CALCIUM AND CALMODULIN-DEPENDENT PROTEIN KINASE

钙和钙调蛋白依赖性蛋白激酶

• 批准号: 2263236
• 负责人: MARY B KENNEDY
• 金额: $ 32.15万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2263236
• 关键词: brain metabolism; calcium flux; calmodulin dependent protein kinase; electron microscopy; hippocampus; immunocytochemistry; intracellular transport; laboratory mouse; laboratory rabbit; laboratory rat; long term potentiation; molecular site; monoclonal antibody; neural transmission; neurochemistry; phosphorylation; synapses; synapsins; synaptic vesicles; tissue /cell culture

The strength of transmission at individual synapses in the central nervous system is often highly plastic and appears to be tightly regulated. This regulation plays a critical role in processing and storage of information. Furthermore, derangements in the regulation of synaptic strength seem likely to contribute to many neurological and psychological disorders including epilepsy, depression, schizophrenia, and Alzheimer's disease. The long-term goal of this study is to understand the molecular mechanisms that control synaptic transmission in the central nervous system and to learn how these mechanisms are distributed among different types of synapses. The proposed experiments will examine local regulation of autophosphorylation of a prominent brain Ca2+/calmodulin-dependent protein kinase in hippocampal neurons and phosphorylation of its substrate proteins, including the presynaptic vesicle protein, synapsin I, and proteins of the postsynaptic density. An immunocytochemical procedure will be developed for visualizing and quantifying phosphorylation of functionally significant sites on these proteins in situ after pharmacological and/or physiological manipulation of organotypic cultures or acute hippocampal slices. The method will make use of an existing monoclonal antibody that recognizes the CaM kinase only when it is autophosphorylated at a specific functional site and a complementary antisera that recognizes it only when it is not autophosphorylated at that site. Changes in autophosphorylation of the kinase in subcellular compartments will be recorded by this method at various times after inducing post-tetanic potentiation or long-term potentiation. We will raise similar antibodies against sites on synapsin I that are phosphorylated by CaM kinase II. We will use the antibodies to visualize changes in phosphorylation of synapsin I during induced changes in synaptic efficacy. Proteins in postsynaptic densities that are phosphorylated in situ by the CaM kinase, the A-kinase, or the C-kinase will be identified by biochemical labeling methods. These proteins will be purified from the postsynaptic density fraction, and the sites on the proteins that are phosphorylated in situ will be sequenced in preparation for immunocytochemical studies of their phosphorylation during induced changes in synaptic efficacy. The proposed immunocytochemical method will provide data about the kinetics of individual regulatory phosphorylation reactions and the sequence of those reactions at defined locations within neurons. Such data will permit testing of predictions made by explicit models of the organization of regulatory pathways in different types of synapses.

中枢神经系统中单个突触的传递强度 该系统通常具有很高的可塑性，似乎受到了严格的监管。这 监管在信息的处理和存储中发挥着关键作用。 此外，突触强度调节的紊乱似乎 可能会导致许多神经和心理障碍 包括癫痫、抑郁症、精神分裂症和阿尔茨海默病。 这项研究的长期目标是了解分子机制。 控制中枢神经系统中的突触传递，并 了解这些机制如何在不同类型的 突触。拟议的实验将检查地方法规对 一种重要的脑内钙/钙调素依赖蛋白的自磷酸化 海马神经元中的激酶及其底物的磷酸化 蛋白质，包括突触前囊泡蛋白、突触素I和 突触后密度的蛋白质。一种免疫细胞化学程序将 被开发用于可视化和量化磷酸化 在这些蛋白质上的功能有意义的位置在 器官培养的药理和/或生理操作 或急性海马区脑片。该方法将利用现有的 一种仅识别CaM激酶的单抗 在特定的功能位点上自动磷酸化和互补 只有当它没有被自动磷酸化时才能识别它的抗血清 地点。亚细胞内蛋白激酶自身磷酸化的变化 车厢将通过这种方法记录在不同的时间后 诱导强直后增强或长期增强。我们会 在突触素I上产生类似的抗体 被CaM激酶II磷酸化。我们将使用抗体来可视化 突触诱导改变过程中突触蛋白I磷酸化的变化 功效。突触后密度中被磷酸化的蛋白质 由CaM激酶、A-激酶或C-激酶原位定位将由 生化标记法。这些蛋白质将从 突触后密度分数，以及蛋白质上的位置 将对原位磷酸化进行测序，以准备 它们在诱导改变过程中磷酸化的免疫细胞化学研究 在突触的功效上。拟议的免疫细胞化学方法将提供 关于个体调节磷酸化反应的动力学数据 以及神经元内特定位置的反应顺序。 这样的数据将允许测试由显式模型做出的预测 不同类型突触中调节通路的组织。