MULTIFUNCTIONAL CA++/CALMODULIN-DEPENDENT PROTEIN KINASE

多功能Ca 2 +/钙调素依赖性蛋白激酶（CaM激酶）是 一种主要的蛋白激酶， 激素、神经递质和生长因子。 它 对升高细胞内Ca 2+和磷酸化的刺激作出反应 定位于细胞质、细胞膜和细胞骨架中的底物 隔间 它作为一般蛋白激酶的名称使我们认识到， 一个重要的问题--细胞如何利用钙调蛋白激酶作为介质 多种钙离子相关激素，但仍然实现其特异性， 细胞反应？ 我们建议测试的假设， CaM激酶的自动调节特性使其能够解码频率， Ca 2+振荡和尖峰，从而增加了一种新的形式的时间 反应特异性 此外，我们建议测试的假设 将CaM激酶亚型空间定位于不同的细胞内 有助于确定激素的特定部位效应。 这种多聚体酶的时间调节通过其独特的 自磷酸化性质。 我们将继续研究 当一个激活的亚基磷酸化， 全酶中也与钙调蛋白结合的近邻。 这些合作性质将被详细阐述，因为它们构成了 一个模型的基础，通过该模型，激酶可以在 适当的钙峰频率。 该模型将在体外进行测试 使用暴露于Ca 2+和Ca 2+浓度的快速波动的固定化激酶， 通过测试激酶和蛋白质的频率依赖性激活， 它调节上皮细胞中的Cl-通道。 我们新克隆的钙调素激酶亚型之一是针对细胞核 和其他人的细胞骨架提出了空间基础的反应 的特异性 我们将确定催化/监管的位置， 通过电子显微镜观察结构内的靶向域。 我们将 鉴定核定位序列并确定是否 酶作为单体中间体或作为多聚体进入细胞核。 通过激活靶向细胞骨架亚型及其易位 也将被审查。 有了这些基本知识，我们将针对 将工程化构建体导入细胞核和细胞骨架的方法和用途 这些作为功能性Ca 2 +/钙调素在这些位点升高的报告基因 通过几种不同的钙离子相关的信号转导途径。

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.