Small-molecule FRET probes for protein kinase activity monitoring in living cells.
In this study, the applicability of fluorescently labeled adenosine analogue-oligoarginine conjugates (ARC-Photo probes) for monitoring of protein kinase A (PKA) activity in living cells was demonstrated. ARC-Photo probes possessing subnanomolar affinity towards the catalytic subunit of PKA (PKAc) and competitive with the regulatory subunit (PKAr), penetrate cell plasma membrane and associate with PKAc fused with yellow fluorescent protein (PKAc-YFP). Detection of inter-molecular Förster resonance energy transfer (FRET) efficiency between the fluorophores of the fusion protein and ARC-Photo probe can be used for both the evaluation of non-labeled inhibitors of PKAc and for monitoring of cAMP signaling via detection of changes in the activity of PKA as a cAMP downstream effector.
Vaasa
Angela
A
Institute of Chemistry, University of Tartu, 50411 Tartu, Estonia.
Lust
Marje
M
Terrin
Anna
A
Uri
Asko
A
Zaccolo
Manuela
M
D000241:Adenosine / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000032:analysis; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000037:antagonists & inhibitors; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000737:chemistry
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D009842:Oligopeptides; C054562:yellow fluorescent protein, Bacteria; D001120:Arginine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000241:Adenosine