Structure and Function of Protein Kinases: Molecular Mechanism of Neuronal Signal Transduction

蛋白激酶的结构和功能：神经信号转导的分子机制

基本信息

批准号：
63440021
负责人：
TANAKA Chikako
金额：
$ 7.74万
依托单位：
Kobe University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

The importance of protein kinases as target molecules of second messengers in the cellular signal transduction mechanisms activated by hormones and neurotransmitters are well established. Protein kinase C and cAMP-dependent protein kinase are thought to be major protein kinases in these mechanisms. In this study, we tried to elucidate the molecular mechanisms of neuronal signal transduction related to these protein kinases.1) Localization and function of protein kinase C isozymes: Light- and electron-microscopic localization of alpha, beta-I, beta-II, and gamma subtypes of protein kinase C in rat brain were elucidated using specific antibodies. Immunohistochemical analysis of developing rat brain showed that each subtype expresses at different stage. Isozymes related to neurorite outgrowth and synaptic formation were identified. Specific expression of these isozymes was also shown by the in situ hybridization technique.2) The role of protein kinase C in the synaptic transmission: Invol … More vement of protein kinase C in calcium-dependent vesicular neurotransmitter release was shown. Gamma subtype of protein kinase C which is activated by arachidonic acid was shown to be involved in the GABA release from Purkinje cell in cerebellum.3) Molecular cloning and expression of regulatory subunit of cAMP-dependent protein kinase: A cDNA clone containing an entire coding region of the regulatory subunit was cloned from a rat brain cDNA library. Wild or mutant regulatory subunits were expressed in E. coli. After incubation with catalytic subunit purified from bovine heart, holo-enzymes were activated by CANW. Although these subunits bound cAMP with high affinity, holo-enzymes contains mutant subunits could not be activated by high concentrations of cAMP, indicating the importance of the presence of intrachain two cAMP-binding sites. These studies suggest that subtypes of protein kinase C have specific roles in neurorite outgrowth, synaptic formation, neurotransmitter release and long term potentiation, and showed the importance of protein phosphorylation in the neuronal signal transduction. Less

蛋白激酶作为第二个信号转导机制中第二信使的靶分子的重要性已得到充分确定。蛋白激酶C和cAMP依赖性蛋白激酶被认为是这些机制中的主要蛋白激酶。在这项研究中，我们试图阐明与这些蛋白激酶相关的神经元信号转导的分子机制。1）蛋白激酶C同工酶的定位和功能：使用蛋白质酶的蛋白质群体特异性的alpha，beta-i，beta-ii，beta-ii，beta-ii，beta-ii，beta-ii，β-i，β-i，β-i，β-i，β-i，β-i，β-i，beta-i，β-i的功能。发育中的大鼠脑的免疫组织化学分析表明，每个亚型在不同阶段表达。鉴定了与神经元出生和突触形成有关的同工酶。这些同工酶的特定表达也通过原位杂交技术显示。2）蛋白激酶C在突触传递中的作用：涉及……更多的蛋白激酶C在钙依赖性的囊泡神经递质释放中的更多属性。 Gamma subtype of protein kinase C which is activated by arachidonic acid was shown to be involved in the GABA release from Purkinje cell in cerebellum.3) Molecular cloning and expression of regulatory subunit of cAMP-dependent protein kinase: A cDNA clone containing an entire coding region of the regulatory subunit was cloned from a rat brain cDNA library.野生或突变的调节亚基在大肠杆菌中表达。与从牛心脏纯化的催化亚基孵育后，CANW激活了全酶。尽管这些亚基与高亲和力结合在一起，但全酶含有突变亚基，不能被高浓度的营地激活，这表明存在内部两个营地结合位点的重要性。这些研究表明，蛋白激酶C的亚型在神经元出生，突触形成，神经递质释放和长期增强中具有特定的作用，并显示了蛋白磷酸化在神经元信号转导中的重要性。较少的