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）：这项研究的长期目标是了解肌球蛋白轻链（MLC）磷酸化在平滑肌中调节的机制，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Ca2+/钙调蛋白依赖性肌球蛋白轻链激酶，因此开始收缩需要Ca2+。然而，已经积累了证据表明Ca2+独立途径也会影响肌球蛋白磷酸化，从而肌肉收缩。在该项目中要检验的假设是激动剂激活CA2+独立的信号系统，从而通过抑制肌球蛋白光链磷酸酶（MLCP）和/或激活平滑肌中的Ca2+独立的MLC激酶来增加MLC磷酸化。 MLCP有两个关键的MLCP调节组件，即MLCP的CPI17和肌球蛋白结合亚基（MB），两者都通过在特定部位的磷酸化来增强其MLCP抑制活性。我们将通过将这些调节剂与平滑肌纤维和细胞中MLC磷酸化的磷酸化与MLC磷酸化的磷酸化相关联，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激酶在平滑肌纤维和细胞中肌球蛋白磷酸化中的作用知之甚少。该提案将解决以下问题，这些激酶在平滑肌中激动剂刺激时是否在MLC磷酸化中起作用。我们将使用两个系统，即α-毒素皮肤纤维以及具有收缩表型的新鲜分离或培养的平滑肌细胞。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调节剂和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/易位之间的相关性，以及激动剂刺激后平滑肌细胞中的Ca2+独立蛋白激酶。这将通过使用超快速的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目标，包括使用磷酸化位点特异性抗体，用dsRNAI或抗稳态的寡核苷酸，基因转染和重组DNA技术沉默的基因沉默。逐项特定的目的是：1。定义CPI17和MB的作用，这是平滑肌收缩激动剂刺激过程中MLCP的两个调节成分； 2。定义负责CPI17和MBS磷酸化的蛋白激酶； 3。定义Ca2+独立的MLC激酶在激动剂刺激时在肌球蛋白磷酸化中的作用； 4。定义CA2+独立MLC激酶和平滑肌中MLCP调节成分的定位和易位。