Biology Reference
In-Depth Information
Abstract
Protein kinases constitute a major class of enzymes underlying essentially all biological
processes. These enzymes present similar structural folds, yet their mechanism of action
and of regulation vary largely, as well as their substrate specificity and their subcellular
localization. Classical approaches to study the function/activity of protein kinases rely on
radioactive endpoint assays, which do not allow for characterization of their dynamic
activity in their native environment. The development of fluorescent biosensors has pro-
vided a whole new avenue for studying protein kinase behavior and regulation in living
cells in real time with high spatial and temporal resolution. Two major classes of bio-
sensors have been developed: genetically encoded single-chain fluorescence reso-
nance energy transfer biosensors and peptide/protein biosensors coupled to small
synthetic fluorophores which are sensitive to changes in their environment.
In this review, we discuss the developments in fluorescent biosensor technology
related to protein kinase sensing and the different strategies employed to monitor pro-
tein kinase activity, conformation, or relative abundance, as well as kinase regulation and
subcellular dynamics in living cells. Moreover, we discuss their application in biomedical
settings, for diagnostics and therapeutics, to image disease progression and monitor
response to therapeutics, in drug discovery programs, for high-throughput screening
assays, for postscreen characterization of drug candidates, and for clinical evaluation
of novel drugs.
ABBREVIATIONS
AFP autofluorescent proteins
AMPK AMP-activated protein kinase
ATM Ataxia telangiectasia mutated kinase
BiFC bimolecular fluorescence complementation
BTF beta-turn-focused
CaMKII calcium/calmodulin-dependent protein kinase II
CDK cyclin-dependent kinase
CFP cyan fluorescent protein
CHEF chelation-enhanced fluorescence
CML chronic myelogenous leukemia
CPP cell-penetrating peptide
EGFP enhanced green fluorescent protein
EGFR epidermal growth factor receptor
DPA dipicolylamine
ELISA enzyme-linked immunosorbent assay
ERK extracellular signal-regulated kinase
FAK focal adhesion kinase
FHA domain forkhead-associated domain
FLIM fluorescence lifetime imaging
FRET fluorescence resonance energy transfer
GFP green fluorescent protein
GLUT Glutamate Transporter
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