Regulation of Myosin Phosphorylation in Smooth Muscle

描述(申请人提供)：这项研究的长期目标是了解肌球蛋白轻链(MLC)磷酸化在平滑肌中被调节从而调节收缩的机制。肌肉收缩主要受肌球蛋白轻链磷酸化的调节。由于负责肌球蛋白磷酸化的蛋白激酶是钙/钙调蛋白依赖的肌球蛋白轻链激酶，因此钙是启动收缩所必需的。然而，越来越多的证据表明，钙不依赖的通路也会影响肌球蛋白的磷酸化，从而影响肌肉收缩。本研究的假设是，激动剂通过抑制肌球蛋白轻链磷酸酶(MLCP)和/或激活非钙依赖的MLC激酶，激活非钙依赖的信号系统，从而增加MLC的磷酸化。MLCP有两个关键的调节成分，即CPI17和MLCP的肌球蛋白结合亚单位(MBS)，这两个成分都可以通过特定位点的磷酸化来增强MLCP的抑制活性。我们将通过将这些调节因子的磷酸化与MLC在平滑肌纤维和细胞中的磷酸化联系起来，来阐明这些MLCP调节成分的生理作用。另一方面，对于非钙依赖的MLC激酶在肌球蛋白在肌球蛋白磷酸化中的作用，人们知之甚少。该提案将解决这样一个问题，即这些激酶是否在MLC激动剂刺激下的MLC磷酸化过程中发挥作用。我们将使用两个系统，即阿尔法毒素皮肤纤维和新鲜分离或培养的具有收缩表型的平滑肌细胞。利用前者，我们将研究力和MLC磷酸化的变化是否与MLCP调节器和钙非依赖性激酶活性的变化有关。利用单细胞系统，我们将研究激动剂刺激后MLC磷酸化的时空变化与MLCP调节成分的定位/移位以及细胞内钙非依赖性蛋白激酶的相关性。这将通过使用超高速3D数字荧光成像技术来实现。我们将使用各种生化和分子生物学技术来实现这一目标，包括使用磷酸化位点特异性抗体、dsRNAi或反义寡核苷酸的基因沉默、基因转染和重组DNA技术。具体的目标是：1.确定MLCP的两个调节成分CPI17和MBS在激动剂刺激平滑肌收缩中的作用；2.确定参与CPI17和MBS磷酸化的蛋白激酶；3.确定钙非依赖性MLC激酶在激动剂刺激下肌球蛋白磷酸化中的作用；4.确定非钙依赖的MLC激酶和MLCP调节成分在平滑肌中的定位和转位。

DESCRIPTION (provided by applicant): The long-term objective of this research is to understand the mechanism by which myosin light chain (MLC) phosphorylation is regulated in smooth muscle thus regulating contraction. Smooth muscle contraction is primarily regulated by myosin light chain phosphorylation. Since the protein kinase responsible for phosphorylation of myosin is Ca2+/calmodulin-dependent myosin light chain kinase, Ca2+ is required for initiation of contraction. However, evidence has been accumulated that Ca2+independent pathways also influences myosin phosphorylation thus muscle contraction. The hypothesis to be tested in this project is that agonists activate the Ca2+independent signaling systems, thus increasing MLC phosphorylation by the inhibition of myosin light chain phosphatase (MLCP) and/or by activating the Ca2+independent MLC kinases in smooth muscle. There are two key MLCP regulatory components, i.e., CPI17 and myosin binding subunit (MBS) of MLCP, both of which are enhanced by their MLCP inhibitory activity by phosphorylation at specific sites. We will clarify the physiological role of these MLCP regulatory components by correlating the phosphorylation of these regulators with MLC phosphorylation in smooth muscle fiber and cells. On the other hand, little is known about the role of Ca2+independent MLC kinases on myosin phosphorylation in smooth muscle fiber and cells. The proposal will address the question whether these kinases play a role in MLC phosphorylation upon agonist stimulation in smooth muscle. We will use the two systems, i.e., alpha-toxin skinned fiber and freshly isolated or cultured smooth muscle cells having contractile phenotype. Using the former system, we will study whether the change in force and MLC phosphorylation correlated with the change in the activity of MLCP regulators and Ca 2+independent kinases. Using the single cell system, we will study the correlation between the spatio-temporal change in MLC phosphorylation and the localization/translocation of the MLCP regulatory components, and the Ca2+independent protein kinases in the smooth muscle cells after agonist stimulation. This will be achieved by using ultra fast 3D digital fluorescence imaging techniques. We will use various biochemical and molecular biological techniques to achieve the goal including the use of phosphorylation site-specific antibodies, gene silencing with dsRNAi or anti-sense oligonucleotides, gene transfection, and recombinant DNA technology. The itemized specific aims are: 1. To define the role of CPI17 and MBS, two regulatory components of MLCP during agonist stimulation of smooth muscle contraction; 2. To define the protein kinases responsible for the phosphorylation of CPI17, and MBS; 3. To define the role of Ca2+independent MLC kinases in myosin phosphorylation upon agonist stimulation; 4. To define the localization and translocation of Ca2+independent MLC kinases and the MLCP regulatory components in smooth muscle.