Smooth muscle contractile state determines vascular tone, and ischemic and hypertensive cardiovascular diseases arise in part from abnormalities in smooth muscle cell function. Contraction and relaxation of smooth muscle depend on the phosphorylation- dephosphorylation of myosin light chains by the myosin light chain kinase and myosin phosphatase (PP1M) respectively. Cyclic GMP-dependent protein kinase (cGK) mediates the physiologic relaxation of vascular smooth muscle in response to nitric oxide and cGMP, but the mechanism by which cGK exerts these important effects remains unclear. Using the yeast two-hybrid system, we recently discovered a direct and specific interaction between cGK, a central physiologic mediator of smooth muscle relaxation, and the myosin binding subunit (MBS) of PP1M, which is the critical phosphatase regulating smooth muscle contractility and is widely recognized to be the common target of signaling pathways that modulate smooth muscle tone. The cGK-PP1M interaction is thus an exciting finding that integrates two large areas of investigation relevant to smooth muscle cell and vascular physiology. Our further preliminary data confirm this interaction in GST-fusion protein, immunoprecipitation and confocal microscopy studies. Furthermore, we show functional PP1 activity associated with cGK and have preliminarily identified several potential substrates of cGK in the PP1M-cGK complex. The goals of this proposal are to further characterize the cGK-PP1M interaction and to analyze the mechanisms whereby cGK regulates PP1M activity. SA1 is to characterize the specific residues mediating the cGK-MBS interaction, using site-directed mutagenesis and protein overexpression studies. SA2 is to identify phosphorylated cGk substrates in the PP1M-cytoskeleton complex using 32P-labeled vascular smooth muscle cells, pharmacologic agonists and antagonists and immunoprecipitation methods. SA3 is to study the role of cGK in the regulation of PP1M activity, and will utilize phosphatase assays from vascular smooth muscle cell lysates and measurements of myosin light chain phosphorylation. These studies are important for the understanding of the mechanism by which cGK, and thus nitric oxide, mediates vascular smooth muscle cell relaxation. This proposal and the training program it contains also will provide me with thorough exposure to molecular vascular biology that will enhance the likelihood of my achieving an independent research career as a physician-scientist.
