CALCIUM/CALMODULIN ACTIVATED KINASES IN SMOOTH MUSCLE

Ca2 +/钙调蛋白依赖性蛋白激酶II（CaM-激酶II）可 磷酸化几种平滑肌收缩和相关调节 蛋白质在体外，影响其功能活性。 这导致 关于其调节平滑肌收缩作用的推测 活性在体内，虽然很少有人知道具体关于其 在这个组织中的发生和活动。 所提议的广泛目标 研究旨在识别和表征Ca2 +/钙调素依赖性 在血管平滑肌中表达的蛋白激酶，而不是MLCK， 肌肉（VSM），并建立其细胞活化特性， 功能底物 我们已经确定了几个池的钙/钙调蛋白依赖性激酶 动脉平滑肌中的活性。 一个主要的池是易溶的 一种类似于脑中CaM-激酶II的CaM-激酶II形式， 其他来源，因为它是一个大的多聚体（550 kDa）的个别50- 60 kDa激酶亚单位。 第二池的钙调蛋白激酶II与一个尚未 未知的亚基组成与富含肌营养素的 颗粒分数 在肌丝提取物中也可识别的是 大量的Ca2 +/钙调蛋白依赖性激酶活性， 与其他CaM-激酶II池分离。 基于其不同寻常的 小尺寸（34 - 35 kDa）、活化特性和免疫反应性 针对CaM-激酶II的抗肽抗体，我们假设， 是一种新的单体激酶，其结构对应于 CaM-激酶II亚基的催化/调节结构域或与之密切相关的 相关激酶 本建议的目的是1）。生物化学纯化 和这种新型激酶活性的分子鉴定，以及 表征其物理和功能特性， 至从VSM纯化的CaM-激酶II，2.）识别所表达的 完整VSM中CaM-激酶II的同工酶和亚基组成 分子和免疫学方法，3.）评估CaM的激活- VSM中的激酶II并确定自磷酸化在其中的作用 活动的规则，以及4.）鉴定蛋白质底物 VSM中表达的CaM激酶活性。 这些研究有助于 阐明CaM激酶II及相关蛋白激酶在VSM中的作用 收缩调节，并提供一般的洞察力的关系， 结构和功能之间的联系。

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.