G Protein Regulation of the PIP3 Signal

PIP3 信号的 G 蛋白调节

• 批准号: 7017636
• 负责人: JAMES Carlton GARRISON
• 金额: $ 28.88万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7017636
• 关键词: Mus musculus; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell line; cell surface receptors; cyclic AMP; enzyme activity; genetically modified animals; laboratory mouse; mass spectrometry; phosphatidylinositol 3 kinase; phosphatidylinositols; phosphomonoesterases; phosphorylation; protein structure function; recombinant proteins; thin layer chromatography

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.

描述（由申请人提供）：G蛋白偶联受体提供造血细胞的正向和负向调节。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-kinase) 的 p110gamma 亚型通过生成磷脂酰肌醇 (3) 在细胞激活、形状变化和迁移中发挥重要作用。 ,4,5) 三磷酸盐 (PIP3)。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，SHIP 受多种机制调节。与 Gi 连接的 G 蛋白偶联受体的激活通过释放特定的 βamma 二聚体刺激 Ptdlns 3-激酶的 p110gamma 亚型 40-60 倍。与 Gs 连接的 G 蛋白偶联受体的激活会提高环 AMP，并能显着抑制造血细胞对刺激性配体的反应。我们的研究提供了激活 Ptdlns 3 激酶 p110gamma 亚型的 βamma 二聚体特定亚型的明确证据。最近的实验揭示了令人兴奋的结果，即 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。 Betagamma 二聚体激活 p101/p110gamma 的能力受到磷酸化的抑制。 SHIP 的磷酸化会激活该酶。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 2 个具体目标来实现。 Aim-1a：确定体外磷酸化对 Ptdlns 3-激酶活性的影响。 Aim-1b：了解 Ptdlns 3-激酶的磷酸化如何调节三种细胞系中酶的功能。 Aim-2a：探讨磷酸化对SHIP体外活性的影响。 Aim-2b：了解 SHIP 的磷酸化如何调节其在完整细胞中的功能。完成这些目标将为调节所有细胞中 PIP3 水平的分子事件提供深入的了解，并揭示 G 蛋白偶联受体刺激和抑制造血细胞功能的机制。