CGMP DEPENDENT PROTEIN KINASE IN VASCULAR BIOLOGY

平滑肌收缩状态决定血管张力，缺血性和高血压性心血管疾病的发生部分是由于平滑肌细胞功能异常。平滑肌的收缩和舒张分别依赖于肌凝蛋白轻链激酶和肌凝蛋白磷酸酶（PP1M）对肌凝蛋白轻链的磷酸化和去磷酸化。环gmp依赖性蛋白激酶（cGK）介导血管平滑肌对一氧化氮和cGMP的生理性松弛，但cGK发挥这些重要作用的机制尚不清楚。利用酵母双杂交系统，我们最近发现了平滑肌松弛的中枢生理介质cGK和PP1M的肌球蛋白结合亚基（MBS）之间的直接和特异性相互作用，PP1M是调节平滑肌收缩性的关键磷酸酶，被广泛认为是调节平滑肌张力的信号通路的共同靶点。因此，cGK-PP1M相互作用是一个令人兴奋的发现，它整合了与平滑肌细胞和血管生理学相关的两个大领域的研究。我们进一步的初步数据在gst融合蛋白、免疫沉淀和共聚焦显微镜研究中证实了这种相互作用。此外，我们发现了与cGK相关的PP1功能活性，并初步确定了PP1M-cGK复合物中cGK的几个潜在底物。本提案的目标是进一步表征cGK-PP1M相互作用，并分析cGK调节PP1M活性的机制。SA1是通过位点定向诱变和蛋白质过表达研究来表征介导cGK-MBS相互作用的特定残基。SA2是利用32p标记的血管平滑肌细胞、药物激动剂和拮抗剂以及免疫沉淀方法，鉴定pp1m -细胞骨架复合物中磷酸化的cGk底物。SA3将研究cGK在PP1M活性调控中的作用，并将利用血管平滑肌细胞裂解物的磷酸酶测定和肌球蛋白轻链磷酸化的测量。这些研究对于理解cGK和一氧化氮介导血管平滑肌细胞松弛的机制非常重要。这份提案和其中包含的培训计划也将使我全面了解分子血管生物学，这将提高我作为一名内科科学家实现独立研究事业的可能性。

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.