Chemical Approaches to Mapping Cell Signaling Pathways

 描述（由申请人提供）：磷酸化是一种普遍存在的蛋白质翻译后修饰，在控制细胞功能的细胞信号传导途径中发挥关键作用。激酶和磷酸酶的动态活性确保健康细胞中的适当磷酸化。然而，当磷酸化变得不受调节时，通常通过异常的激酶或磷酸酶活性，细胞变得患病。事实上，已经开发了各种针对激酶和磷酸酶的药物来治疗从癌症到阿尔茨海默氏症的疾病。因此，对激酶和磷酸酶及其控制的信号通路的监测是表征疾病状态和开发新药的重要目标。不幸的是，现有的方法来监测细胞磷酸化和地图的复杂的信号通路控制的磷酸化是具有挑战性的。新的方法是必要的，以充分表征磷酸化，细胞信号和疾病的形成。 该项目的长期目标是严格表征细胞中的激酶和磷酸酶活性，以绘制健康和疾病状态下的信号通路。在前一个资助期建立的科学基础是我们发现激酶混杂地接受γ-磷酰基修饰的ATP类似物作为共底物。基于这种共底物混杂性，我们开创了几种激酶催化的标记方法。我们的目标是将激酶催化标记应用于细胞信号研究中的几个关键挑战。首先，我们将开发激酶催化的生物素化，以区分动态与非动态磷酸化事件（具体目标1）。其次，将建立激酶催化的交联，以监测瞬时磷酸化介导的蛋白质-蛋白质相互作用，这些相互作用难以通过传统方法表征，重点是激酶-底物对（特定目标2）。磷酸化依赖性交联具有提供关于信号转导途径的网络水平细节的令人兴奋的潜力，这是细胞信号传导研究中的关键但尚未开发的需求。该项目的一个大胆的新方向是将激酶催化标记应用于Ser/Thr磷酸酶底物鉴定（特异性目标3）。很少有策略可用于表征Ser/Thr磷酸酶的底物，这使得我们的创新方法对于鼓励细胞信号传导研究至关重要。通过研究激酶和磷酸酶，激酶催化的标记实验将建立一个更详细和完整的图片复杂的细胞信号转导途径控制疾病。总的来说，本项目建立的化学工具 将提供创新和前所未有的方法来监测磷酸化在生物学中的作用。

 DESCRIPTION (provided by applicant): Phosphorylation is a ubiquitous protein post-translational modification that plays a critical role in the cell signaling pathways governing cellular functions. The dynamic activities of kinase and phosphatase enzymes ensure proper phosphorylation in healthy cells. However, when phosphorylation becomes unregulated, generally through aberrant kinase or phosphatase activities, cells become diseased. In fact, various drugs targeting kinases and phosphatases have been developed to treat diseases from cancers to Alzheimer's. As a result, the monitoring of kinases and phosphatases, and the signaling pathways that they control, represents an important goal towards characterizing disease states and developing new drugs. Unfortunately, the available methods to monitor cellular phosphorylation and map the complex signaling pathways controlled by phosphorylation are challenging. New methods are necessary to fully characterize phosphorylation, cellular signaling, and disease formation. The long-term goal of this project is to rigorously characterize kinase and phosphatase activities in cells to map signaling pathways in healthy and diseased states. The scientific foundation established in the prior funding period is our revelation that kinases promiscuously accept γ-phosphoryl modified ATP analogs as cosubstrates. Based on this cosubstrate promiscuity, we pioneered several kinase-catalyzed labeling methods. We aim here to apply kinase-catalyzed labeling to several critical challenges in cell signaling research. First, we will develop kinase-catalyzed biotinylation to distinguish dynamic versus nondynamic phosphorylation events (Specific Aim 1). Second, kinase-catalyzed crosslinking will be established to monitor transient phosphorylation-mediated protein-protein interactions that are challenging to characterize by traditional methods, focusing on kinase-substrate pairs (Specific Aim 2). Phosphorylation-dependent crosslinking has the exciting potential to provide network-level details about signal transduction pathways, which is a critical yet underdeveloped need in cell signaling studies. A bold new direction for the project is application of kinase-catalyzed labeling to Ser/Thr phosphatase substrate identification (Specific Aim 3). Few strategies are available to characterize substrates of Ser/Thr phosphatases, making our innovative approach crucial to embolden cell signaling research. By studying both kinases and phosphatases, kinase-catalyzed labeling experiments will build a more detailed and complete picture of the complex cell signaling pathways governing disease. In total, the chemical tools established in this project will provide innovative and unprecedented approaches to monitor the role of phosphorylation in biology.