Moleculap biological and Pustochemical study on phospholipid me tabolic enzymes inviolved in signal transduction

参与信号转导的磷脂代谢酶的分子生物学和化学研究

基本信息

批准号：
07457001
负责人：
KONDO Hisatake
金额：
$ 4.93万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Phosphoinositides (PI) turnover produces second messengers such as ciacylglycerol (DG) and inositol triphosphate in response to external stimuli. In addition, its intermediate products such as PI 4-monophosphate (PI4-P), PI4,5-bisphosphate (PI4,5-P) play roles in the membrane transport. It is thus important to clarify the molecular identity and characteristics of enzymes involved in the PI turnover for understanding the signal transduction and membrane transport. The present study addressed this point and revealed the molecular feature of DG kinase and PI 4-kinase, and localized them at mRNA levels in the brain of developing and adult rats by in situ hybridization histochemistry. DG kinase converts DG to phosphatidic acid (PA), and PI 4-kinase phosphorylates phosphatidyl-inositol to produce PI 4,5-P.Four subtypes of DG kinase (termed type I-IV) were identified by gene cloning from rat brain. The DG kinase type I is of soluble form and localized in the oligodendrocyte, but not neurons, … More and the absence of its expression is seen in the brain of myelin-deficient (shiverer) mice, suggesting the role in formation and maintenance of the myelin. The type II DG kinase is of membrane-associated form and localized in the medium-spiny neurons, neurons in the nucleus accumbens and olfactory tubercle, and in the endocrine cells of the pituitary inermediate lobe, suggesting the involvement in the dopaminergic transmission. The type III is localized dominantly in the Purkinije neurons and substantially in the granule cells of the cerebellum, suggesting its role in the cerebellar motor-control. The type IV is localized in the cerebral and cerebellar cortical neurons, but peculiar in the molecular structure : while the former three contain two EF-hand and zinc-finger motifs in addition to the ATP-binding domain, the type IV contains no EF-hand motifs, but possesses ankyrin-like repeats. This structure has a high homology to rdgA (a gene of Drosophila inducing the retinal degeneration), but this rat molecule is localized in the bipolar cell, but not the photoreceptor cell of the retina, suggesting some different roles for this homologous molecule between different animal species.Two subtypes of PI 4-kinase were idenfified with the molecular weight of 230kDa and 92kDa. The former has a high homology to Pikl and the latter to Stt4, both of which are yeast PI 4-kinases and has previously been cloned by other authors. Both PI 4-kinases are localized at mRNA levels widely in almost all neurons with much higher expression in immature cells. When overexpressed in COS-cells, these molecules are localized in the Golgi apparatus. It is thus suggested that these PI 4 kinases are involved in the vesicle-transport within cells. Less

磷酸肌醇（PI）响应于外部刺激，产生了第二个使者，例如西贝酰基甘油（DG）和三磷酸肌醇。此外，它的中间产品，例如4-磷酸PI（PI4-P），PI4,5-双磷酸（PI4,5-P）在膜转运中起着作用。因此，重要的是要阐明参与PI转换的酶的分子身份和特征，以了解信号转移和膜转运。本研究解决了这一点，并揭示了DG激酶和PI 4-激酶的分子特征，并通过原位杂交组织化学将它们定位在发育和成年大鼠大脑中的mRNA水平。 DG激酶将DG转化为磷脂酸（PA），PI 4-激酶磷酸化磷脂酰肌醇可通过基因从大鼠脑中鉴定出DG激酶（称为I-IV）的DG激酶（称为I-IV）的PI 4,5-P。 DG激酶I型具有固体形式，并局部在少突胶质细胞中，而不是神经元，而不是……更多，并且缺乏其表达在髓磷脂缺乏症（Sheverer）小鼠的大脑中，这表明在形成和维持髓磷脂中的作用。 II型DG激酶是与膜相关的形式的，并位于中棘神经元，伏隔核和嗅觉结节中的神经元中，以及垂体无源叶的内分泌细胞中，表明在多巴胺剂传输中累及。 III型位于Purkinije神经元中，并且基本上在小脑的颗粒细胞中，表明其在小脑运动控制中的作用。 IV型位于脑和小脑皮质神经元中，但在分子结构中很奇特：虽然前三个含有两个EF手和锌指基序除了ATP结合域外，IV型不含EF手的基序，但不具有ANKYRIN类Ankyrin like Repoys。这种结构与RDGA具有很高的同源性（果蝇的基因诱导了视网膜变性），但是该大鼠分子位于双极细胞中，但没有视网膜的光感受器细胞，表明这种同源分子在不同动物物种之间的某些不同作用。前者与PIKL具有很高的同源性，而后者对STT4具有很高的同源性，这两者都是酵母菌Pi 4-激酶，以前曾被其他作者克隆。在未成熟细胞中，几乎所有具有更高表达的神经元中，这两种PI 4-激酶均位于mRNA水平宽。当在COS细胞中过表达时，这些分子位于高尔基体中。因此，这些PI 4激酶与细胞内的囊泡传输有关。较少的