Rac1 in Smooth Muscle

平滑肌中的 Rac1

• 批准号: 8505529
• 负责人: Mitsuo Ikebe
• 金额: $ 43.22万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-05 至 2013-11-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505529
• 关键词: Actin-Binding Protein; Actins; Actomyosin; Address; Adhesions; Agonist; Arteries; Asthma; Binding; Biochemical; Biological; Blood Pressure; Blood Vessels; Cell membrane; Cells; Cytoskeletal Modeling; Cytoskeleton; DNA Sequence Rearrangement; Disease; Dominant-Negative Mutation; Down-Regulation; Electron Microscopy; Extracellular Matrix; Filament; Fluorescence; Fluorescent Antibody Technique; Gene Silencing; Goals; Hypertension; Image; Laboratories; Link; Measures; Mechanics; Membrane; Microfilaments; Microscope; Molecular; Monitor; Movement; Muscle Contraction; Myosin ATPase; Myosin Light Chains; Organ; Pathway interactions; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Protein phosphatase; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Relaxation; Research; Resolution; Rho-associated kinase; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Stream; Structure; Techniques; Tissues; base; constriction; digital; enzyme activity; fluorescence microscope; genetic regulatory protein; improved; inhibitor/antagonist; insight; myosin phosphatase; novel; polymerization; tomography; tool; transmission process; two-photon

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.

描述（由申请人提供）：该项目的目的是阐明Rac1途径调节平滑肌收缩的新型调节机制。平滑肌收缩是调节空心器官（例如气道和动脉）收缩的关键组成部分，因此控制气流和血压，因此，拟议的研究将为血管和气道疾病提供新的见解。平滑肌收缩主要由肌球蛋白轻链（MLC）磷酸化调节，但是，最近的研究表明，肌动蛋白细胞骨架重排可能部分造成收缩的变化。在此提案中，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协调地控制平滑肌收缩。 MLC磷酸化受CA2+依赖性和CA2+独立途径的调节，MLC磷酸酶（MLCP）在后一种机制中起关键作用。 MLCP活性受MYPT1（MLCP的肌球蛋白结合调节亚基）和MLCP特异性抑制剂CPI-17的磷酸化调节。过去的研究集中在负责MYPT1和CPI-17磷酸化（例如Rho激酶和PKC）的激酶上，但对将MyPT1和CPI-17脱磷酸化的蛋白磷酸酶尚无方面了解。根据我们的发现，我们假设RAC途径在激动剂刺激过程中调节MYPT1/CPI-17磷酸酶，该刺激调节MLCP，部分负责RAC依赖于RAC的收缩性调节。 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.我们将首先确定激动剂刺激后是否激活Rac1。评估角色 Rac1的收缩，我们将使用药理学特定的RAC抑制剂，分子生物学工具和基因沉默。此外，我们将阐明RAC激活调节收缩的机制。 MyPT1磷酸酶和/或CPI-17磷酸酶活性的变化将与RAC活性使用生物化学方法一起确定。我们还将检查Rac1激活是否通过波和ARP2/3转移到细胞周围刺激肌动蛋白细胞骨架变化。我们将测量肌动蛋白聚合以及RAC及其下流蛋白的结合。 RAC1及其下游靶标的易位还将使用动脉组织和单个细胞研究，使用两光子数字显微镜，3D数字共聚焦显微镜以及具有超级分辨率分析的总内反射荧光（TIRF）显微镜。 RAC1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实现3D结构图像。