A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo.
We have developed a method for visualizing phosphorylation of proteins in living cells using a novel fluorescent indicator composed of two green fluorescent protein (GFP) variants joined by the kinase-inducible domain (KID) of the transcription factor cyclic adenosine monophosphate (cAMP)-responsive element binding protein (CREB). Phosphorylation of KID by the cAMP-dependent protein kinase A (PKA) decreased the fluorescence resonance energy transfer (FRET) among the flanking GFPs. By transfecting COS-7 cells with an expression vector encoding this indicator protein (termed ART for cAMP-responsive tracer), we were able to visualize activation dynamics of PKA in living cells.
Nagai
Y
Y
Department of Functional Genomics, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Tokyo 113-8510, Japan.
Miyazaki
M
M
Aoki
R
R
Zama
T
T
Inouye
S
S
Hirose
K
K
Iino
M
M
Hagiwara
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014162:Transfection
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases