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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.
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 optical imaging reporters to measure the kinase activity of various oncologically important kinases ([1–11], Table 1) and have utilized these reporters in subsequent studies that lead to the identification of new inhibitors and discovery of novel signaling mechanisms [12, 13].
Bioluminescence is a chemical reaction where light is emitted by a living organism. Luciferases are a large family of light-generating enzymes that catalyze the oxidation of a substrate, generically called luciferin, to yield oxyluciferin with the concomitant production of light. For in vivo bioluminescence imaging of malignancy, tumor cells or cancer-related genes are tagged with a reporter gene that encodes a light-generating enzyme, luciferase [14–16]. When this reporter is in the presence of the substrate it emits a blue to yellow-green light with an emission spectra peaking at a wavelength between 490 and 620 nm . An extremely sensitive cooled charged-coupled device (CCD) camera or a photomultiplier detects any low light that is emitted during the bioluminescence reaction. Due to its extreme sensitivity, broad dynamic range and exceptionally large signal-to-noise ratio, this type of noninvasive imaging permits a real-time analysis of an ample amount of various biological events . Although there are more than 30 luciferase-luciferin systems that were derived independently of each other, the most frequently used luciferase for in vivo molecular imaging is the ATP-dependent firefly (Photinus pyralis) luciferase . The reason for this is that 30% of the light produced by firefly luciferase has an emission spectra above 600 nm, a region in which the signal attenuation by the absorbing and scattering properties of live mammalian tissue is at a minimum [15, 17]. Recently, a very bright and smaller luciferase (NanoLuc; NLuc) from deep sea shrimp (Oplophorus gracilirostris) has been successfully used for dual luciferase imaging in a mouse model .
A significant advantage of cell-based bioluminescent kinase reporter is its adaptability for high-throughput screening. Bioluminescence generated in luciferase assays offers higher sensitivity than FRET-based systems due to amplification of the signal. In addition, luciferase is less susceptible to inference from nonspecific fluorescence of compounds. Thus, bioluminescence-based assays are highly suited for high-throughput screening. Furthermore, luciferase activity can be monitored dynamically and noninvasively, allowing bioluminescence-based cell assays to provide a unique method for identifying specific compounds that interact with the target in the correct cellular compartment and under normal cellular physiological conditions of that compartment (pH, concentrations of specific ions, etc). Reporters wherein the firefly luciferase enzyme has been divided into two halves (N-Luc and C-Luc) were originally developed to study protein-protein interaction . These split- luciferase reporters were based on either the inter-molecular or intra-molecular complementation of the luciferase fragments to generate signal in response to cellular cues.
Ataxia Telangiectasia Mutated (ATM) is a member of the PI3- like family of serine/threonine kinases. It is a very large 370 KDa protein encoded by human chromosome 11q22-23. It plays a critical role in repair of DNA double-stranded breaks (DSBs) thereby maintaining genomic stability. These processes include, but are not limited to, DNA replication, DNA repair, cell cycle progression, apoptosis, and senescence. ATM exists in its inactive form as a noncovalently linked dimer where the kinase domain of one monomer is bound to the internal domain of another monomer covering the S1981 residue. In response to DSBs, the kinase domain of one monomer phosphorylates S1981 of the other interacting ATM resulting in subunit dissociation, ATM activation, and recruitment to DNA break sites . Ionizing radiation-induced ATM activation results in the activation of a large number of ATM substrates [21-26] including P53, MDM2, SMC1, KAP1, BRCA1, γH2AX, and CHK2. The activated ATM triggers a sequence of events including cell cycle arrest, allowing time for the repair of the damaged DNA in sync with circadian rhythm . If damaged DNA is left unrepaired it can lead to cell death, genomic instability, cancer, and/or other pathologies . The 2015 award of the Nobel Prize in Chemistry for the discovery of DNA repair mechanisms highlights the importance of this pathway. Because of the important role ATM plays in cancer, therapeutics have been devised to target it .
In vitro kinase assays using purified substrate and kinase are routinely used to evaluate kinase activity. For traditional cell-based studies, immunohistological and biochemical techniques have been utilized for evaluating the kinase activity of ATM, such as counting pATM foci, γH2AX foci, immunofluorescence, or immunoprecipitation-western blotting [26, 30, 31]. Johnson, You, and Hunter  described a fluorescence resonance energy transfer (FRET)-based biosensor for monitoring ATM kinase activity in live cells. Although this reporter provides direct measurement of the ATM kinase activity, it has a limitation of usability in mouse model due to tissue penetration and autofluorescence in the CFP-YFP range. In this book chapter, we provide detailed methods of use for the recently developed split firefly-based bioluminescence reporter to noninvasively, dynamically, and sensitively measure ATM kinase activity in live cells and mouse models .
The method described herein is an adaptation of the traditional protein complementation assay for the detection of protein-protein interaction in live cells. Instead of monitoring the interaction of two proteins through the use of split reporter molecules, we have adapted the assay such that the interaction between the “bait” and the “prey” occurs in response to the activity of a specific kinase. The kinase can be a serine/threonine- or a tyrosine-kinase. The reporter has also been engineered such that increased complementation (and therefore reporter activity) occurs in response to decreased kinase activity. This approach is therefore very well suited for high-throughput screens for kinase inhibitor libraries since a positive hit would be detected as an increase in bioluminescence activity, thereby less likely to result in false positives. We have also used analogous reporters for whole genome siRNA screens. As an example, a reporter for TGF-β receptor serine/threonione kinase activity was used in a human kinome siRNA screen to yield a number of novel genes as regulators of the TGF- β receptor function . Regulation of the molecular events that lead to the activation and/or inactivation of the ATM kinase activity is yet to be defined; therefore, it is anticipated that analogous whole genome siRNA screens against the ATMR will most likely yield new insights into the role of novel genes in the regulation of the cellular response to DNA damage.
We would like to acknowledge members of the Center for Molecular Imaging at the University of Michigan for their valuable input and support for the studies. This work was supported by the National Institutes of Health grant R01CA193690 (AR), P01CA087634 (BDR, AR and SN) as well as a P30CA046592 award to the University of Michigan Cancer Center.