MULTIFUNCTIONAL CA++/CALMODULIN-DEPENDENT PROTEIN KINASE

多功能钙/钙调蛋白依赖的蛋白激酶(CaM)是 一种主要的蛋白激酶，它协调着 许多组织中的激素、神经递质和生长因子。它 对升高细胞内钙离子和磷酸化的刺激作出反应 位于细胞质、膜和细胞骨架中的底物 车厢。它被指定为一种普通的蛋白激酶，使我们能够 一个重要的问题--细胞如何利用CaM激酶作为介体 多种与钙离子相关的激素，但仍在其 细胞反应？我们建议检验这一假设 CaM激酶的自身调节特性使其能够解码频率 钙振荡和尖峰，从而增加了一种新的时间形式 反应的特异性。此外，我们建议检验这一假设 CaM激酶亚型在细胞内的空间靶向性 SITES有助于确定激素对特定部位的影响。 这种多聚体酶的时间调节是通过其独特的 自动磷酸化特性。我们将扩大我们的研究范围，展示 当被激活的亚基磷酸化时，这种自动磷酸化就会发生 全酶中的近邻，也与钙调蛋白结合。 我们将详细介绍这些合作属性，因为它们构成了 一种模型的基础，通过该模型可以选择性地在 适当的钙离子尖峰频率。该模型将在体外进行测试 在钙离子和钙离子快速波动的情况下使用固定化激酶 通过测试依赖于频率的激酶的激活和对 它在上皮细胞中调节的CL-通道。 我们新克隆的一种CaM激酶亚型是针对细胞核的 而其他的对细胞骨架的影响表明了反应的空间基础 专一性。我们将确定催化/调节和 通过电子显微镜定位结构中的结构域。我们会 确定核定位序列并确定是否 酶以单体中间体或多聚体的形式进入细胞核。 细胞骨架异构体的靶向及其激活易位 还将接受检查。有了这些基本知识，我们将针对 构建到细胞核和细胞骨架的工程化结构并使用 这些作为功能性钙离子/钙调蛋白在这些部位升高的报告 通过几个不同的钙信号转导通路。

Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is a major protein kinase orchestrating the physiological effects of hormones, neurotransmitters and growth factors in numerous tissues. It responds to stimuli that elevate intracellular Ca2+ and phosphorylates substrates which are localized in the cytosol, membrane and cytoskeletal compartments. Its designation as a general protein kinase brings us to an important question--how does a cell utilize CaM kinase as a mediator of multiple Ca2+-linked hormones, yet still achieve specificity in its cellular responses? We propose to test the hypothesis that the autoregulatory properties of CaM kinase enable it to decode the frequency of Ca2+ oscillations and spikes, thus adding a novel form of temporal response specificity. Furthermore, we propose to test the hypothesis that spatial targeting of CaM kinase isoforms to distinct intracellular sites helps to determine site-specific effects of hormones. Temporal regulation of this multimeric enzyme occurs by its unique autophosphorylation properties. We will extend our studies demonstrating that autophosphorylation occurs when an activated subunit phosphorylates a proximate neighbor in the holoenzyme which also has calmodulin bound. These cooperative properties will be elaborated, since they form the basis for a model by which the kinase may be selectively activated at appropriate Ca2+ spike frequencies. The model will be tested in vitro using immobilized kinase exposed to rapid fluctuations in Ca2+ and in situ by testing frequency dependent activation of the kinase and of the Cl-channel that it regulates in epithelial cells. One of our newly cloned CaM kinase isoforms is targeted to the nucleus and others to the cytoskeleton suggesting a spatial basis for response specificity. We will identify the position of catalytic/regulatory and targeting domains within the structure by electron microscopy. We will identify the nuclear localization sequence and determine whether the enzyme enters the nucleus as a monomeric intermediate or as a multimer. Targeting of the cytoskeletal isoform and its translocation by activation will also be examined. With this basic knowledge, we will target engineered constructs to the nucleus and to the cytoskeleton and use these as reporters of functional Ca2+/calmodulin elevated at these sites by several distinct Ca2-linked signal transduction pathways.