Rac1 in Smooth Muscle

项目描述（由申请人提供）：本项目旨在阐明Rac1通路调控平滑肌收缩的新调控机制。平滑肌收缩是调节气道和动脉等中空器官收缩的重要组成部分，从而控制气流和血压，因此本研究将为血管和气道疾病的研究提供新的视角。平滑肌收缩主要受肌球蛋白轻链（MLC）磷酸化调节，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。在本研究中，我们假设Rac信号通路通过改变MLC磷酸化和细胞骨架重排来共同控制平滑肌收缩。MLC磷酸化受Ca2+依赖性和Ca2+非依赖性两种途径的调控，MLC磷酸酶（MLCP）在后一种机制中起关键作用。MLCP活性受MLCP肌球蛋白结合调节亚基MYPT1和MLCP特异性抑制剂CPI-17的磷酸化调控。过去的研究主要集中在MYPT1和CPI-17磷酸化的激酶，如Rho激酶和PKC，但对MYPT1和CPI-17去磷酸化的蛋白磷酸酶一无所知。根据我们的研究结果，我们假设Rac途径在激动剂刺激期间调节MYPT1/CPI-17磷酸酶，从而调节MLCP，并部分负责Rac依赖的收缩调节。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来招募粘附连接蛋白，这些蛋白加强了膜粘附连接和肌动球蛋白细丝之间的连接以传递力。我们将首先确定Rac1是否在激动剂刺激后被激活。来评估这个角色

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.