Analyzing protein kinase dynamics in living cells with FRET reporters.
Genetically encoded reporters based on fluorescence resonance energy transfer (FRET) are being developed for analyzing spatiotemporal dynamics of kinase activities in living cells, as the activities of this class of enzymes are often dynamically regulated and spatially compartmentalized within specific signaling context. Here we describe a general modular design and engineering strategies for the development of activity reporters for kinases of interest, using A-kinase activity reporter (AKAR) as an illustrative example. Discussed here are basic structure of such reporters, design considerations, reporter gene construction, cellular and in vitro characterization. Strategies for improving specificity, dynamic range or sensitivity, reversibility and integrity of the reporter as well as basic methods for live-cell time-lapse imaging using these reporters are summarized. Discussion of using this approach in the study of MAPK cascades is also provided. These FRET-based kinase activity reporters, along with analogous probes based on alternative designs, provide real-time tracking of kinase dynamics with subcellular resolution, which should complement other methods and offer great opportunities to delineate the molecular mechanisms underlying the complex regulation of kinases.
Ni
Qiang
Q
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Titov
Denis V
DV
Zhang
Jin
J
D000818:Animals; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D020935:MAP Kinase Signaling System; D019606:Phosphoamino Acids / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D019606:Phosphoamino Acids; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases