GTPase Regulation of Smooth Muscle Contraction

小的GTP结合蛋白传递控制一系列细胞反应的信号。由GTP结合形式的小G蛋白激活的蛋白激酶和高度调控的肌动蛋白细胞骨架的变化似乎是特定GTP酶信号特性的重要特征。肌球蛋白II是一种基于肌动蛋白的分子马达，可以将化学能转化为机械功。肌动蛋白-肌球蛋白II的相互作用受钙-钙调蛋白依赖酶肌球蛋白轻链蛋白轻链上SER19的磷酸化调节。最近的报道表明，GTP酶和肌球蛋白磷酸化相互作用，调节肌动蛋白细胞骨架和平滑肌收缩。Somlyo、Kaibuhi和Narumiya及其同事进行的实验表明，GTP酶调节平滑肌收缩的钙敏感性。我们最近发现，肌球蛋白轻链激酶受PAK1的磷酸化调节，PAK1是由p21GTPase结合激活的蛋白激酶家族中的一个成员。虽然PAK通常被认为与应激反应有关，但PAK的底物还没有得到很好的描述，肌球蛋白轻链激酶是一个重要的底物。基于大量的初步数据，我们认为PAKs通过涉及肌球蛋白轻链激酶磷酸化的机制来调节平滑肌收缩的钙敏感性。我们现在提议用实验来检验这一假设。在特定目的1中描述的实验将在体外研究PAK 1对肌球蛋白轻链激酶磷酸化的动力学。《特定目标2》将通过对皮肤光滑的肌肉进行研究来检验这一假说。具体目标3将通过在完整的血管上进行实验来检验这一假设。这些实验是对我们假设的有力检验，它们将为调节平滑肌收缩的钙敏感性的分子机制提供重要的见解。

Small GTP binding proteins transduce signals that control a host of cellular responses. The activation of protein kinases by the GTP-bound form of small G proteins and highly regulated changes in the actin cytoskeleton appear to be important characteristics of the signalling properties of specific GTPases. Myosin II is an actin based molecular motor that converts chemical energy into mechanical work. The actin- myosin II interaction in smooth muscles is regulated by the phosphorylation of ser 19 on the 20 kDa light chain of smooth muscle myosin by the calcium-calmodulin dependent enzyme myosin light chain kinase. Recent reports have suggested that GTPases and myosin phosphorylation interact to regulate the actin cytoskeleton and smooth muscle contraction. Experiments performed by Somlyo, Kaibuchi and Narumiya and their co-workers have shown that GTPases regulate the calcium sensitivity of smooth muscle contraction. We have recently discovered that myosin light chain kinase is regulated by phosphorylation by PAK 1, a member of a family of protein kinases that is activated by the binding of the p21 GTPase. Although PAKs are generally thought to be involved in responding to stress, substrates for PAK's are not well characterized and myosin light chain kinase represents an important one. Based on extensive preliminary data, we propose that PAKs regulate the calcium sensitivity of smooth muscle contraction by a mechanism that involves the phosphorylation of myosin light chain kinase. We now propose experiments to test this hypothesis. The experiments described in Specific Aim 1 will investigate the kinetics of myosin light chain kinase phosphorylation by PAK 1, in vitro. Specific Aim 2 will test the hypothesis by performing studies on skinned smooth muscles. Specific Aim 3 will test the hypothesis by performing experiments on intact blood vessels. These experiments represent a powerful test of our hypothesis and they will provide important insights into the molecular mechanisms that regulate the calcium sensitivity of smooth muscle contraction.