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的血管功能至关重要。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，MLC是收缩的生化决定因素。反过来，MLC的磷酸化状态又受到反向调节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是一种关键的磷酸酶，通过去磷酸化MLCs来介导VSMC的松弛。现已了解到，PP1M受激活PP1M的血管扩张通路和抑制磷酸酶的血管收缩通路的调节，前者导致VSMC松弛，后者导致VSMC收缩。RhoA/Rho-Kinase通路被血管紧张剂激活，抑制PP1M活性，导致VSMC收缩。最近的研究表明，RhoA/Rhokinase活性可以影响血管疾病的发病机制，包括高血压、冠状动脉痉挛和新生内膜形成。尽管有数据支持RhoA/Rho-Kinase介导的PP1M抑制作用，但RhoA/Rho-Kinase与PP1M之间的直接相互作用尚未见报道。RhoA/Rho-Kinase定位于收缩装置以及介导Rho依赖的抑制PP1M的细胞底物和事件的机制尚不清楚。我的长期目标是识别肌原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活动，以响应血管扩张剂和血管收缩信号通路。我最近克隆了一种令人兴奋的新分子，它直接与RhoA和MBS结合，组装了这三种蛋白质的复合体，并对其进行了部分表征。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与肌原纤维共存于VSMC中，MRIP本身是一种Rho-Kinase底物。这一建议的中心假设是，M-RIP通过靶向PP1M复合体靶向RhoA/Rho-Kinase来介导Rho依赖的肌球蛋白磷酸酶抑制反应。 在SA 1中，将研究M-RIP、RhoA和MBS之间的络合物形成。利用体外相互作用研究和定点突变，将确定RhoA-M-RIP-MBS结合的分子决定因素，并将开发出破坏性多肽和非结合M-RIP突变体。 在SA 2中，将通过(A)M-RIP磷酸化在PP 1M调节中的作用和(B)M-RIP在PP 1M的Rho激酶磷酸化中的作用来研究磷酸化在RhoA/Rho激酶依赖的PP 1M抑制中的作用。 在SA 3中，将通过对(A)缺乏M-RIP的细胞和(B)M-RIP-RhoA或M-RIP-MBS相互作用中断的细胞中血管收缩因子介导的PP 1M抑制的生化分析，研究MRIP在调节PP 1M中的功能作用。这些研究有望为RhoA/Rho-Kinase抑制PP1M的分子机制提供有价值的新见解。RhoA/Rho-Kinase是VSMC收缩的中心调节因子，调节血管张力。阐明调节血管张力的分子途径对于了解正常的血管功能和我们社会中的主要血管疾病都是至关重要的。

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.