MULTIFUNCTIONAL CA2+/CALMODULIN-DEPENDENT PROTEIN KINASE

细胞内钙离子作为第二信使介导细胞内钙离子的释放。 许多激素、生长因子、神经递质和 其他细胞外信号。 虽然钙的作用 似乎不是由普遍的生化机制介导的， 这项建议旨在测试某些 钙的生物化学和生理学作用可能是通过 通过多种底物蛋白的磷酸化 由多功能钙/钙调素依赖性 蛋白激酶（CaM激酶）。 该提案将审查其 钙的调节可以通过自身磷酸化， 哪种类型的受体受激酶的控制， 激酶在调节 碳水化合物和氨基酸代谢以及细胞核内 膜破裂 定点诱变将用于 阻断或模拟参与的自磷酸化位点， 激活或抑制CaM激酶。 机制 将研究通过自磷酸化的调节。 的 激酶调节抑制性“假底物”假说将 用缺乏抑制结构域的截短突变体进行测试。 这些实验将产生一种不依赖钙的形式， 该激酶将用于检查其许多细胞中的两个， 功能协调发展的 其中一个功能，钙依赖性调节 碳水化合物和氨基酸代谢的研究将在 H35肝癌细胞。 激酶的抑制性抗体，或 钙非依赖性激酶制剂将被显微注射 以及激素刺激的能力， 调节丙酮酸激酶和苯丙氨酸等酶 检查羟化酶。 类似的研究建议审查 激酶在钙触发的细胞分裂中的作用 海胆卵的核膜，另一个假设是 功能协调发展的 最后，CaM激酶参与介导 受体的作用，评估钙通过 磷脂酰肌醇和其他信号系统将是 考察 为了实现这一点，激酶的模型底物， 酪氨酸羟化酶，将转染到瑞士3 T3细胞中 它有许多受体，具有不同的机制， 提高钙含量 细胞会受到信号的刺激， 血管加压素、蛙皮素和血小板衍生生长因子， 通过其磷酸化监测激酶的活化， 酪氨酸羟化酶

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.