MYOSIN LIGHT CHAIN KINASE FUNCTION IN SMOOTH MUSCLE

平滑肌中肌球蛋白轻链激酶的功能

• 批准号: 6388871
• 负责人: JAMES T STULL
• 金额: $ 39万
• 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 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6388871
• 关键词: actins; affinity chromatography; aorta; biosensor device; calcium ion; calmodulin; chemical binding; crosslink; cytoskeletal proteins; enzyme activity; genetic promoter element; genetically modified animals; intermolecular interaction; laboratory mouse; laboratory rabbit; 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

The overall objectives of the research projects described in this proposal are to provide insights into how myosin light chain kinase (MLCK) is regulated by Ca2+ / calmodulin in vivo and in vitro and to establish the importance of kinase binding to actin-containing filaments in smooth muscle. Specific aim I will test the hypothesis that Ca2+ / calmodulin activation of MLCK involves sequential binding steps between the two domains of calmodulin with the calmodulin-binding sequence and catalytic core. Low-angle X-ray and neutron-scattering studies will be combined with protein fragment complementation and protein cross-linking to identify sites of interactions between the catalytic core, the regulatory segment, and calmodulin. Specific aim II will determine the temporal and spatial distributions of calmodulin binding to MLCK in vivo with a biosensor MLCK containing fluorescent indicator proteins. The relationship between calmodulin-bound kinase and the extent of myosin regulatory light chain phosphorylation will be established. A biosensor MLCK will be expressed in transgenic mice with a smooth muscle-specific promoter for physiological studies on aortic and bladder tissues. Specific aim III will determine the biochemical mechanism for MLCK binding to actin-containing filaments. We will test the hypothesis that all three motifs cooperatively confer high-affinity binding and that spacing between the motifs is important. Specific aim IV will investigate the importance of MLCK binding to actin-containing filaments in vivo. We will test the hypothesis that the bound kinase is not translocated from F-actin filaments to the cytosol with increases in [Ca2+] in smooth muscle cells in culture and in tissues. Smooth muscle tissues play important roles in many body functions and are crucial for maintaining the homeostatic environment. The investigations proposed in this application address fundamental mechanisms involved in contractile regulation of smooth muscle. Investigations dealing with the primary biochemical pathway controlling smooth muscle contractility are essential for understanding derangements in smooth muscle-based diseases such as asthma, hypertension, erectile dysfunction and irritable bowl syndrome.

本提案中描述的研究项目的总体目标是深入了解肌球蛋白轻链激酶 (MLCK) 在体内和体外如何受 Ca2+/钙调蛋白调节，并确定激酶与平滑肌中含肌动蛋白丝结合的重要性。 具体目标我将测试以下假设：MLCK 的 Ca2+/钙调蛋白激活涉及钙调蛋白的两个结构域与钙调蛋白结合序列和催化核心之间的连续结合步骤。 低角度 X 射线和中子散射研究将与蛋白质片段互补和蛋白质交联相结合，以确定催化核心、调节片段和钙调蛋白之间的相互作用位点。 具体目标 II 将使用含有荧光指示蛋白的生物传感器 MLCK 确定体内钙调蛋白与 MLCK 结合的时间和空间分布。 将建立钙调蛋白结合激酶与肌球蛋白调节轻链磷酸化程度之间的关系。 生物传感器 MLCK 将在带有平滑肌特异性启动子的转基因小鼠中表达，用于主动脉和膀胱组织的生理研究。具体目标 III 将确定 MLCK 与含肌动蛋白丝结合的生化机制。 我们将测试以下假设：所有三个基序共同赋予高亲和力结合，并且基序之间的间距很重要。 具体目标 IV 将研究 MLCK 在体内与含肌动蛋白丝结合的重要性。 我们将测试以下假设：随着培养物和组织中平滑肌细胞中 [Ca2+] 的增加，结合激酶不会从 F-肌动蛋白丝转移至胞质溶胶。 平滑肌组织在许多身体功能中发挥着重要作用，对于维持体内平衡环境至关重要。 本申请中提出的研究涉及平滑肌收缩调节所涉及的基本机制。 研究控制平滑肌收缩力的主要生化途径对于了解平滑肌疾病（如哮喘、高血压、勃起功能障碍和碗易激综合征）的紊乱至关重要。