Chemical Approaches to Mapping Cell Signaling Pathways

 描述(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通路中发挥关键作用。激酶和磷酸酶的动态活性确保了健康细胞中适当的磷酸化。然而，当磷酸化变得不受调控时，通常是通过异常的激酶或磷酸酶活性，细胞就会患病。事实上，针对激酶和磷酸酶的各种药物已经被开发出来，用于治疗从癌症到阿尔茨海默氏症的各种疾病。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一个重要目标。不幸的是，现有的监测细胞磷酸化和绘制由磷酸化控制的复杂信号通路的方法是具有挑战性的。需要新的方法来充分表征磷酸化、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的活性，以绘制出健康和疾病状态下的信号通路。在之前的资助阶段中建立的科学基础是我们揭示了激酶混杂地接受γ-磷酸化修饰的三磷酸腺苷类似物作为共底物。基于这种共底物的混杂，我们开创了几种由激酶催化的标记方法。我们的目标是在细胞信号研究的几个关键挑战中应用激酶催化的标记。首先，我们将开发激酶催化的生物素化来区分动态和非动态磷酸化事件(特定目标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，这些相互作用很难用传统方法来表征，重点是激酶-底物对(特定目标2)。依赖于磷酸化的交联具有提供有关信号转导通路的网络水平细节的令人兴奋的潜力，这是细胞信号研究中一个关键而又不发达的需求。该项目的一个大胆的新方向是应用激酶催化的标记来识别丝氨酸/苏氨酸磷酸酶底物(特定目标3)。很少有策略可用于表征丝氨酸/苏氨酸磷酸酶底物，这使得我们的创新方法对于鼓励细胞信号研究至关重要。通过对激酶和磷酸酶的研究，激酶催化的标记实验将对控制疾病的复杂细胞信号通路建立更详细和完整的图景。总体而言，该项目中建立的化学工具 将提供前所未有的创新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.