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The role of phosphoinsitides in cell responses

磷酸肌醇在细胞反应中的作用

基本信息

批准号：
07456046
负责人：
FUKUI Yasuhisa
金额：
$ 4.67万
依托单位：
The University of Tokyo
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

We have explored the role of Phosphatidylinositol (PI)-3 kinase by two methods. 1) Search for PIP3 (the product of PI-3 kinase) binding protein 2) Expression of activated PI-3 kinase in the cells. For 1), we made a resin that binds PIP3 binding protein specifically. Using this resin, we searched for the PIP3 binding protein. We purified one with the molecular weight of 40kD,cloned the gene for the protein, and analyzed the nucleotide sequence of the gene. We found that the gene encoded a protein with a homology to ARF-GAP protein which is involved in vesicle trafficking suggesting that PI-3 kinase might be involved in membrane trafficking. For 2), we used adenovirus mediated sequence specific recombination. Since constitutively active PI-3 kinase is toxic to the cells, we needed an inducible gene system. In the adenovirus method, genes are completely silent before it is activated by recombination. Often, the gene suppression is leaky when an inducible promotor is used for this type of experiment not allowing us to get a cell line. Using the adenovirus system, we introduced active PI-3 kinase gene into some cell lines. We obteined intersting results suggesting that PI-3 kinase may be involved in cytoskeleton reorganization and membrane trafficking suggesting that we have established cell lines useful of analysis of these kinds of experiments.

我们用两种方法探讨了磷脂酰肌醇（PI）-3激酶的作用。1)PI-3激酶产物PIP 3结合蛋白的研究2）活化PI-3激酶在细胞中的表达。对于1），我们制备了特异性结合PIP 3结合蛋白的树脂。使用这种树脂，我们寻找PIP 3结合蛋白。我们纯化了一个分子量为40 kD的蛋白，克隆了该蛋白的基因，并对该基因的核苷酸序列进行了分析。我们发现，该基因编码的蛋白质与ARF-GAP蛋白，这是参与囊泡运输的同源性表明PI-3激酶可能参与膜运输。对于2），我们使用腺病毒介导的序列特异性重组。由于组成型活性PI-3激酶对细胞是有毒的，我们需要一个诱导型基因系统。在腺病毒方法中，基因在被重组激活之前完全沉默。通常，当诱导型启动子用于这种类型的实验时，基因抑制是泄漏的，不允许我们获得细胞系。利用腺病毒系统，我们将活性PI-3激酶基因导入一些细胞系。我们获得了有趣的结果，表明PI-3激酶可能参与细胞骨架重组和膜运输，这表明我们已经建立了用于分析这类实验的细胞系。