MOLECULAR INTERACTIONS OF MYOSIN PHOSPHATASE SUBUNITS

肌球蛋白的活性是通过调节轻链的磷酸化来调节的，而平滑肌的正常功能需要精确地控制磷酸化的程度。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。虽然MLCK及其受钙-钙调蛋白的调节已被广泛研究，但直到分离出一种异源三聚体蛋白，似乎是主要的肌原纤维肌球蛋白磷酸酶(MP)，人们对相应的磷酸酶知之甚少。MP由靶向亚基、催化亚基和小的非催化亚基(分别为MPT、MPC和MPS)组成。这三个亚基都已被克隆。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度较低，深入的生化和结构分析受到阻碍。我们实验室已经成功地在细菌中表达了MPT和Mpc.MPt复合体。在这一应用中，我们建议通过分析超速离心法、交联法和电子显微镜来全面表征MP及其相互作用。具体地说，我们将1)表征MP亚基之间以及MPT和肌球蛋白之间的相互作用。2)确定MP亚基之间以及MPT与肌球蛋白相互作用的部位。3)识别MPT旋转阴影电子显微图像中的区域。这些研究的结果将开始检验这样的假设，即MPT功能将MPC的催化位置定位在RLC重建电子显微镜、X射线结晶学等。从长远来看，来自该项目的信息可能有助于高血压和动脉粥样硬化等心血管疾病的预防、诊断和治疗。

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.