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

描述(申请人提供)：血小板激活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反应，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。这些调节血小板生理事件的信号分子的信号级联和细胞内相互作用尚不完全清楚。在这一应用中，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激活过程中下游事件的相互作用。已知PDK1可磷酸化多种激酶，但其在血小板中的功能尚未阐明。目的1)我们推测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的选择性药理抑制剂和条件性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及其下游信号事件的活性至关重要。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的产生。目的2)我们推测，细胞内PAK与Akt的结合是其被PDK1磷酸化的关键。我们建议使用药理学方法和PAK1和2基因敲除小鼠来评估PAK在Akt到膜转位中的作用。在初步的数据中，我们发现Akt的膜转位和磷酸化遵循不同的动力学。我们推测，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的磷酸化。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产生、凝块回缩和在固定化纤维蛋白原上扩散方面的作用。我们的初步数据显示，Pyk2在这些血小板功能反应中起着重要作用。这些研究将加深我们对信号分子在细胞内的相互作用及其在血小板激活中的作用的理解，并可能确定潜在的治疗血栓形成的新靶点。

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.