G Protein Regulation of the PIP3 Signal

描述（申请人提供）：G蛋白偶联受体对造血细胞有正调控和负调控作用。在中性粒细胞、巨噬细胞、肥大细胞和血小板等细胞中，磷脂酰肌醇（4,5）3-激酶（Ptdlns 3-激酶）的p110γ亚型通过生成磷脂酰肌醇（3,4,5）三磷酸（PIP3），在细胞活化、形状改变和迁移中发挥重要作用。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的磷酸化和一系列细胞反应。这些细胞膜中PIP3的水平也受到含有肌醇5-磷酸酶（SHIP）的SH2结构域的严格控制，该结构域受多种机制调节。与Gi相连的G蛋白偶联受体的激活通过释放特异性β二聚体刺激Ptdlns 3-激酶的p110gamma亚型40-60倍。G蛋白偶联受体的激活可提高环AMP，并可显著抑制造血细胞对刺激配体的反应。我们的研究已经提供了明确的证据，证明β - γ二聚体的特定异构体可以激活Ptdlns 3激酶的p110γ异构体。最近的实验揭示了令人兴奋的结果，Ptdlns 3-激酶的p110gamma亚型和SHIP都可以被环AMP依赖性蛋白激酶磷酸化。β二聚体激活p101/p110gamma的能力被磷酸化抑制。SHIP的磷酸化激活了该酶。这些结果为环状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：体外测定磷酸化对Ptdlns 3-激酶活性的影响。Aim-1b：了解Ptdlns 3-激酶磷酸化如何调节三种细胞系中该酶的功能。Aim-2a：探讨体外磷酸化对SHIP活性的影响。Aim-2b：了解SHIP在完整细胞中磷酸化如何调控其功能。这些目标的完成将对所有细胞中调节PIP3水平的分子事件提供相当大的理解，并揭示G蛋白偶联受体刺激和抑制造血细胞功能的机制。

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.