Regulation of Myosin Phosphorylation in Smooth Muscle

平滑肌肌球蛋白磷酸化的调节

• 批准号: 6873033
• 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/6873033
• 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调节肌球蛋白轻链（MLC）磷酸化从而调节收缩的机制。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 Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶，因此启动收缩需要 Ca2+。然而，已有证据表明 Ca2+ 独立途径也会影响肌球蛋白磷酸化，从而影响肌肉收缩。本项目要测试的假设是激动剂激活不依赖 Ca2+ 的信号系统，从而通过抑制肌球蛋白轻链磷酸酶 (MLCP) 和/或激活平滑肌中不依赖 Ca2+ 的 MLC 激酶来增加 MLC 磷酸化。 MLCP 有两个关键的调控元件，即 CPI17 和 MLCP 的肌球蛋白结合亚基 (MBS)，这两个元件均通过特定位点的磷酸化来增强其 MLCP 抑制活性。我们将通过将这些调节因子的磷酸化与平滑肌纤维和细胞中的 MLC 磷酸化相关联来阐明这些 MLCP 调节成分的生理作用。另一方面，关于 Ca2+ 独立 MLC 激酶对平滑肌纤维和细胞中肌球蛋白磷酸化的作用知之甚少。该提案将解决这些激酶是否在平滑肌激动剂刺激后 MLC 磷酸化中发挥作用的问题。我们将使用两个系统，即α-毒素皮纤维和新鲜分离或培养的具有收缩表型的平滑肌细胞。使用前一个系统，我们将研究力和 MLC 磷酸化的变化是否与 MLCP 调节器和 Ca 2+ 独立激酶的活性变化相关。使用单细胞系统，我们将研究MLC磷酸化的时空变化与MLCP调节成分的定位/易位以及激动剂刺激后平滑肌细胞中Ca2+非依赖性蛋白激酶之间的相关性。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、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。详细的具体目标是： 1. 明确 CPI17 和 MBS（MLCP 的两个调节成分）在激动剂刺激平滑肌收缩过程中的作用； 2. 定义负责CPI17和MBS磷酸化的蛋白激酶； 3. 明确Ca2+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. 定义Ca2+独立MLC激酶和MLCP调节成分在平滑肌中的定位和易位。