many fundamental processes of cellular signaling in cancer as well as normal
cells. Biochemical methods have been widely used to investigate
whether or not a protein kinase of interest is active. Although biochemical methods are robust in vitro, they generally do not provide information about protein kinase activity in specific subcellular
compartments; nor do they provide information about activity
changes at the single-cell level. We and others have developed