Myosin Light Chain Kinase Function in Smooth Muscle

肌球蛋白轻链激酶在平滑肌中的功能

• 批准号: 7013143
• 负责人: JAMES T STULL
• 金额: $ 38.08万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7013143
• 关键词: actins; affinity chromatography; biosensor device; calcium ion; calmodulin; chemical binding; cytoskeletal proteins; enzyme activity; genetic promoter element; genetically modified animals; inhibitor /antagonist; intermolecular interaction; laboratory mouse; low angle X ray diffraction analysis; molecular site; muscle contraction; muscle pharmacology; muscle proteins; myosin light chain kinase; neutron diffraction; phosphoproteins; urinary bladder; vascular smooth muscle

DESCRIPTION (provided by applicant): The key signal that contracts bladder smooth muscle is an increase in [Ca]i which activates Ca2+/calmodulin-dependent myosin light chain kinase (MLCK), thereby promoting phosphorylation of myosin regulatory light chain (RLC) and initiating contraction. Emerging evidence suggests that functional derangements associated with chronic obstruction of the urinary bladder are associated with significant changes in the contractile protein signaling system. We propose to investigate mechanisms necessary for activation of the bladder smooth muscle contractile apparatus to unravel the complexities of interacting signaling networks. Specific Aim 1. Is MLCK the only kinase that phosphorylates RLC in a Ca -dependent manner in bladder smooth muscle? Transgenic mice expressing biosensor MLCK will be used to determine Ca2+-dependency of activation relative to [Ca2+]i, RLC phosphorylation and contraction. Protein transduction domain inhibitors will be used to test involvement of specific kinases. Smooth muscle MLCK gene ablation will be restricted to smooth muscle cells by a tamoxifen-controlled ablation system. Contractile responsiveness will be measured in isolated bladder tissues and in vivo. Specific Aim 2. Is MLCK targeting to the contractile domain of smooth muscle cells necessary and sufficient for RLC phosphorylation? Gene exons containing the actin-binding motifs and the myosin binding module will be deleted by knock-in procedures and contractile performance of bladder smooth muscle will be characterized in vitro and in vivo. Specific Aim 3. What are the roles ofnonmuscle myosin in smooth muscle? Is it regulated selectively by Rho kinase and PKC? Blebbistatin, a selective inhibitor of nonmuscle myosin II will be used to evaluate contributions of nonmuscle myosin to contractile properties. Nonmuscle RLC phosphorylation will be measured along with smooth muscle RLC under different modes of signaling. We consider the possibility that nonmuscle myosin may contribute to the stability of membrane adhesion sites necessary for cellular force transmission by intracellular contractile domains in bladder myocytes.

描述（由申请人提供）：收缩膀胱平滑肌的关键信号是[Ca]i的增加，其激活Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶（MLCK），从而促进肌球蛋白调节轻链（RLC）的磷酸化并启动收缩。新的证据表明，与慢性膀胱梗阻相关的功能紊乱与收缩蛋白信号系统的显着变化有关。我们建议研究激活膀胱平滑肌收缩装置所需的机制，以揭示相互作用的信号网络的复杂性。具体目标 1. MLCK 是膀胱平滑肌中唯一以 Ca 依赖性方式磷酸化 RLC 的激酶吗？表达生物传感器 MLCK 的转基因小鼠将用于确定相对于 [Ca2+]i 的 Ca2+ 激活依赖性、RLC 磷酸化和收缩。蛋白转导域抑制剂将用于测试特定激酶的参与。平滑肌 MLCK 基因消融将通过他莫昔芬控制的消融系统仅限于平滑肌细胞。将在离体膀胱组织和体内测量收缩反应性。具体目标 2. MLCK 靶向平滑肌细胞的收缩结构域对于 RLC 磷酸化来说是必要且充分的吗？含有肌动蛋白结合基序和肌球蛋白结合模块的基因外显子将通过敲入程序删除，并且将在体外和体内表征膀胱平滑肌的收缩性能。具体目标 3. 非肌肉肌球蛋白在平滑肌中的作用是什么？它是否受 Rho 激酶和 PKC 选择性调节？布雷他汀是一种非肌肉肌球蛋白 II 的选择性抑制剂，将用于评估非肌肉肌球蛋白对收缩特性的贡献。非肌肉 RLC 磷酸化将在不同的信号传导模式下与平滑肌 RLC 一起测量。我们认为非肌肉肌球蛋白可能有助于膀胱肌细胞中细胞内收缩域传递细胞力所必需的膜粘附位点的稳定性。