MYOSIN LIGHT CHAIN KINASE FUNCTION IN SMOOTH MUSCLE

这项研究项目的总体目标是深入了解肌球蛋白轻链激酶(MLCK)是如何在体内和体外受钙/钙调蛋白调节的，并确定肌球蛋白轻链激酶与肌动蛋白微丝结合的重要性。具体目的我将验证MLCK的钙/钙调蛋白激活涉及钙调蛋白的两个结构域与钙调素结合序列和催化核心之间的顺序结合步骤的假设。低角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部位。特定目的II将利用含有荧光指示蛋白的生物传感器MLCK在体内测定与MLCK结合的钙调蛋白的时空分布。钙调蛋白结合的激酶和肌球蛋白调节轻链磷酸化的程度之间的关系将被建立。一种生物传感器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学研究。特殊目的III将确定MLCK与含有肌动蛋白的细丝结合的生化机制。我们将检验这样的假设，即所有三个基序都协同提供高亲和力结合，并且基序之间的间距很重要。具体目的IV将在体内研究MLCK与含肌动蛋白的细丝结合的重要性。我们将检验这样一种假设，即结合的激酶不会随着培养和组织中平滑肌细胞[Ca~(2+)]的增加而从F-肌动蛋白细丝转移到胞浆中。平滑肌组织在许多身体功能中起着重要作用，对维持体内平衡环境至关重要。在这项申请中提出的研究涉及到涉及平滑肌收缩调节的基本机制。对控制平滑肌收缩的主要生化途径的研究对于理解以平滑肌为基础的疾病，如哮喘、高血压、勃起功能障碍和肠易激综合征的紊乱是必不可少的。

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.