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阳性和阴性调节。 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).该脂质是一个重要信号，它激活磷脂酰肌醇依赖性蛋白激酶PDK-1，导致蛋白激酶B的磷酸化和许多细胞反应。这些细胞的膜中的PIP3水平也由含有含有多种机制调节的肌醇5-磷酸酶的SH2结构域紧密控制。与GI相关的G蛋白偶联受体的激活通过释放特定的betagamma二聚体，刺激PTDLNS 3-激酶40-60倍的P110GAMMA同工型。与GS相关的G蛋白偶联受体的激活会引起环状AMP，并明显抑制造血细胞对刺激配体的反应。我们的研究为Betagamma二聚体的特定同工型提供了明确的证据，该二聚体激活了PTDLNS 3激酶的P110GAMMA同工型。最近的实验发现了PTDLNS 3-激酶的P110GAMMA同工型和船舶都可以通过环状AMP依赖性蛋白激酶磷酸化的令人兴奋的结果。磷酸化抑制了β二聚体激活p101/p110gamma的能力。船的磷酸化激活酶。这些结果为循环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。该目标将通过2个特定目标实现。 AIM-1A：确定磷酸化对体外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个细胞系中酶的功能。 AIM-2A：探索磷酸化对体外船舶活性的影响。 AIM-2B：了解如何调节其在完整细胞中的功能如何调节其功能。这些目标的完成将提供对调节所有细胞中PIP3水平的分子事件的充分了解，并应揭示G蛋白偶联受体刺激并抑制造血细胞功能的机制。