The role of phosphoinsitides in cell responses

我们通过两种方法探讨了磷脂酰肌醇(PI)-3激酶的作用。1)寻找PI-3蛋白结合蛋白的产物PIP3；2)在细胞中表达活化的PI-3蛋白。对于1)，我们制作了一种能与PIP3结合蛋白特异结合的树脂。使用该树脂，我们寻找了PIP3结合蛋白。我们纯化了一个相对分子质量为40kD的蛋白，克隆了该蛋白的基因，并对该基因进行了核苷酸序列分析。我们发现该基因编码的蛋白与参与囊泡转运的ARF-GAP蛋白具有同源性，提示PI-3蛋白可能参与膜转运。2)我们使用腺病毒介导的序列特异性重组。由于固有活性的PI-3激酶对细胞有毒性，我们需要一个可诱导的基因系统。在腺病毒方法中，基因在被重组激活之前是完全沉默的。通常，当可诱导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激酶基因导入了一些细胞系。我们获得了有趣的结果，表明PI-3激酶可能参与细胞骨架的重组和膜的转运，这表明我们已经建立了有助于分析这类实验的细胞系。

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.