G Protein Regulation of the PIP3 Signal

描述（由申请人提供）：G蛋白偶联受体提供造血细胞的正性和负性调节。在细胞如嗜中性粒细胞、巨噬细胞、肥大细胞和血小板中，磷脂酰肌醇（4，5）3-激酶（PtdIns 3-激酶）的p110 γ同种型通过产生磷脂酰肌醇（3，4，5）三磷酸（PIP 3）在细胞活化、形状改变和迁移中起主要作用。这种脂质是激活磷脂酰肌醇依赖性蛋白激酶PDK-1的重要信号，导致蛋白激酶B磷酸化和许多细胞反应。这些细胞膜中的PIP 3水平也受到含有肌醇5-磷酸酶SHIP的SH 2结构域的严格控制，SHIP受多种机制调节。与Gi连接的G蛋白偶联受体的活化通过释放特异性β-γ二聚体刺激Ptdlns 3-激酶的p110 γ同种型40-60倍。与Gs相连的G蛋白偶联受体的激活可升高环AMP，并可显著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，β-γ二聚体的特定亚型激活Ptdlns 3激酶的p110 γ亚型。最近的实验揭示了令人兴奋的结果，即Ptdlns 3-激酶的p110 γ亚型和SHIP都可以被环AMP依赖性蛋白激酶磷酸化。β-γ二聚体激活p101/p110 γ的能力受到磷酸化的抑制。SHIP的磷酸化激活酶。这些结果为环AMP抑制造血细胞反应的能力提供了可能的分子解释。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：体外研究磷酸化对Ptdlns 3-激酶活性的影响。目的-1b：了解Ptdlns 3-激酶的磷酸化如何在三种细胞系中调节该酶的功能。目的-2a：探讨磷酸化对SHIP活性的影响。目的-2b：了解完整细胞中SHIP的磷酸化如何调节其功能。这些目标的完成将提供相当多的了解的分子事件调节的水平PIP 3在所有细胞中，并应揭示机制，通过G蛋白偶联受体刺激和抑制造血细胞的功能。

DESCRIPTION (provided by applicant): G protein coupled receptors provide both positive and negative regulation of hematopoietic cells. In cells such as neutrophils, macrophages, mast cells and platelets, the p110gamma 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 (PIP3). 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 p110gamma isoform of Ptdlns 3-kinase 40-60 fold by releasing specific betagamma 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 betagamma dimer which activate the p110gamma isoform of Ptdlns 3 kinase. Recent experiments uncover the exciting result that both the p110gamma isoform of Ptdlns 3-kinase and SHIP can be phosphorylated by the cyclic AMP dependent protein kinase. The ability of the betagamma dimer to activate p101/p110gamma 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.