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深入了解肌球蛋白轻链激酶（MLCK）是如何在体内和体外由Ca 2 + /钙调蛋白调节的，并建立激酶结合平滑肌中含肌动蛋白的细丝的重要性。 具体目标我将测试的假设，钙/钙调蛋白激活MLCK涉及钙调蛋白的两个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X射线和中子散射研究将结合蛋白质片段互补和蛋白质交联，以确定催化核心，调节段和钙调蛋白之间的相互作用的网站。 具体目标II将使用含有荧光指示蛋白的生物传感器MLCK来确定钙调素与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将在具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。具体目标III将确定MLCK结合到肌动蛋白纤维的生化机制。 我们将测试的假设，所有三个图案合作赋予高亲和力结合，图案之间的间距是很重要的。 具体目标IV将调查MLCK结合肌动蛋白在体内的重要性。 我们将测试的假设，即结合激酶是不是从F-肌动蛋白丝易位到胞质溶胶中的[Ca 2 +]在平滑肌细胞培养和组织中的增加。 平滑肌组织在许多身体功能中起重要作用，并且对于维持稳态环境至关重要。 本申请中提出的研究解决了涉及平滑肌收缩调节的基本机制。 控制平滑肌收缩性的主要生化途径的研究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.