Regulation of Myosin Phosphorylation in Smooth Muscle

平滑肌肌球蛋白磷酸化的调节

• 批准号: 6719089
• 负责人: Mitsuo Ikebe
• 金额: $ 39.75万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6719089
• 关键词: antisense nucleic acid; calcium ion; fluorescent dye /probe; immunologic assay /test; laboratory rabbit; molecular site; muscle contraction; muscle stimulant; myosin light chain kinase; myosins; phosphorylases; phosphorylation; protein localization; protein structure function; smooth muscle; three dimensional imaging /topography; transfection

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.

描述（由申请人提供）：本研究的长期目标是了解平滑肌中肌球蛋白轻链（MLC）磷酸化的调节机制，从而调节收缩。平滑肌收缩主要由肌球蛋白轻链磷酸化调节。由于负责肌球蛋白磷酸化的蛋白激酶是Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶，因此收缩起始需要Ca 2+。然而，已经积累的证据表明，Ca 2+非依赖性途径也影响肌球蛋白磷酸化，从而肌肉收缩。本项目中待检验的假设是激动剂激活Ca 2+非依赖性信号传导系统，从而通过抑制肌球蛋白轻链磷酸酶（MLCP）和/或激活平滑肌中的Ca 2+非依赖性MLC激酶来增加MLC磷酸化。MLCP监管有两个关键组成部分，即，CPI 17和MLCP的肌球蛋白结合亚基（MBS），这两者都通过在特定位点磷酸化而增强其MLCP抑制活性。我们将阐明这些MLCP调节组分的生理作用，通过将这些调节剂的磷酸化与平滑肌纤维和细胞中的MLC磷酸化相关联。另一方面，鲜为人知的是，在平滑肌纤维和细胞中的肌球蛋白磷酸化的作用，钙离子非依赖性MLC激酶。该提案将解决这些激酶是否在平滑肌激动剂刺激后的MLC磷酸化中发挥作用的问题。我们将使用两个系统，即，α-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。利用前一个系统，我们将研究力和MLC磷酸化的变化是否与MLCP调节剂和Ca 2+非依赖性激酶活性的变化相关。利用单细胞系统，我们将研究MLC磷酸化的时空变化和MLCP调节组分的定位/易位之间的相关性，以及激动剂刺激后平滑肌细胞中的Ca 2+非依赖性蛋白激酶。这将通过使用超快速3D数字荧光成像技术来实现。我们将使用各种生物化学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，使用dsRNAi或反义寡核苷酸进行基因沉默，基因转染和重组DNA技术。具体目标如下：1.明确MLCP的两种调节成分CPI 17和MBS在激动剂刺激平滑肌收缩过程中的作用。确定负责CPI 17和MBS磷酸化的蛋白激酶; 3.明确钙离子非依赖性MLC激酶在激动剂刺激下肌球蛋白磷酸化中的作用; 4.明确平滑肌中钙非依赖性MLC激酶和MLCP调节组分的定位和转位。