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Molecular cytobiological study on CaィイD12+ィエD1 signaling in the cells with cultured cells

培养细胞中CaiD12+CaiD1信号传导的分子细胞生物学研究

基本信息

批准号：
09480223
负责人：
MIYAMOTO Eishichi
金额：
$ 9.34万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480223/
关键词：
Receptor Signal transduction Calcium ion Cultured cell CaM kinase II CaM kinase IV Calcineurin Cellular functions グルタミン酸受容体 CaMキナーゼキナーゼ初代培養神経細胞受容体刺激細胞内応答 δサブユニット cDNA 核移行シグナル MAPキナーゼ脳由来神経成長因子

项目摘要

Cell stimulants such as nerve impulses, neurotransmitters, active peptides, hormones, autacoids, cytokines, growth factors etc stimulate receptors and ion channels in plasma membranes, and mediate extracellular information into cells. Consequently, second messengers are produced in the cells. Intracellular calcium ion (CaィイD12+ィエD1) increases in the cells by stimulation. CaィイD12+ィエD1 is now established to be one of second messengers and involved in many cellular functions such as various metabolic reactions, biosynthesis of neurotransmitters, stimulus-secretion coupling, stimulus-contraction coupling, neuronal plasticity, cellular morphology, axonal flow, membrane ion conductance, down-regulation of receptors and gene expression. A low concentration of intracellular CaィイD12+ィエD1 is mainly regulated by CaィイD12+ィエD1 channels and pumps. Homeostasis of intracellular CaィイD12+ィエD1 is strictly maintained.In the present study, we attempted to elucidate cellular functions through CaィイD12+ィエD1 signaling, using primary cultures such as hippocampal neurons, cerebral neurons, cerebellar granule cells and astrocytes and established cell lines such as PC12 cells, NG108-15 cells and MIN6 cells. We found that CaM kinases II and IV are activated through elevation of intracellular CaィイD12+ィエD1 in response to the extracellular stimuli such as glutamate. CaM kinase II is localized in both the cytosol and nucleus, while CaM kinase IV is exclusively localized in the nucleus. Addition of cyclosporin A increased glutamate-induced phosphorylation of CaM kinase IV, but did not increase autophosphorylation of CaM kinase II. GST-CaM kinase IV phosphorylated by CaM kinase kinase was dephosphorylated by protein phosphatases 1, 2A and 2C and calcineurin. We characterized the isoforms of CaM kinase II and synapsin I in MIN6 cells. We continuously have a plan to study on in situ dynamic activation of the enzymes in response to receptor stimulation with concomitant cellular functions.

细胞刺激物如神经冲动、神经递质、活性肽、激素、自育素、细胞因子、生长因子等刺激质膜上的受体和离子通道，并将细胞外信息传递到细胞内。因此，第二信使在细胞中产生。细胞内钙离子（Ca ~（2+）D_12 + Ca ~（2+）D_1）在细胞内通过刺激而增加。Ca ~（2+）D_12 + Ca ~（2+）D_1被认为是第二信使，参与多种细胞功能，如各种代谢反应、神经递质的生物合成、刺激-分泌偶联、刺激-收缩偶联、神经元可塑性、细胞形态、轴突流动、膜离子电导、受体下调和基因表达等。细胞内低浓度的Ca ~（2+）D_1主要受Ca ~（2+）D_1通道和泵的调节。本研究以原代培养的海马神经元、脑神经元、小脑颗粒细胞和星形胶质细胞为材料，以已建立的细胞系PC 12细胞、NG 108 -15细胞和MIN 6细胞为研究对象，通过对细胞内Ca ~（2+）D_12 + D_1D_1信号通路的研究，探讨了细胞内Ca ~（2+）D_12 + D_1D_1信号通路的功能。我们发现，钙调素激酶II和IV的激活，通过升高细胞内的钙调素D12+钙调素D1响应细胞外的刺激，如谷氨酸。CaM激酶II定位于细胞质和细胞核，而CaM激酶IV仅定位于细胞核。此外，环孢菌素A增加谷氨酸诱导的磷酸化钙调素激酶IV，但不增加钙调素激酶II的自磷酸化。GST-CaM激酶IV被CaM激酶激酶磷酸化，被蛋白磷酸酶1、2A和2C以及钙调神经磷酸酶去磷酸化。我们的特点是钙调蛋白激酶II和突触蛋白I在MIN 6细胞的亚型。我们一直计划研究酶对受体刺激的原位动态激活以及伴随的细胞功能。