Regulation of Myosin Phosphorylation in Smooth Muscle

摘要：本项目的目的是了解Rho/MLCP(肌球蛋白轻链磷酸酶)途径调节血管内肌球蛋白轻链(MLC)磷酸化和收缩的机制。 平滑的肌肉。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许多研究，但对Rho激酶在血管中的调节作用知之甚少 另一种RhoA下游激酶--PKN的功能。我们的初步研究提出了PKN在RhoA/MLCP通路中发挥调节作用的可能性，并将研究PKN在激动剂诱导的平滑肌收缩调节中的作用和调节。在前期研究的基础上，我们对PKN和p116Rip的调控功能提出了如下假说。关于激动剂 刺激后，RhoA移位到膜上，胞内与活性RhoA有亲和力的PKN被募集到膜上。PKN被激活，维持与RhoA的膜结合，从而延长RhoA的活性。另一方面，p116Rip在肌动球蛋白纤维上与肌球蛋白和MBS结合，激活MLCP反应，从而促进MLC去磷酸化。P116Rip，肌动球蛋白 细丝，与胞浆中的RhoA相互作用，防止移位到膜上，从而减弱RhoA的激活。这些作用导致平滑肌细胞MLCP活性增加，MLC磷酸化水平下调。在这项提议中，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，并研究特定的siRNA在激动剂诱导的MLC20磷酸化和肌肉收缩变化中的作用。一旦我们确定了p116Rip和PKN在调节MLC20磷酸化中的功能，我们将研究p116Rip和PKN活性的调控机制。 已经知道，Rho及其下游分子的激活与这些分子向膜上的移位密切相关。为了进一步解决我们的假设，我们将通过对分化的平滑肌细胞进行3D数字成像分析，来研究刺激后这些分子的定位的时空变化。为了实现这一点，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结构域的变化。 此外，我们还将通过FRET分析来研究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。

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.