Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins.
A-kinase anchoring proteins tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of PKA (RII) and the RII-binding domain of the human thyroid RII anchoring protein (Ht31), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to enhanced yellow fluorescent protein (EYFP), Ht31 was linked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP, an increase in emission of the acceptor fluorophore, RII.EYFP, and a decrease in emission from Ht31.ECFP were observed. The emission ratio (acceptor/donor) was increased 2-fold (p < 0.05) in cells expressing Ht31.ECFP and RII.EYFP compared with cells expressing Ht31P.ECFP, the inactive form of Ht31, and RII.EYFP. These results provide the first in vivo demonstration of RII/Ht31 interaction in living cells and confirm previous in vitro findings of RII/Ht31 binding. Using surface plasmon resonance, we also showed that the green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the PKA signaling pathway in living cells.
Ruehr
M L
ML
Department of Molecular Cardiology, Lerner Research Institute, Case Western Reserve University School of Medicine, Cleveland, Ohio 44195, USA.
Zakhary
D R
DR
Damron
D S
DS
Bond
M
M
D054758:A Kinase Anchor Proteins; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014162:Transfection
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D048868:Adaptor Proteins, Signal Transducing; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases