GTPase Regulation of Smooth Muscle Contraction

小的GTP结合蛋白转导控制许多细胞反应的信号。小G蛋白的gtp结合形式激活蛋白激酶和肌动蛋白细胞骨架的高度调节变化似乎是特定gtp酶信号特性的重要特征。肌凝蛋白II是一种基于肌动蛋白的分子马达，它将化学能转化为机械能。平滑肌肌动蛋白-肌球蛋白II相互作用受钙钙调蛋白依赖的肌球蛋白轻链激酶对平滑肌肌球蛋白20 kDa轻链上丝氨酸19的磷酸化调控。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、Kaibuchi和Narumiya及其同事进行的实验表明，gtpase调节平滑肌收缩时钙的敏感性。我们最近发现肌球蛋白轻链激酶受PAK 1的磷酸化调控，PAK 1是蛋白激酶家族的一员，由p21 GTPase结合激活。虽然PAKs通常被认为参与应激反应，但PAK的底物尚未被很好地表征，肌球蛋白轻链激酶是其中一个重要的底物。基于广泛的初步数据，我们提出PAKs通过一种涉及肌球蛋白轻链激酶磷酸化的机制调节平滑肌收缩的钙敏感性。我们现在提出实验来验证这一假设。在Specific Aim 1中描述的实验将研究PAK 1在体外磷酸化肌球蛋白轻链激酶的动力学。具体目标2将通过对皮肤平滑肌进行研究来验证这一假设。具体目标3将通过对完整血管进行实验来验证这一假设。这些实验对我们的假设进行了有力的检验，并将为研究平滑肌收缩时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.