Calcium/Calmodulin Activated Kinases in Smooth Muscle

描述（由申请人提供）：由本基金资助的正在进行的工作主要集中在Ca2+/钙调素依赖性蛋白激酶II （CaMKII）的作用，CaMKII是一种普遍存在的多功能丝氨酸/苏氨酸激酶，具有独特的结构和自我调节特性，在控制血管平滑肌（VSM）收缩和生长反应中。我们在这一领域的前期工作导致了CaMKII同工酶在VSM中的表达的生化和分子表征。利用这些知识和经验，我们开发了许多特定的免疫化学工具和分子方法，首次能够解剖VSM中的CaMKII活性和功能。最近的研究得出了一个普遍的假设，即CaMKIIdelta2是培养大鼠主动脉VSM中表达的主要亚型，它具有独特的结构特征，导致其与非受体酪氨酸激酶和细胞迁移和生长所必需的erk1 /2依赖性信号通路偶联。提出生化和分子方法：（目的1）在培养的VSM细胞中，检测CaMKIIdelta2同工酶在调节ERK1/2的Ca2+依赖性激活、迁移和增殖中的需求和特异性。（目的2）确定新型CaMKIIdelta2/Fyn酪氨酸激酶物理相互作用在camkll依赖性信号传导和VSM细胞迁移和增殖方面的功能意义；（目的3）确定自磷酸化在调节CaMKlIdelta2支架功能和细胞功能方面对Ca2+依赖性信号，迁移和增殖的影响。基于已发表的对VSM中CaMKII基因表达的分析，我们提出CaMKII γ -同工酶的表达变化与VSM收缩的控制有关，而与delta - 2同工酶的表达变化是血管损伤反应的重要组成部分，并使Ca2+依赖性控制VSM细胞的迁移和生长。目的4在大鼠颈动脉损伤再狭窄模型中验证这一假设。建议的工作包括开发新的方法和工具来解决动脉平滑肌中特定CaMKII同工酶的功能和调节，重点是细胞迁移和生长。从这些研究中获得的知识可以潜在地应用于开发新的治疗方法，以治疗已知涉及血管平滑肌增殖和迁移的疾病，如动脉粥样硬化和再狭窄。

DESCRIPTION (provided by applicant): Ongoing work funded by this grant has focused on the role of Ca2+/calmodulin-dependent protein kinase II (CaMKII), a ubiquitous multifunctional serine/threonine kinase with unique structural and autoregulatory properties, in the control of vascular smooth muscle (VSM) contractile and growth responses. Our prior work in this area resulted in the biochemical and molecular characterization of CaMKII isozyme expression in VSM. Using this knowledge and experience, we have developed a number of specific immunochemical tools and molecular approaches that, for the first time, are capable of dissecting CaMKII activity and function in VSM. Recent studies have led to the general hypothesis that CaMKIIdelta2, the principal isoform expressed in cultured rat aortic VSM, has unique structural features that result in its coupling to nonreceptor tyrosine kinases and ERK1/2-dependent signaling pathways necessary for cell migration and growth. Biochemical and molecular approaches are proposed to: (Aim 1) test the requirement for and specificity of CaMKIIdelta2 isozymes in regulating Ca2+depedent activation of ERK1/2, migration, and proliferation in cultured VSM cells. (Aim 2) determine the functional significance of a novel CaMKIIdelta2/Fyn tyrosine kinase physical interaction with respect to CaMKll-dependent signaling and VSM cell migration and proliferation; (Aim 3) determine the effect of autophosphorylation in regulating CaMKlIdelta2 scaffolding functions and cellular function with respect to Ca2+dependent signaling, migration, and proliferation. Based on published analyses of CaMKII gene expression in VSM, we propose that a change in expression from CaMKII gamma-isozymes, which have been associated with control of VSM contraction, to the delta2-isozyme is an important component of the response to vascular injury and enables Ca2+-dependent control of VSM cell migration and growth. Aim 4 tests this hypothesis in the rat carotid artery injury and restenosis model. The proposed work includes the development of novel approaches and tools to address the function and regulation of specific CaMKII isozymes in arterial smooth muscle, focusing on cell migration and growth. Knowledge gained from these studies can potentially be applied to developing new therapeutic approaches for diseases that are known to involve vascular smooth muscle proliferation and migration such as atherosclerosis and restenosis.