Heterogeneity of lipid second messenger-metabolizing enzyme, diacylglycerol kinase, in the brain in terms of molecule, localization, and function.

大脑中脂质第二信使代谢酶二酰甘油激酶在分子、定位和功能方面的异质性。

基本信息

批准号：
11480225
负责人：
GOTO Kaoru
金额：
$ 8.96万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

Diacylglycerol kinase (DGK), which catalyzes phosphorylation of lipid second messenger, DG to phosphatidic acid (PA), is thought to be a key enzyme in the regulation of DG levels and, as a result, to be responsible for attenuating the activation of PKC. We have so far cloned several isozymes from rat brain cDNA library, and performed detailed examination of their gene expression in the brain by in situ hybridization histochemistry. In this study we analyzed precise subcellular localization of the isozymes in cDNA-transfected COS cells using green fluorescent protein-fusion expression vector. DGK-alpha was localized diffusely in both cytoplasm and nucleus in the cells. DGK-beta was distributed throughout the cytoplasm in the shape of filamentous structure, which is quite similar to the location of actin filament. DGK-gamma was seen as a reticular structure in the juxtanuclear position, which is coincided with Golgi apparatus marker. DGK-zeta was located mostly in the nucleus, as suggested by the presence of nuclear localizing signal in its primary structure. DGK-epsilon was widespread to the perinuclear area, which resembles the location pattern of endoplasmic reticulum. These data clearly show the heterogeneity of DGK in subcellular localization as well as molecular structure, suggesting that each isozyme may be responsible for the metabolism of DG, which is produced at specific subcellular site upon stimulation. Therefore it is highly conceivable that each DGK isozyme plays a unique role in the signal transduction.

二酰甘油激酶 (DGK) 催化脂质第二信使 DG 磷酸化为磷脂酸 (PA)，被认为是调节 DG 水平的关键酶，因此负责减弱 PKC 的激活。迄今为止，我们已经从大鼠脑cDNA文库中克隆了几种同工酶，并通过原位杂交组织化学对其脑内基因表达进行了详细检查。在本研究中，我们使用绿色荧光蛋白融合表达载体分析了 cDNA 转染的 COS 细胞中同工酶的精确亚细胞定位。 DGK-α 广泛定位于细胞的细胞质和细胞核中。 DGK-β以丝状结构分布在整个细胞质中，与肌动蛋白丝的位置非常相似。 DGK-γ在核旁位置被视为网状结构，与高尔基体标记一致。 DGK-zeta 主要位于细胞核中，如其一级结构中存在核定位信号所示。 DGK-epsilon广泛分布于核周区域，类似于内质网的位置模式。这些数据清楚地显示了 DGK 在亚细胞定位和分子结构中的异质性，表明每种同工酶可能负责 DG 的代谢，DG 在刺激后在特定的亚细胞位点产生。因此，很可能每种 DGK 同工酶在信号转导中都发挥着独特的作用。