MYOSIN LIGHT CHAIN KINASE FUNCTION IN SMOOTH MUSCLE

本提案中描述的研究项目的总体目标是提供关于体内和体外Ca2+ /钙调蛋白如何调节肌球蛋白轻链激酶（MLCK）的见解，并确定激酶与平滑肌中含有肌动蛋白的细丝结合的重要性。具体目的：我将验证Ca2+ / calmodulin激活MLCK的假设，即钙调蛋白的两个结构域之间的顺序结合步骤与钙调蛋白结合序列和催化核心。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。特异性目的II将通过含有荧光指示蛋白的生物传感器MLCK确定钙调素在体内与MLCK结合的时空分布。钙调素结合激酶与肌球蛋白调节轻链磷酸化程度之间的关系将被建立。一个生物传感器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。特异性目的III将确定MLCK与含肌动蛋白丝结合的生化机制。我们将测试所有三个基序合作赋予高亲和力结合的假设，并且基序之间的间距很重要。特异性目的IV将在体内研究MLCK与含肌动蛋白的纤维结合的重要性。我们将通过培养和组织中平滑肌细胞中[Ca2+]的增加来验证结合激酶不会从f -肌动蛋白丝转移到细胞质的假设。平滑肌组织在许多身体功能中发挥重要作用，对维持体内平衡环境至关重要。在本应用中提出的研究涉及平滑肌收缩调节的基本机制。研究控制平滑肌收缩的主要生化途径对于理解平滑肌疾病如哮喘、高血压、勃起功能障碍和肠易激综合征的紊乱至关重要。

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.