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

G蛋白偶联受体提供造血细胞的正性和负性调节。细胞中 如中性粒细胞、巨噬细胞、肥大细胞和血小板，磷脂酰肌醇的p110 y亚型（4，5） 3-蛋白激酶（Ptdlns 3-kinase）在细胞活化、形状改变和通过生成的迁移中起主要作用 磷脂酰肌醇（3，4，5）三磷酸（PIP）。这种脂质是一种重要的信号， 磷脂酰肌醇依赖性蛋白激酶，PDK-1，导致蛋白激酶B磷酸化， 宿主细胞反应。这些细胞膜中PIP 3的水平也受到SH 2的严格控制。 结构域含有肌醇5-磷酸酶，SHIP，这是由多种机制调节。G的激活 与Gi连接的蛋白偶联受体通过释放p110 γ刺激Ptdlns 3-激酶的p110 γ同种型40-60倍 特异性Py二聚体。与Gs相连的G蛋白偶联受体的激活可升高环AMP， 抑制造血细胞对刺激性配体的反应。我们的研究提供了明确的证据 激活Ptdlns 3激酶的p110 y同种型的Py二聚体的特异性同种型。最近 实验揭示了一个令人兴奋的结果，即Ptdlns 3-激酶的p110 y亚型和SHIP都可以被 由环AMP依赖性蛋白激酶磷酸化。Py二聚体激活 p101/p110 y被磷酸化抑制。SHIP的磷酸化激活酶。这些结果 为环腺苷酸抑制造血反应的能力提供了一种可能的分子解释。 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：确定磷酸化对细胞凋亡的影响。 Ptdlns 3-激酶的体外活性。目的-1b：了解Ptdlns 3-激酶的磷酸化如何调节 酶在三种细胞系中的功能。目的-2a：探讨磷酸化对细胞凋亡的影响。 SHIP的体外活性。目的-2b：了解SHIP的磷酸化如何调节其在细胞内的功能。 完整细胞这些目标的完成将提供相当多的分子事件的理解 调节所有细胞中PIP 3的水平，并应揭示G蛋白偶联受体的机制， 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.