A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo

We have developed a method for visualizing phosphorylation of proteins in l iving cells using a novel fluorescent indicator composed of two green fluor escent protein (GFP) variants joined by the kinase-inducible domain (KID) o f the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-depende nt protein kinase A (PKA) decreased the fluorescence resonance energy trans fer (FRET) among the flanking GFPs, By transfecting COS-7 cells with an exp ression vector encoding this indicator protein (termed ART for cAMP-respons ive tracer), we were able to visualize activation dynamics of PKA in living cells.