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

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，导致形状改变，aIIbb3整合素激活，致密颗粒释放和血栓素A2 （TXA2）的产生，放大初始信号。调控血小板生理事件的信号级联和这些信号分子的细胞内相互作用尚未完全了解。在本应用中，我们提出验证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的相互作用和激动剂诱导的血小板活化的下游事件。PDK1已知可使许多激酶磷酸化，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调控作用。我们建议通过PDK1的选择性药理抑制剂和条件PDK1缺失小鼠来验证这一假设。我们的初步数据表明，Thr308的PDK1磷酸化对Akt的活性和下游信号事件至关重要。此外，我们的初步数据显示，PDK1抑制影响激动剂诱导的血小板整合素激活和TXA2的生成。目的2)我们假设细胞内PAK与Akt的结合对于PDK1磷酸化Akt至关重要。我们建议通过药理学方法和PAK1和2敲除小鼠来评估PAK在Akt转位到细胞膜中的作用。在初步数据中，我们发现Akt易位到膜和磷酸化遵循不同的动力学。我们假设PYK2通过酪氨酸磷酸化PI3激酶和PDK1，调节血小板功能反应和Akt磷酸化。我们还假设Pyk2被G12/13通路激活，并调节血栓素的产生。我们将利用敲除小鼠和药物抑制剂评估Pyk2在aIIbb3整合素激活、TXA2生成、凝块收缩和固定纤维蛋白原扩散中的功能。我们的初步数据表明，Pyk2在这些血小板功能反应中起着重要作用。这些研究将增强我们对细胞内信号分子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。

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.