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.

细胞刺激剂，例如神经脉冲，神经递质，活性肽，激素，异构体，细胞因子，生长因子等，刺激质膜中的受体和离子通道以及细胞外信息中的细胞外信息。因此，在细胞中产生了第二个使者。细胞内钙离子（CAII D12+IE D1）通过刺激增加细胞。 CAII D12+IE D1现在被确定为第二使者之一，并参与了许多细胞功能，例如各种代谢反应，神经递质的生物合成，刺激 - 分泌耦合，刺激 - 刺激 - 共同接触，神经塑性，神经塑性，细胞神经形式，轴突流动，轴突流动，轴突流经，轴突流动，敏感性，敏感性，敏感性，敏感性，敏感性，敏感性。低浓度的细胞内CAIY D12+IY D1主要由CAIY D12+IY D1通道和泵调节。严格维护了细胞内CAM激酶II和IV的稳态。在本研究中，我们尝试使用海马神经元，例如脑神经元等原发性培养物，通过CAM激酶D12+IV信号传导阐明细胞功能，小脑神经元，小脑细胞和星细胞细胞和pc12细胞等细胞等细胞等细胞等细胞等细胞等细胞等细胞等细胞等细胞。我们发现，通过升高细胞内CAM激酶II的升高将CAM激酶II和IV激活，而cAM激酶IV均位于核溶胶和核中，而CAM激酶IV仅位于核中。添加了环孢菌素A增加了谷氨酸诱导的CAM激酶IV的磷酸化，但并未增加CAM激酶II的自磷酸化。通过蛋白磷酸酶1、2a和2c和钙调蛋白磷酸化，通过CAM激酶激酶磷酸化的GST-CAM激酶IV磷酸化。我们表征了MIN6细胞中CAM激酶II和突触I的同工型。我们继续制定一个计划，研究酶的原位动态激活，以响应受体刺激，并具有伴随的细胞功能。