Rac1 in Smooth Muscle

描述（由申请人提供）：本项目的目标是阐明Rac 1通路调节平滑肌收缩的新调节机制。平滑肌收缩是调节气道和动脉等中空器官收缩的关键组成部分，从而控制气流和血压，因此，拟议的研究将为血管和气道疾病提供新的见解。平滑肌的收缩主要受肌球蛋白轻链（MLC）磷酸化的调节，然而，最近的研究表明肌动蛋白细胞骨架重排可能是收缩变化的部分原因。在这个提议中，我们假设Rac信号通路通过改变MLC磷酸化和细胞骨架重排协调控制平滑肌收缩。MLC磷酸化受Ca ~（2+）依赖性和非Ca ~（2+）依赖性两种途径的调节，MLC磷酸酶（MLCP）在后者的调节机制中起关键作用。MLCP活性受MYPT 1（MLCP的肌球蛋白结合调节亚基）和CPI-17（MLCP特异性抑制剂）的磷酸化调节。过去的研究主要集中在负责MYPT 1和CPI-17磷酸化的激酶，如Rho激酶和PKC，但对MYPT 1和CPI-17去磷酸化的蛋白磷酸酶一无所知。基于我们的研究结果，我们假设Rac通路在激动剂刺激期间调节MYPT 1/CPI-17磷酸酶，其调节MLCP并且部分负责Rac依赖性收缩调节。由于平滑肌经历了涉及肌动蛋白细胞骨架变化的快速机械可塑性，我们假设激动剂刺激诱导Rac易位到膜，在那里它激活其下游靶点，如WAVE和PAK，以招募粘附连接蛋白，从而加强膜粘附连接和肌动球蛋白丝之间的连接，以传递力。我们将首先确定激动剂刺激后Rac 1是否被激活。为了评估角色， 我们将使用药理学特异性Rac抑制剂和分子生物学工具以及基因沉默。此外，我们将阐明Rac激活调节收缩的机制。MYPT 1磷酸酶和/或CPI-17磷酸酶活性的变化将使用生物化学方法与Rac活性变化一起沿着测定。我们还将研究Rac 1激活是否通过WAVE和ARP 2/3易位到细胞外周来刺激肌动蛋白细胞骨架的变化。我们将测量肌动蛋白聚合，以及Rac与其下游蛋白的结合。还将使用双光子数字显微镜、3D数字共聚焦显微镜和具有超分辨率分析的全内反射荧光（TIRF）显微镜，对动脉组织和单细胞研究Rac 1及其下游靶标的易位。Rac 1依赖性超微结构变化将通过电子显微镜使用断层扫描技术获得3D结构图像来实现。

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the novel regulatory mechanism by which the Rac1 pathway regulates smooth muscle contraction. The smooth muscle contraction is a critical component for the regulation of constriction of hollow organs such as airway and arteries, thus controlling airflow and blood pressure, therefore, the proposed study will provide a novel insight into vascular and airway diseases. Smooth muscle contraction is primarily regulated by myosin light chain (MLC) phosphorylation, however, recent studies have suggested that actin cytoskeletal rearrangement may be in part responsible for the change in contraction. In this proposal, we hypothesize that the Rac signaling pathway concertedly controls smooth muscle contraction by changing MLC phosphorylation and cytoskeletal rearrangement. MLC phosphorylation is regulated by both Ca2+ dependent and Ca2+ independent pathways, and MLC phosphatase (MLCP) plays a key role in the latter mechanism. MLCP activity is regulated by the phosphorylation of MYPT1, a myosin binding regulatory subunit of MLCP, and CPI-17, a MLCP specific inhibitor. The research in the past has focused on the kinases responsible for MYPT1 and CPI-17 phosphorylation, such as Rho kinase and PKC, but nothing is known about the protein phosphatases that dephosphorylate MYPT1 and CPI-17. Based upon our findings, we hypothesize that the Rac pathway regulates MYPT1/CPI-17 phosphatases during agonist stimulation, which regulates MLCP and is in part responsible for the Rac dependent contractile regulation. Since smooth muscle undergoes rapid mechanical plasticity involving actin cytoskeletal change, we hypothesize that agonist stimulation induces Rac translocation to the membrane, where it activates its down-stream targets such as WAVE and PAK to recruit adhesion junction proteins, which strengthen the connections between the membrane adhesion junctions and actomyosin filaments to transmit force. We will first determine if Rac1 is activated after agonist stimulation. To evaluate the role of Rac1 in contraction, we will use pharmacological specific Rac inhibitors and molecular biological tools and gene silencing. Furthermore, we will clarify the mechanism by which Rac activation regulates the contraction. The change in MYPT1 phosphatase and/or CPI-17 phosphatase activities will be determined along with the Rac activity change using biochemical means. We will also examine if Rac1 activation stimulates the actin cytoskeletal change via WAVE and ARP2/3 translocation to the cell periphery. We will measure actin polymerization, and the binding of Rac and its down stream proteins. Translocation of Rac1 and its down-stream targets will also be studied with arterial tissues and single cells using a two-photon digital microscope, 3D digital confocal microscope, and a total internal reflection fluorescence (TIRF) microscope with super resolution analysis. The Rac1 dependent ultrastructural change will be achieved by electron microscopy using tomography technique to obtain 3D structural images.