Small-molecule FRET probes for protein kinase activity monitoring in living cells

Biochem Biophys Res Commun. 2010 Jul 9;397(4):750-5. doi: 10.1016/j.bbrc.2010.06.026. Epub 2010 Jun 10.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine / chemistry
  • Arginine / chemistry
  • Bacterial Proteins / chemistry
  • Cell Membrane / enzymology
  • Cyclic AMP / analysis
  • Cyclic AMP-Dependent Protein Kinases / analysis*
  • Cyclic AMP-Dependent Protein Kinases / antagonists & inhibitors
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / chemistry*
  • Humans
  • Luminescent Proteins / chemistry
  • Microscopy, Fluorescence
  • Oligopeptides / chemistry

Substances

  • Bacterial Proteins
  • Fluorescent Dyes
  • Luminescent Proteins
  • Oligopeptides
  • yellow fluorescent protein, Bacteria
  • Arginine
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases
  • Adenosine