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：该项目的目标是了解 Rho/MLCP（肌球蛋白轻链磷酸酶）通路调节血管中肌球蛋白轻链 (MLC) 磷酸化和收缩的机制。 平滑肌。在本提案中，我们将根据我们最近对 MBS/RhoA 结合分子的发现，阐明 RhoA/MLCP 途径的新型调节蛋白 p116Rip 的功能。虽然已经对 Rho 激酶在平滑肌中的调节作用进行了大量研究，但对于其作用尚不清楚。 PKN（另一种 RhoA 下游激酶）的功能。我们的初步研究提出了 PKN 在 RhoA/MLCP 通路调节中发挥作用的可能性，我们将研究 PKN 在激动剂诱导的平滑肌收缩调节中的功能和调节。根据我们的初步研究，我们对 PKN 和 p116Rip 的调节功能提出以下假设。激动剂后 在刺激下，RhoA 易位到膜上，并且对活性 RhoA 具有结合亲和力的胞质 PKN 被招募到膜上。 PKN 被激活并维持 RhoA 的膜结合，从而延长 RhoA 活性。另一方面，p116Rip在肌动球蛋白纤维处与肌球蛋白和MBS结合并激活MLCP反应，从而促进MLC去磷酸化。 P116Rip，肌动球蛋白 丝，与胞质 RhoA 相互作用以防止易位至膜，从而减弱 RhoA 激活。这些作用导致平滑肌中 MLCP 活性增加和 MLC 磷酸化下调。在本提案中，我们将验证这一假设。我们将首先使用 siRNA 方法分别消除 p116Rip 和 PKN，并研究特定 siRNA 对激动剂诱导的 MLC20 磷酸化和肌肉收缩变化的影响。一旦我们确定了p116Rip和PKN在MLC20磷酸化调节中的功能，我们将研究p116Rip和PKN活性的调节机制。 已知Rho及其下游分子的激活与这些分子向膜的易位密切相关。为了进一步验证我们的假设，我们将通过使用分化的平滑肌细胞的 3D 数字成像分析来研究刺激后这些分子定位的时空变化。为了实现这一目标，我们将引入荧光蛋白 (FP) 标记 使用蛋白质递送技术的调节蛋白质。我们还将使用我们新开发的全内反射荧光（TIRF）显微镜来监测细胞近膜区域的变化。 此外，我们将通过FRET分析研究p116Rip和PKN与其靶分子的结合，从而监测活细胞中分子相互作用的时空变化。该项目将阐明激动剂诱导血管平滑肌收缩的机制，这将为血管收缩的调节提供新的见解，并有助于心血管疾病的发病机制。