MOLECULAR INTERACTIONS OF MYOSIN PHOSPHATASE SUBUNITS

平滑肌肌球蛋白的活性由调节轻链的磷酸化调节，并且平滑肌的正常功能需要精确控制磷酸化的程度。这种控制是通过调节肌球蛋白轻链激酶（MLCK）和相应的磷酸酶的活性来实现的。而MLCK和它的调节Ca 2 +-钙调蛋白被广泛研究，鲜为人知的是，直到分离的异源三聚体蛋白，似乎是主要的肌原纤维肌球蛋白磷酸酶（MP）的相应的磷酸酶。MP由靶向亚基、催化亚基和非催化亚基（分别为Mpt、Mpc和Mps）组成。所有三个亚单位都已克隆。虽然文献中已经报道了这种MP的性质，但由于其天然丰度和纯化产率低，阻碍了深入的生化和结构分析。我们的实验室已经成功地在细菌中表达Mpt和Mpc.MPt复合物。在此应用中，我们建议充分表征MP及其相互作用的分析超离心，交联和电子显微镜。具体而言，我们将1）表征MP亚基之间以及Mpt与肌球蛋白之间的相互作用。2)确定MP亚基之间以及Mpt和肌球蛋白之间的相互作用位点。3)识别Mpt旋转阴影电子显微照片中的区域。从这些研究的结果将开始，以测试这样的假设，即Mpt功能定位Mpc的催化位点在RLC重建电子显微镜，X射线晶体学等，从长远来看，来自这个项目的信息可能有助于预防，诊断和治疗心血管疾病，如高血压和动脉粥样硬化。

The activity of smooth muscle myosin is regulated by phosphorylation of the regulatory light chain, and proper functioning of smooth muscles requires precise control of the extent of the phosphorylation. This control is achieved by regulating the activities of myosin light chain kinase (MLCK) and the corresponding phosphatase. Whereas MLCK and its regulation by Ca2+-calmodulin is extensively studied, little was known about the corresponding phosphatase until the isolation of a heterotrimeric protein that appears to be the major myofibrillar myosin phosphatase (MP). MP is composed of a targeting, catalytic and small non-catalytic subunits (Mpt, Mpc and Mps respectively). All three subunits have been cloned. Although reports of the properties of this MP have appeared in the literature, incisive biochemical and structural analyses is hampered by its low natural abundance and purification yield. Our laboratory has succeeded in expressing Mpt and the Mpc.MPt complex in bacteria. In this application we propose to fully characterize MP and its interactions by analytical ultracentrifugation, crosslinking and electron microscopy. Specifically, we will 1) Characterize the interactions between the subunits of MP and between Mpt and myosin. 2) Identify the sites of interactions between the MP subunits and between Mpt and myosin. 3) Identify the regions in the rotary shadowing electron micrographs of Mpt. Results from these studies will begin to test such hypotheses that Mpt functions to position the catalytic site of Mpc at the RLC reconstruction electron microscopy, X-ray crystallography etc. In the long term, information derived from this project may contribute toward the prevention, diagnosis and treatment of such cardiovascular diseases as hypertension and atherosclerosis.