Cyclic AMP-dependent protein kinase binding to A-kinase anchoring proteinsin living cells by fluorescence resonance energy transfer of green fluorescent protein fusion proteins

A-kinase anchoring proteins tether cAMP-dependent protein kinase (PRA) to s pecific subcellular locations. The purpose of this study was to use fluores cence resonance energy transfer to monitor binding events in Living cells b etween the type II regulatory subunit of PKA (RII) and the RII-binding doma in 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 baked to enhanced cyan fluorescent protein (ECFP), and these constructs were coexpressed in Chine se hamster ovary cells. Upon excitation of the donor fluorophore, Ht31.ECFP , an increase in emission of the acceptor fluorophore, RII.EYFP, and a decr ease 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 R II.EYFP compared with cells expressing Ht31.ECFP, the inactive form of Ht31 , and RII.EYFP. These results provide the first in vivo demonstration of RI I/Ht31 interaction in Living cells and confirm previous in vitro findings o f RII/Ht31 binding. Using surface plasmon resonance, we also showed that th e green fluorescent protein tags did not significantly alter the binding of Ht31 to RII. Thus, fluorescence resonance energy transfer can be used to d irectly monitor protein-protein interactions of the PKA signaling pathway i n living cells.