PHOSPHATIDYLINOSITOL KINASES

磷脂酰肌醇激酶

• 批准号: 2178461
• 负责人: LEWIS C. CANTLEY
• 金额: $ 25.55万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-12-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178461
• 关键词: antibody; antibody formation; cell membrane; complementary DNA; diacylglycerols; enzyme mechanism; enzyme reconstitution; enzyme structure; enzyme substrate; gel electrophoresis; growth factor receptors; high performance liquid chromatography; inositol phosphates; laboratory rabbit; laboratory rat; liver; molecular cloning; nucleic acid hybridization; nucleic acid sequence; oncogenes; phosphatidylinositols; phosphorylation; phosphotransferases; platelet derived growth factor; protein purification; protein sequence; site directed mutagenesis

The long term goal of this laboratory is to understand the role of phosphoinositides in cellular regulation. The objective of this grant is to understand the mechanism of regulation of the enzymes that produce the various phosphoinositides. During the previous granting period it was discovered that the phosphatidylinositol kinase that co-purifies with protein-tyrosine kinases is in a new pathway. This enzyme phosphorylates the D-3 position of phosphoinositides to produce three lipids not previously known to exist, phosphatidylinositol (3)phosphate [PtdIns(3)P], phosphatidylinositol(3,4)bisphosphate [PtdIns(3,4)P2] and phosphatidylinositol(3,4,5)trisphosphate [PtdIns(3,4,5)P3]. Mutational studies of growth factor receptors and oncogenes indicate that production of these lipids is essential for stimulation of cell growth and transformation. PtdIns(3,4)P2 can be produced by phosphorylation of PtdIns(4)P at the D-3 position by the PtdIns 3-kinase or in some cells by phosphorylation of PtdIns(3)P at the D-4 position by a recently discovered PtdIns(3)P 4-kinase. In order to understand the importance of these lipids for cell signalling and the regulation of their synthesis, the phosphoinositide kinases will be purified to homogeneity. The purified enzymes will be characterized with respect to subunit composition, substrate utilization, and association with and phosphorylation by purified protein-tyrosine kinases. The proteins will be sequenced and oligonucleotide probes based on the sequence will be used to obtain cDNA clones. Antibodies will be raised against the purified proteins. Once clones are available the proteins will be expressed and the role of phosphorylation in regulation of activity can be evaluated by site specific mutations. The PtdIns 3-kinase has been highly implicated in platelet derived growth factor responses and in cell transformation and might be a useful target for drug intervention of vascular smooth muscle growth and tumor growth.

该实验室的长期目标是了解 细胞调节中的磷酸肌醇。 这笔赠款的目的是 了解产生的酶调节机制 各种磷酸肌醇。 在上一个授予期内 发现与与 蛋白质酪氨酸激酶处于新途径。 这种酶磷酸化 磷酸肌醇的D-3位置可产生三种脂质而不是 以前存在的磷脂酰肌醇（3）磷酸盐[PTDINS（3）P]， 磷脂酰肌醇（3,4）双磷酸[PTDINS（3,4）P2]和 磷脂酰肌醇（3,4,5）三磷酸[PTDINS（3,4,5）P3]。 突变 生长因子受体和致癌基因的研究表明生产 这些脂质对于刺激细胞生长和 转型。 PTDINS（3,4）P2可以通过磷酸化产生 ptdins（4）p在ptdins 3-激酶或某些细胞中的D-3位置。 PTDINS（3）P在D-4位置的磷酸化被最近发现的 Ptdins（3）P 4-激酶。 为了了解这些脂质的重要性 对于细胞信号传导及其合成的调节， 磷酸肌醇激酶将被纯化为均匀性。 纯化 酶将以亚基组成的特征来表征 底物利用，并通过纯化与磷酸化相关联 蛋白质酪氨酸激酶。 蛋白质将进行测序，并 基于序列的寡核苷酸探针将用于获得cDNA 克隆。 将对纯化的蛋白质提出抗体。 一次 克隆可用，将表达蛋白质，并将其作用 可以通过特定地点评估活性调节的磷酸化 突变。 PTDINS 3-激酶高度与血小板有关 得出的生长因子反应和细胞转化，可能是 血管平滑肌生长的药物干预和 肿瘤生长。