Regulation and function of PDK1-Akt-Pyk2 axis in platelets

血小板 PDK1-Akt-Pyk2 轴的调节和功能

基本信息

批准号：
9088501
负责人：
Satya P. Kunapuli
金额：
$ 47.41万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9088501
关键词：
1-Phosphatidylinositol 3-Kinase Affect Agonist Biochemical Blood Platelets Cells Clot retraction Complex Cytoplasmic Granules Data Event Fibrinogen Fibrinogen Receptors G-Protein Signaling Pathway GTP-Binding Proteins Generations Health Hemostatic function Integrins Kinetics Knockout Mice Lead Mediating Membrane Molecular Genetics Mus PTK2B gene Pathway interactions Phosphatidylinositide 3-Kinase Inhibitor Phosphatidylinositols Phosphorylation Phosphotransferases Physiological Platelet Activation Play Proteinase-Activated Receptors Proto-Oncogene Proteins c-akt Receptor Activation Regulation Role Shapes Signal Pathway Signal Transduction Signaling Molecule Surface System Testing Thrombin Thrombosis Thromboxane A2 Thromboxane Receptor Thromboxanes Tyrosine Phosphorylation Work genetic approach inhibitor/antagonist knockout gene p21 activated kinase receptor response src-Family Kinases

项目摘要

DESCRIPTION (provided by applicant): Platelet activation plays a major role in hemostasis and thrombosis. Platelet agonists activate complex signaling cascades resulting in shape change, aIIbb3 integrin activation, and dense granule release and thromboxane A2 (TXA2) generation, amplifying the initial signal. The signaling cascades and the intracellular interaction of these signaling molecules regulating platelet physiological events have not been completely understood. In this application, we propose to test the overall hypothesis that PDK1-Akt-Pyk2 axis regulates platelet functional responses. We propose to understand the interaction of these signaling molecules and the downstream events in the agonist-induced platelet activation using complimentary biochemical, pharmacological, and gene knockout approaches. PDK1 is known to phosphorylate a number of kinases, but its function in platelets has not been elucidated. Aim 1) We hypothesize that PDK1 plays an important positive regulatory role in platelets through selective phosphorylation and activation of Akt. We propose to test this hypothesis through selective pharmacological inhibitors of PDK1 and using conditional PDK1 null mice. Our preliminary data shows that PDK1 phosphorylation of Thr308 is crucial for the activity of Akt and downstream signaling events. Furthermore our preliminary data shows that PDK1 inhibition affects agonist-induced platelet integrin activation and TXA2 generation. Aim 2) We hypothesize that intracellular association of PAK with Akt is crucial for its phosphorylation by PDK1. We propose to evaluate the role of PAK in translocation of Akt to the membrane using pharmacological approaches and PAK1 and 2 knockout mice. In preliminary data, we show that Akt translocation to the membrane and phosphorylation follows different kinetics. We postulate that PYK2, through tyrosine phosphorylation of PI3 kinases and PDK1, regulates platelet functional responses and Akt phosphorylation. We also postulate that Pyk2 is activated by G12/13 pathways and regulates thromboxane generation. We will evaluate the function of Pyk2 in aIIbb3 integrin activation, TXA2 generation, clot retraction, and spreading on immobilized fibrinogen, using knockout mice and pharmacological inhibitors. Our preliminary data shows that Pyk2 plays an important role in these platelet functional responses. These studies will enhance our understanding of the intracellular interactions of signaling molecules and their role in platelet activation, and might identify potential newer targets for the treatmentof thrombosis.

描述（由申请人提供）：血小板活化在止血和血栓形成中起主要作用。血小板激动剂激活复杂的信号级联，导致形状变化、aIIbb3 整合素激活、致密颗粒释放和血栓素 A2 (TXA2) 生成，从而放大初始信号。这些信号分子调节血小板生理事件的信号级联和细胞内相互作用尚未完全了解。在此应用中，我们建议检验 PDK1-Akt-Pyk2 轴调节血小板功能反应的总体假设。我们建议使用互补的生化、药理学和基因敲除方法来了解这些信号分子与激动剂诱导的血小板活化中下游事件的相互作用。已知 PDK1 可使多种激酶磷酸化，但其在血小板中的功能尚未阐明。目的 1) 我们假设 PDK1 通过选择性磷酸化和激活 Akt 在血小板中发挥重要的正向调节作用。我们建议通过 PDK1 的选择性药理学抑制剂并使用条件性 PDK1 缺失小鼠来检验这一假设。我们的初步数据表明，PDK1 Thr308 的磷酸化对于 Akt 和下游信号转导事件的活性至关重要。此外，我们的初步数据表明，PDK1 抑制会影响激动剂诱导的血小板整合素激活和 TXA2 生成。目标 2) 我们假设 PAK 与 Akt 的细胞内关联对于 PDK1 对其进行磷酸化至关重要。我们建议使用药理学方法和 PAK1 和 2 敲除小鼠来评估 PAK 在 Akt 易位至膜上的作用。在初步数据中，我们表明 Akt 易位至膜和磷酸化遵循不同的动力学。我们假设 PYK2 通过 PI3 激酶和 PDK1 的酪氨酸磷酸化来调节血小板功能反应和 Akt 磷酸化。我们还假设 Pyk2 被 G12/13 途径激活并调节血栓素的产生。我们将使用基因敲除小鼠和药物抑制剂来评估 Pyk2 在 aIIbb3 整合素激活、TXA2 生成、凝块收缩和在固定纤维蛋白原上扩散中的功能。我们的初步数据表明 Pyk2 在这些血小板功能反应中发挥着重要作用。这些研究将增强我们对信号分子的细胞内相互作用及其在血小板活化中的作用的理解，并可能确定治疗血栓形成的潜在新靶点。