CALCIUM/CALMODULIN ACTIVATED KINASES IN SMOOTH MUSCLE

钙/钙调蛋白依赖的蛋白激酶II(CaM-Kinase II)可以 几种平滑肌收缩的磷酸化及其相关调节 蛋白质在体外，影响其功能活性。这导致了 关于它在调节平滑肌收缩中作用的推测 体内活性，尽管对其具体情况知之甚少 在这个组织中的出现和活动。拟议中的总体目标 研究旨在识别和表征钙/钙调蛋白依赖 在血管中表达的蛋白激酶，而不是MLCK 肌肉(VSM)，并建立其细胞激活特性和 功能底物。 我们已经确定了几个钙离子/钙调蛋白依赖的激酶的池 动脉平滑肌的活动。一个主要的水池是一种可自由溶解的 与大脑中的CaM-Kinase II相似的CaM-Kinase II的形式和 其他来源，因为它是一个巨大的多聚体(550 KDa)，个体50- 60 kDa的激酶亚基。第二个CaM-Kinase II池，到目前为止 未知的亚基组成与富含肌丝的 颗粒物分数。在肌丝提取液中也可以鉴定出 大量的钙/钙调蛋白依赖的激酶活性，可以是 从其他的CaM-Kinase II池中分离出来。基于它不同寻常的 体积小(34-35 kDa)、激活特性和免疫反应性 针对CaM-Kinase II的抗肽抗体，我们假设这是 是一种新型的单体激酶，其结构与 CaM-Kinase II亚单位或紧密结合的催化/调节结构域 相关的激酶。这项提议针对的是1.)生化提纯 和对这种新的激酶活性的分子鉴定，以及 比较其物理和功能性质的表征 到从VSM中纯化的CaM-Kinase II，2)识别所表达的 完整VSM中CaM-Kinase II同工酶及其亚基组成的研究 分子和免疫学方法。评估CaM的激活- 蛋白激酶II在血管紧张素转换酶中的作用及其机制的研究 对活动的监管，以及4.)鉴定蛋白质底物 钙调素-激酶活性在VSM中表达。这些研究应该有助于 阐明CaM-Kinase II及相关蛋白激酶在VSM中的作用 收缩调节，并提供对关系的一般洞察 这些蛋白激酶的结构和功能之间的关系。

Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) can phosphorylate several smooth muscle contractile and associated regulatory proteins in vitro, affecting their functional activity. This has led to speculation regarding its role in regulating smooth muscle contractile activity in vivo, though very little is known specifically about its occurrence and activity in this tissue. The broad goals of the proposed research are to identify and characterize Ca2+/calmodulin-dependent protein kinases, other than MLCK, which are expressed in vascular smooth muscle (VSM), and to establish their cellular activation properties and functional substrates. We have identified several pools of Ca2+/calmodulin-dependent kinase activity in arterial smooth muscle. One major pool is a freely soluble form of CaM-kinase II which is similar to CaM-kinase II from brain and other sources in that it is a large multimer (550 kDa) of individual 50- 60 kDa kinase subunits. A second pool of CaM-kinase II with an as yet unknown subunit composition is associated with a myofilament-rich particulate fraction. Also identifiable in the myofilament extract is a large pool of Ca2+/calmodulin dependent kinase activity which can be resolved from the other pools of CaM-kinase II. Based on its unusually small size (34-35 kDa), activation properties, and immunoreactivity with anti-peptide antibodies to CaM-kinase II, we have hypothesized that this is a novel monomeric kinase with a structure corresponding to the catalytic/regulatory domain of a CaM-kinase II subunit or a closely related kinase. This proposal is aimed at 1.) biochemical purification and molecular identification of this novel kinase activity, and characterization of its physical and functional properties as compared to CaM-kinase II purified from VSM, 2.) identifying the expressed isozymes and subunit composition of CaM-kinase II in intact VSM using molecular and immunological approaches, 3.) assessing activation of CaM- kinase II in VSM and determining the role of autophosphorylation in the regulation of activity, and 4.) identifying protein substrates for the CaM-kinase activities expressed in VSM. These studies should help to clarify the role of CaM-kinase II and related protein kinases in VSM contractile regulation and provide general insight into the relationship between structure and function in these protein kinases.