MULTIFUNCTIONAL CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE

细胞内钙离子起第二信使的作用 许多激素、生长、因子、神经递质和 其他细胞外信号。尽管钙的作用 似乎不是由一种普遍的生化机制来调节的， 这项提议旨在测试某些人是否可能 钙的生物化学和生理效应可能是由 通过磷酸化一系列不同的底物蛋白质 由多功能钙/钙调蛋白依赖性催化 蛋白激酶(CaM)。该提案将审查其 钙的调节可以通过自动磷酸化，将决定 哪种类型的受体被激酶取代，并将扩大 肌动蛋白在细胞周期调控中的作用 碳水化合物和氨基酸的代谢和核内 膜破裂。定点突变将被用于 阻断或模拟涉及的自动磷酸化位点 在激活或抑制CaM激酶的过程中。该机制的目的是 将对自动磷酸化的调节进行研究。这个 蛋白激酶调节的抑制性“伪底物”假说 用缺乏抑制结构域的截断突变体进行测试。 这些实验将产生一种钙不依赖的形式 用于检测其众多细胞中的两个的激酶 功能。一种这样的功能是钙依赖调节 碳水化合物和氨基酸的代谢将在 鲁伯H35肝癌细胞株。激酶抑制性抗体或 不依赖钙的激酶制剂将被显微注射 进入肝癌细胞和荷尔蒙刺激能力 调节丙酮酸激酶和苯丙氨酸等酶 羟基酶检测。类似的研究也被提出来检验 磷酸肌酸激酶在钙离子引发的心肌细胞损伤中的作用 海胆卵中的核膜，它的另一个假设 功能。最后，钙调素蛋白激酶参与调节 受体通过钙离子通道评估钙离子的作用 磷脂酰肌醇和其他信号系统将 检查过了。为了实现这一点，一种激酶的模型底物， 酪氨酸羟化酶基因将被导入瑞士3T3细胞 它们有许多受体，具有不同的作用机制 提升钙质。细胞会受到信号的刺激，比如 血管加压素、蛙皮素和血小板衍生生长因子 通过其磷酸化来监测该激酶的激活 酪氨酸羟基酶。

Intracellular calcium serves as a second messenger to mediate the effects of many hormones, growth, factors, neurotransmitters and other extracellular signals. Although the actions of calcium do not appear to be mediated by a universal biochemical mechanism, this proposal aims to test the possibility that certain of the biochemical and physiological effects of calcium may be mediated by phosphorylation of a diverse array of substrate proteins catalyzed by the multifunctional calcium/calmodulin-dependent protein kinase (CaM kinase). The proposal will examine its regulation by calcium can by autophosphorylation, will determine which type of receptor is subserved by the kinase and will expand on the putative role of the kinase in the regulation of carbohydrate and amino acid metabolism and in nuclear membrane breakdown. Site directed mutagenesis will be used to either block or mimic autophosphorylation sites that are involved in activating or inhibiting the CaM kinase. The mechanism for regulation by autophosphorylation will be investigated. The inhibitory "pseudosubstrate" hypothesis of kinase regulation will be tested with truncation mutants lacking the inhibitory domain. These experiments will generate a calcium-independent form of the kinase to be used in examining two of its many cellular functions. One such function, the calcium-dependent regulation of carbohydrate and amino acid metabolism will be examined in Reuber H35 hepatoma cells. Inhibitory antibodies to the kinase or calcium-independent kinase preparations will be microinjected into the hepatoma cells and the ability of hormonal stimulation to regulate enzymes such as pyruvate kinase and phenylalanine hydroxylase examined. Similar studies are proposed to examine the role of the kinase in the calcium-triggered breakdown of nuclear membrane in sea urchin eggs, another of its postulated functions. Finally, involvement of the CaM kinase in mediating the effect of receptors that evaluate calcium via the phosphatidylinositol and other signalling systems will be examined. To achieve this, a model substrate of the kinase, tyrosine hydroxylase, will be transfected into Swiss 3T3 cells which have numerous receptors with diverse mechanisms for elevating calcium. Cells will be stimulated by signals such as vasopressin, bombesin and platelet-derived growth factor and the activation of the kinase monitored by its phosphorylation of tyrosine hydroxylase.