133
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 [19]. These splitluciferase 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 PI3like 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 [20]. 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 [27]. If damaged DNA is
left unrepaired it can lead to cell death, genomic instability, cancer,
and/or other pathologies [28]. 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 [29].
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,
Molecular Imaging of ATM Kinase Activity
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 [19]. These splitluciferase 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 PI3like 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 [20]. 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 [27]. If damaged DNA is
left unrepaired it can lead to cell death, genomic instability, cancer,
and/or other pathologies [28]. 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 [29].
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,
Molecular Imaging of ATM Kinase Activity