Spatio-temporal signaling mechanism of protein kinase C

蛋白激酶C的时空信号机制

基本信息

批准号：
13470023
负责人：
SAITO Naoaki
金额：
$ 7.49万
依托单位：
KOBE UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470023/
关键词：
protein kinase C targeting subtype green fluorescent protein apoptosis transgenic mice 可視化脂質

项目摘要

PKC consists of more than 10 isoforms and the existence of multiple isoforms strongly suggests that each isoform has individual functional role in signal transduction. We have been investigated "When, Where, Which isoform of PKC is activated and How the cellular function is regulated by the activation" by using green fluorescent protein(GFP)-tagged PKC isoforms. We have found that PKC translocation is dynamic and reversible in living cells and that PKC targeting is isoform-specific, stimulus-specific and cell-specific.In the present study, we have demonstrated that 1) arachidonic acid and ceramide act on CIB domains of epsilon and delta PKC and translocate these PKC isoforms to Golgi complex in isoform-specific manner, 2) ceramide induces translocation of deltaPKC to Golgi complex and activates this PKC isoform through tyrosine-phosphorylation., 3) Activation of deltaPKC by ceramide results in apoptosis which is distinctly different cell response from that induced by phorbol ester, another activator of PKC., 4) endogenous PKC can be knock-down by siRNA in isoform and species specifically., 5) Multiple PKC isoforms are involved in phagocytosis of microglial cells. Finally, we produced transgenic mice expressing GFP-tagged PKC isoforms using tetracyclin-regulated system. The expression of PKC-GFP can be regulated spatially and temporally. These transgenic mice enable us to monitor PKC targeting in brain slices by physiological condition and we observed PKC translocation in living brain which was different from that observed in cultured cells.

PKC由10多种同工型组成，多种同工型的存在强烈表明，每个同工型在信号转导中具有个体功能作用。我们已经通过使用绿色荧光蛋白（GFP）标记的PKC同工型来调查“何时，何时，何时何时激活PKC的同工型”。 We have found that PKC translocation is dynamic and reversible in living cells and that PKC targeting is isoform-specific, stimulus-specific and cell-specific.In the present study, we have demonstrated that 1) arachidonic acid and ceramide act on CIB domains of epsilon and delta PKC and translocate these PKC isoforms to Golgi complex in isoform-specific manner, 2) ceramide诱导Deltapkc转移到高尔基体复合物，并通过酪氨酸 - 磷酸化激活该PKC同工型。3）Ceramide通过cer酰胺激活DeltapkC会导致凋亡，该细胞的反应明显不同，该细胞反应与Phorbol酯，PKC的另一个pkc的另一个pkc and sirt and sirn and sirf and sirnne insne and sirnne sirf and sirnne and sirnn.同工型参与小胶质细胞的吞噬作用。最后，我们使用四环素调节的系统产生了表达GFP标记的PKC同工型的转基因小鼠。 PKC-GFP的表达可以在空间和时间上受到调节。这些转基因小鼠使我们能够通过生理条件监测脑切片中的PKC靶向，我们观察到活体大脑中的PKC易位，这与在培养细胞中观察到的PKC易位。