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Myosin Phosphatase Regulation in Vascular Smooth Muscle

血管平滑肌中肌球蛋白磷酸酶的调节

基本信息

批准号：
7459932
负责人：
HOWARD K SURKS
金额：
$ 30.91万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7459932
关键词：
Appendix Atherosclerosis Binding Biochemical Blood Vessels Cells Complex Coronary artery Cyclic GMP Cyclic GMP-Dependent Protein Kinases Data Enzymes Event Figs - dietary Goals Hypertension In Vitro Investigation Localized Maintenance Mediating Mediator of activation protein Molecular Muscle Contraction Myofibrils Myosin ATPase Myosin Light Chain Kinase Myosin Light Chains Pathogenesis Pathway interactions Peptides Phosphoric Monoester Hydrolases Phosphorylation Physiological Protein Dephosphorylation Proteins Regulation Relative (related person)Relaxation Reporting Rho-associated kinase Role Signal Pathway Site-Directed Mutagenesis Smooth Muscle Myocytes Societies Spasm Testing Vascular Diseases Vascular Smooth Muscle Vasoconstrictor Agents Vasodilator Agents in vivo insight mutant myosin phosphatase response rho rhoA GTP-Binding Protein

项目摘要

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.

描述（由申请人提供）：血管张力的生理调节对于维持正常血管功能至关重要。血管紧张性降低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）磷酸化程度决定，这是收缩的生化决定因素。MLC磷酸化状态反过来又受到反调节酶肌球蛋白轻链激酶（MLCK）和肌球蛋白磷酸酶（PP 1 M）的相对活性的严格调节。PP 1 M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，PP 1 M受两种血管舒张剂途径和血管收缩剂途径的调节，所述血管舒张剂途径激活PP 1 M，引起VSMC松弛，所述血管收缩剂途径抑制磷酸酶，引起VSMC收缩。RhoA/Rho激酶途径被血管收缩剂激活，抑制PP 1 M活性，导致VSMC收缩。近年来的研究表明RhoA/Rhokinase的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的形成。尽管数据支持RhoA/Rho激酶介导的PP 1 M抑制，但没有报道RhoA/Rho激酶和PP 1 M之间的直接相互作用。RhoA/Rho激酶定位于收缩器和细胞底物的机制以及介导Rho依赖性PP 1 M抑制的事件尚不清楚。我的长期目标是识别肌原纤维收缩器官中的分子，并了解它们的相互作用如何调节PP 1 M活性，以响应血管舒张和血管收缩信号通路。我最近克隆并部分表征了一种令人兴奋的新分子，它直接结合RhoA和MBS，组装了所有三种蛋白质的复合物。这种肌球蛋白磷酸酶-Rho相互作用蛋白（M-RIP）存在于VSMC的PP 1 M复合物中。进一步的初步研究表明，M-RIP与平滑肌细胞中的肌原纤维共定位，并且MRIP本身是Rho激酶底物。该建议的中心假设是M-RIP通过将RhoA/Rho激酶靶向PP 1 M复合物来介导Rho依赖性肌球蛋白磷酸酶抑制，以响应血管收缩剂。在SA 1中，将研究M-RIP、RhoA和MBS之间的复合物形成。使用体外相互作用研究和位点特异性诱变，RhoA-M-RIP-MBS结合的分子决定因素将被定义，并将开发破坏肽和非结合M-RIP突变体。在SA 2中，将通过（a）研究M-RIP磷酸化对PP 1 M调节的作用和（B）M-RIP在PP 1 M的Rho激酶磷酸化中的作用，研究磷酸化在RhoA/Rho激酶依赖性PP 1 M抑制中的作用。在SA 3中，将通过在（a）不含M-RIP的细胞和（B）M-RIP-RhoA或M-RIP-MBS相互作用被破坏的细胞中对血管收缩剂介导的PP 1 M抑制进行生物化学分析，在体内研究MRIP在PP 1 M调节中的功能作用。这些研究有望为RhoA/Rho激酶抑制PP 1 M的分子机制提供有价值的新见解，RhoA/Rho激酶是调节血管张力的VSMC收缩的中心介质。阐明调节血管张力的分子途径对于理解正常血管功能和我们社会中的主要血管疾病至关重要。