G-protein Regulation of the Phosphatidyl Inositol (3,4,5) Trisphosphate Signal

G蛋白偶联受体对造血细胞有正向调节和负向调节作用。在单元格中 如中性粒细胞、巨噬细胞、肥大细胞和血小板，磷脂酰肌醇的p110y亚型(4，5) 3-激酶(Ptdlns 3-Kinase，Ptdlns 3-Kinase)在细胞的激活、形态变化和代间迁移中发挥着重要作用 磷脂酰肌醇(3，4，5)三磷酸(PIPs)。这种脂质是一个重要的信号，它激活了 磷脂酰肌醇依赖的蛋白激酶，PDK-1，导致蛋白激酶B和a的磷酸化 细胞反应的宿主。这些细胞膜中的PIP3水平也受到SH2的严格控制 含有肌醇5-磷酸酶的结构域，SHIP，受多种机制调节。G的激活 与Gi相连的蛋白偶联受体通过释放Ptdlns 3-Kinase40-60倍刺激Ptdlns 3-Kinase的p110Y亚型 特定的PY二聚体。激活与Gs相关的G蛋白偶联受体可显著提高环磷酸腺苷 抑制造血细胞对刺激配体的反应。我们的研究提供了明确的证据 Py二聚体的特定异构体激活Ptdlns 3激酶的p110y异构体。近期 实验发现，Ptdlns 3-K的p110y亚型和SHIP都可以是 被环状AMP依赖的蛋白激酶磷酸化。Py二聚体的激活能力 P101/p110y被磷酸化抑制。SHIP的磷酸化激活了该酶。这些结果 为cAMP抑制造血细胞反应的能力提供可能的分子解释 细胞。该项目的目标是确定这些磷酸化事件在细胞功能中的重要性。 这一目标将通过两个具体目标来实现。目的-1a：确定磷酸化对肌动蛋白表达的影响。 Ptdlns 3-Kinase的体外活性。目的-1b：了解Ptdlns 3-Kinase的磷酸化如何调节 该酶在三种细胞系中的功能。目的：探讨磷酸化对细胞外信号转导通路的影响。 SHIP的体外活性。AIM-2b：了解SHIP的磷酸化如何调节其在 完好无损的细胞。这些目标的完成将提供对分子事件的相当大的理解 调节所有细胞中PIP3的水平，并揭示G蛋白与受体偶联的机制 刺激和抑制造血细胞的功能。

G protein coupled receptors provide both positive and negative regulation of hematopoietic cells. In cells such as neutrophils, macrophages, mast cells and platelets, the p110y isoform of phosphatidylinositol (4,5) 3-kinase (Ptdlns 3-kinase) plays a major role in cell activation, shape changes and migration via generation of phosphatidylinositol (3,4,5) trisphosphate (PIPs). This lipid is an important signal that activates the phosphatidylinositol dependent protein kinase, PDK-1, leading to phosphorylation of protein kinase B and a host of cell responses. The levels of PIP3 in the membrane of these cells are also tightly controlled by a SH2 domain containing inositol 5-phosphatase, SHIP, that is regulated by multiple mechanisms. Activation of G protein coupled receptors linked to Gi stimulates the p110y isoform of Ptdlns 3-kinase 40-60 fold by releasing specific py dimers. Activation of G protein coupled receptors linked to Gs raise cyclic AMP and can markedly inhibit the response of hematopoietic cells to stimulatory ligands. Our research has provided clear evidence of the specific isoforms of the Py dimer which activate the p110y isoform of Ptdlns 3 kinase. Recent experiments uncover the exciting result that both the p110y isoform of Ptdlns 3-kinase and SHIP can be phosphorylated by the cyclic AMP dependent protein kinase. The ability of the Py dimer to activate p101/p110y is inhibited by phosphorylation. Phosphorylation of SHIP activates the enzyme. These results provide a possible molecular explanation for the ability of cyclic AMP to inhibit the response of hematopoietic cells. The goal for this project is to determine the importance of these phosphorylation events in cell function. This goal will be approached via 2 Specific Aims. Aim-1a: To determine the effects of phosphorylation on the activity of Ptdlns 3-Kinase in vitro. Aim-1b: To understand how phosphorylation of Ptdlns 3-Kinase regulates the function of the enzyme in three cell lines. Aim-2a: To explore the effects of phosphorylation on the activity of SHIP in vitro. Aim-2b: To understand how phosphorylation of SHIP regulates its function in the intact cell. Completion of these Aims will provide considerable understanding of the molecular events regulating the levels of PIP3 in all cells and should reveal mechanisms by which G protein coupled receptors stimulate and inhibit the function of hematopoietic cells.