Myosin Phosphatase Regulation in Vascular Smooth Muscle

描述（申请人提供）：血管张力的生理调节对维持正常血管功能至关重要。血管张力异常是动脉粥样硬化性心血管疾病和高血压的标志。血管平滑肌细胞（VSMC）是血管的主要细胞成分，其收缩状态调节血管张力。VSMC的收缩状态由肌球蛋白轻链（MLC）的磷酸化程度决定，这是收缩的生化决定因素。反过来，MLC的磷酸化状态受到反调控酶肌球蛋白轻链激酶（MLCK）和肌球蛋白磷酸酶（PP1M）的相对活性的严格调控。PP1M是通过MLCs的去磷酸化介导VSMC松弛的关键磷酸酶。现在了解到，PP1M受激活PP1M的血管舒张途径和抑制磷酸酶的血管收缩途径的调节，导致VSMC收缩。RhoA/ rho激酶通路被血管收缩剂激活，抑制PP1M活性，导致VSMC收缩。最近的研究表明，RhoA/ rho激酶活性可以影响血管疾病的发病机制，包括高血压、冠状动脉痉挛和新内膜形成。尽管有数据支持RhoA/ rho激酶介导的PP1M抑制，但RhoA/ rho激酶与PP1M之间没有直接相互作用的报道。RhoA/ rho激酶定位于收缩器官的机制以及介导rho依赖性PP1M抑制的细胞底物和事件尚不清楚。我的长期目标是确定肌纤维收缩装置中的分子，并了解它们的相互作用如何调节PP1M活性，以响应血管舒张剂和血管收缩剂信号通路。我最近克隆并部分描述了一个令人兴奋的新分子，它直接结合RhoA和MBS，组装了这三种蛋白质的复合体。这种肌球蛋白磷酸酶- rho相互作用蛋白（M-RIP）存在于VSMC的PP1M复合体中。进一步的初步研究表明，M-RIP与VSMCs中的肌原纤维共定位，并且MRIP本身是rho激酶的底物。该建议的中心假设是M-RIP通过靶向RhoA/ rho激酶到PP1M复合体，介导rho依赖性肌球蛋白磷酸酶对血管收缩剂的抑制。

DESCRIPTION (provided by applicant): The physiological regulation of vascular tone is critical to the maintenance of normal vascular function. Abnormalities of vascular tone are a hallmark of atherosclerotic cardiovascular diseases and hypertension. Vascular smooth muscle cells (VSMC) are the principal cellular component of blood vessels and their contractile state regulates vascular tone. VSMC contractile state is determined by the degree of myosin light chain (MLC) phosphorylation, the biochemical determinant of contraction. MLC phosphorylation state, in turn, is tightly regulated by the relative activities of the counter-regulatory enzymes myosin light chain kinase (MLCK) and myosin phosphatase (PP1M). PP1M is the critical phosphatase that mediates VSMC relaxation by dephosphorylation of MLCs. It is now understood that PP1M is regulated by both vasodilator pathways that activate PP1M, causing VSMC relaxation, and vasoconstrictor pathways that inhibit the phosphatase, causing VSMC contraction. The RhoA/Rho-kinase pathway, which is activated by vasoconstrictors, inhibits PP1M activity, leading to VSMC contraction. Recent studies have shown that RhoA/Rhokinase activity can influence the pathogenesis of vascular diseases, including hypertension, coronary artery spasm and neointimal formation. Despite the data supporting RhoA/Rho-kinase-mediated PP1M inhibition, no direct interaction between RhoA/Rho-kinase and PP1M has been reported. The mechanism by which RhoA/Rho-kinase localizes to the contractile apparatus and the cellular substrates and events that mediate Rho-dependent inhibition of PP1M are unknown. My long-term goal is to identify molecules in the myofibrillar contractile apparatus and to understand how their interactions regulate PP1M activity in response to vasodilator and vasoconstrictor signaling pathways. I have recently cloned and partially characterized an exciting new molecule that directly binds both RhoA and MBS, assembling a complex of all three proteins. This Myosin Phosphatase-Rho Interacting Protein (M-RIP) is found in the PP1M complex in VSMC. Further preliminary studies show that M-RIP colocalizes with myofibrils in VSMCs, and MRIP itself is a Rho-kinase substrate. The central hypothesis of this proposal is that M-RIP mediates Rho-dependent myosin phosphatase inhibition in response to vasoconstrictors by targeting RhoA/Rho-kinase to the PP1M complex. In SA 1, the complex formation between M-RIP, RhoA and MBS will be investigated. Using in vitro interaction studies and site-specific mutagenesis, the molecular determinants of RhoA-M-RIP-MBS binding will be defined and both disrupting peptides and non-binding M-RIP mutants will be developed. In SA 2, the role of phosphorylation in RhoA/Rho-kinase-dependent PP 1M inhibition will be studied by (a) investigating the role of M-RIP phosphorylation on PP 1M regulation and (b) the role of M-RIP in Rho-kinase phosphorylation of PP 1M. In SA 3, the functional role of MRIP in regulation of PP 1M will be studied in vivo by biochemical analysis of vasoconstrictor-mediated PP 1M inhibition in (a) cells made devoid of M-RIP and in (b) cells in which M-RIP-RhoA or M-RIP-MBS interactions are disrupted. These studies are expected to provide valuable new insights into the molecular mechanism of PP1M inhibition by RhoA/Rho-kinase, a central mediator of VSMC contraction that regulates blood vessel tone. Elucidating the molecular pathways that regulate blood vessel tone is of utmost importance for understanding both normal vascular function and the major vascular diseases in our society.