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

描述(由申请人提供)：平滑肌肉被微调，以执行特定于不同的中空器官和它们周围的血管系统的机械功能。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与哮喘相关的呼吸道高反应性。与这些疾病状态有关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。在每一种情况下，近端的原因都是肌动蛋白-肌球蛋白活性的变化引起的收缩亢进。肌动蛋白-肌球蛋白的活性主要通过肌球蛋白(SMM)的磷酸化来调节。具体地说，肌球蛋白轻链激酶(Mylk)对SMM的磷酸化激活了肌动蛋白-肌球蛋白ATPase活性和肌肉收缩。通过这种方式，SMM的活性与Mylk的活性直接相关；事实上，Mylk的活性似乎对平滑肌的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，令人惊讶的是，人们对影响Mylk活性的因素知之甚少。我们的初步研究表明，Mylk-SMM相互作用限制了Mylk的活性。在这项提议中，我们继续检验这一假设，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磷酸化和机械激活。我们将使用溶液动力学来建立更详细的动力学机制，并通过细胞研究来建立体内相关性。这项提议将提供对Mylk调节正常和疾病状态下的平滑肌收缩的机制的直接测量。此外，通过这一提议开发的洞察力将扩展关于非肌肉细胞中非肌肉肌球蛋白如何被激活的知识。

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.