Regulation of Myosin Phosphorylation in Smooth Muscle

平滑肌肌球蛋白磷酸化的调节

• 批准号: 8488458
• 负责人: Mitsuo Ikebe
• 金额: $ 13.2万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2013-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488458
• 关键词: Actomyosin; Address; Affinity; Agonist; Attenuated; Binding; Blood Vessels; Cardiovascular Diseases; Cells; Down-Regulation; Fiber; Filament; Fluorescence Resonance Energy Transfer; Goals; Hypertension; Image Analysis; Life; Link; Membrane; Monitor; Muscle Contraction; Myosin ATPase; Myosin Light Chains; Pathogenesis; Pathway interactions; Phosphorylation; Phosphotransferases; Play; Protein Dephosphorylation; Proteins; Reaction; Recruitment Activity; Regulation; Rho-associated kinase; Role; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Stream; Techniques; Vascular Diseases; Vascular Smooth Muscle; base; blood pressure regulation; constriction; digital imaging; fluorescence microscope; genetic regulatory protein; improved; insight; myosin phosphatase; novel; prevent; rho

ABSTRACT: The goal of this project is to understand the mechanism by which the Rho/MLCP (myosin light chain phosphatase) pathway regulates myosin light chain (MLC) phosphorylation and contraction in vascular smooth muscle. In the present proposal, we will clarify the function of p116Rip, the novel regulatory protein of the RhoA/MLCP pathway, based upon our recent findings of this MBS/RhoA binding molecule. While a number of studies have been done on the regulatory role of Rho kinase in smooth muscle, nothing is known about the function of PKN, another RhoA down-stream kinase. Our PRELIMINARY STUDIES raise the possibility that PKN plays a role in the regulation of the RhoA/MLCP pathway, and we will study the function and regulation of PKN in the agonist induced regulation of smooth muscle contraction. Based upon our PRELIMINARY STUDIES, we propose the following hypothesis of the regulatory function of PKN and p116Rip. Upon agonist stimulation, RhoA translocates to the membrane and cytosolic PKN, which has a binding affinity for active RhoA, is recruited to the membrane. PKN becomes activated and sustains the membrane binding of RhoA, thus prolongs RhoA activity. On the other hand, p116Rip associates with myosin and MBS at the actomyosin fiber and activates MLCP reaction, thus facilitating MLC dephosphorylation. P116Rip, at the actomyosin filaments, interacts with cytosolic RhoA to prevent translocation to the membrane, thus attenuating RhoA activation. These effects result in the increase in MLCP activity and down-regulation of MLC phosphorylation in smooth muscle. In this proposal, we will verify this hypothesis. We will first use a siRNA approach to eliminate p116Rip and PKN, respectively, and study the effects of the specific siRNAs in the agonist induced change in MLC20 phosphorylation and muscle contraction. Once we identify the function of p116Rip and PKN in the regulation of MLC20 phosphorylation, we will study the regulatory mechanism of p116Rip and PKN activity. It has been known that the activation of Rho and its downstream molecules are closely related to the translocation of these molecules to the membrane. To further address our hypothesis, we will study the spatio-temporal change in the localization of these molecules after stimulation by using 3D digital imaging analysis of the differentiated smooth muscle cells. To achieve this, we will introduce fluorescent protein (FP)-tagged regulatory proteins using the protein delivery technique. We will also use our newly developed total internal reflection fluorescence (TIRF) microscope to monitor the change in the near-membrane domain of the cells. Furthermore, we will study the binding of p116Rip and PKN with their target molecules by using FRET analysis, thus monitoring the spatio-temporal change in the interaction of the molecules in living cells. The proposed project will clarify the mechanism by which agonists induce vascular smooth muscle contraction, which should provide novel insights into the regulation of vascular constriction and contribute to the pathogenesis of cardiovascular diseases.

摘要：本项目旨在了解肌球蛋白轻链磷酸酶（myosin light chain phosphatase, Rho/MLCP）通路调控血管中肌球蛋白轻链（myosin light chain， MLC）磷酸化和收缩的机制