The cAMP dependent-protein kinase (PKA) signaling pathway may be regulated via compartmentalization of its components, whereby A- kinase anchoring proteins (AKAPs) tether PKA to subcellular areas giving each receptor binding event specificity. The purpose of this study is to investigate the binding kinetics between (1) the regulatory subunit of PKA (R) and AKAP100 or Ht31, the RII binding region of an AKAP, (2) between RII and the catalytic subunit (C) of PKA, or (3) AKAP100 or C and the ryanodine receptor (RyR). Each protein will be fused to a derivative of the green fluorescent protein (GFP) so that the excitation- emission spectra of the two fluorescent proteins overlap, and these fusion proteins will be expressed in CHO cells. Fluorescence resonance energy transfer (FRET) will be used to monitor binding events between each pair of these proteins. As the fusion proteins bind, energy will be transferred from one fluorophore (donor) to the other (acceptor). Binding can be determined by measuring the emission of the acceptor protein. This will allow for real-time measurement of PKA-dependent binding events in live cells. Intracellular Ca2+ cycling will also be measured in cells transfected with AKAP100 and the RyR to determine what effect this AKAP has on PKA-dependent alterations in Ca2+ efflux from the RyR. This study will help to elucidate the time course by which R interacts with AKAP and what influence the C subunit has on this binding. Also, these experiments will aid in determining if AKAP100 influences Ca2+ efflux from the RyR. Studying the mechanisms by which PKA is sequestered into specific subcellular pools may provide valuable information for cases where the PKA signaling pathway no longer functions properly, such as during cardiac hypertrophy.
| Ruehr, Mary L; Russell, Mary A; Ferguson, Donald G et al. (2003) Targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation and function of the skeletal muscle ryanodine receptor. J Biol Chem 278:24831-6 |
| Ruehr, M L; Zakhary, D R; Damron, D S et al. (1999) 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. J Biol Chem 274:33092-6 |
