Myosin light chain kinase interactions and the rate of smooth muscle activation

描述（由申请人提供）：平滑肌被精细调节，以执行其周围不同中空器官和脉管系统的特定机械功能。改变平滑肌细胞的收缩行为可导致多种病理生理状态，如高血压导致心力衰竭和哮喘相关的气道高反应性。涉及这些疾病状态的机制包括平滑肌可塑性、肌球蛋白异构体移位和调节受损。在每种情况下，近端原因是由肌动蛋白-肌球蛋白活性的变化产生的过度收缩。肌动蛋白-肌球蛋白活性主要通过平滑肌肌球蛋白（SMM）的磷酸化调节。具体来说，肌球蛋白轻链激酶（MYLK）磷酸化SMM可激活肌动蛋白-肌球蛋白atp酶活性和肌肉收缩。这样，SMM的活性与MYLK的活性直接相关；事实上，MYLK活性似乎对平滑肌活动施加了严格的控制。因此，MYLK活性的微小变化与许多慢性和急性人类疾病直接相关也就不足为奇了。然而，我们对影响MYLK活性的因素所知甚少。我们的初步研究表明，MYLK- smm相互作用限制了MYLK的活性。在本研究中，我们将继续验证这一假设，并确定影响MYLK- SMM相互作用的因素。我们将使用体外模型系统来控制我们系统的成分，并使用广泛的生化，动力学和成像技术来同时测量MLYK-SMM相互作用，SMM磷酸化和力学激活。我们将使用溶液动力学来建立更详细的动力学机制和细胞研究来建立体内相关性。这一提议将为MYLK调节正常和疾病状态下平滑肌收缩的机制提供直接测量。此外，通过这一建议发展的见解将扩展非肌肉肌球蛋白如何在非肌肉细胞中被激活的知识。

DESCRIPTION (provided by applicant): Smooth muscle is finely tuned to carry out mechanical functions specific to the different hollow organs and vasculature they surround. Altering the contractile behaviors of smooth muscle cells can lead to a variety of pathophysiological states, such as hypertension resulting in cardiac failure and airway hyperresponsiveness associated with asthma. Mechanisms implicated in these disease states include smooth muscle plasticity, myosin isoform shifts, and impaired regulation. In each case, the proximal cause is hypercontractility generated by a change in actin-myosin activity. Actin-myosin activity is primarily regulated through the phosphorylation of smooth muscle myosin (SMM). Specifically, phosphorylation of SMM by myosin light chain kinase (MYLK) activates actin-myosin ATPase activity and muscle contraction. In this way, the activity of SMM is directly linked to the activity of MYLK; in fact MYLK activity appears to exert tight control over smooth muscle activity. It is thus no surprise that small changes in MYLK activity have been directly linked to many chronic and acute human diseases. Yet remarkably little is known about the factors that influence MYLK activity. Our preliminary studies indicate that MYLK-SMM interactions limit MYLK activity. In this proposal we continue to test this hypothesis and determine the factors that influence MYLK- SMM interactions. We will use an in vitro model system to control the constituents of our system and use a wide range of biochemical, kinetic, and imaging techniques to simultaneously measure MLYK-SMM interactions, SMM phosphorylation, and activation of mechanics. We will use solution kinetics to establish more detailed kinetic mechanisms and cell studies to establish in vivo relevance. This proposal will provide direct measurements of the mechanisms by which MYLK tunes smooth muscle contraction in normal and disease states. Moreover, the insights developed through this proposal will extend of knowledge of how non-muscle myosin is activated in non-muscle cells.