Phosphoproteomic Analysis of T Cell Activation Pathways

描述（由申请人提供）：信号网络对于响应刺激的细胞功能的协调至关重要。这些网络的结构知识提供了一个基础，了解其故障的病理后果，并提供了设计治疗干预的机会。这些网络的复杂性和信号在小区中传输的速度使得映射它们成为一个艰巨的挑战。阐明细胞信号网络结构的典型方法涉及创建信号蛋白破坏、结构域突变体和定点突变体的迭代过程，然后通过一系列细胞活化测定来表征每个突变体。作为一种补充方法，现代磷酸化蛋白质组学方法在质谱可以促进假说驱动的信号通路的表征，通过提供一个全局视图的细胞磷酸化通过各种激活状态或扰动在特定的途径蛋白质或磷酸化位点。这些信息提供了一个合理的基础，产生关于信号通路结构的假设。然后，我们通过监测破坏网络中特定节点（蛋白质或磷酸化位点）的全局后果来测试由此产生的假设。 T细胞在针对病毒、各种微生物和癌症的细胞介导的免疫中发挥核心作用。本提案的重点是阐明T细胞信号通路的分子细节。为了获得对导致T细胞活化的途径的新见解，将新型磷酸化蛋白质组学技术与传统方法相结合，以提供通过T细胞受体活化的T细胞中磷酸化事件网络的详细视图。这种独特的方法的承诺是说明在初步的磷酸化蛋白质组学研究的T细胞与破坏受体近端蛋白酪氨酸激酶，Zap-70。预期的T细胞信号通路结构被复制，并发现了96个新的磷酸化事件。这些新的磷酸化位点位于先前与T细胞途径相关的蛋白质以及功能上未表征的蛋白质上。我们现在将测试的假设，这些新的网站可以放置在特定的位置内的途径，通过定量磷酸化蛋白质组学分析的T细胞与破坏途径蛋白LCK，PLC 1，VAV，和ERK。特别是，这些磷酸化事件相对于关键途径蛋白SLP 76和LAT的位置将通过一系列结构域和点突变体进行详细检查，允许在从这些蛋白质起始的不同信号传导途径分支内精确放置新的磷酸化位点。测试一个新的假设，磷酸化蛋白质组学数据启发的假设Zap-70依赖性调节Fyn激酶通过PTP将探索与经典的分子方法。 公共卫生相关性：T细胞信号网络的全面定义是绝对需要了解激活和抑制途径之间的平衡，联合收割机建立正常的生理和破坏这种相互作用，导致各种疾病的状态，包括免疫缺陷，1型糖尿病，系统性红斑狼疮和类风湿性关节炎。这些网络的细胞内结构的知识为理解其功能障碍的病理后果提供了基础，并为设计治疗干预措施提供了机会。在这项提案中，我们应用现代质谱分析方法，通过提供正常和突变细胞激活状态的全局视图，促进T细胞信号通路的假设驱动表征。

DESCRIPTION (provided by applicant): Signaling networks are crucial for the orchestration of cellular functions in response to stimuli. Knowledge of the structure of these networks provides a basis for understanding the pathological consequences of their malfunction and offers opportunities for designing therapeutic interventions. The complexity of these networks and the speed with which signals are transmitted in cells makes mapping them a formidable challenge. The typical approach for elucidating the structure of cellular signaling networks involves an iterative process of creating signaling protein disruptions, domain mutants and site-directed mutants followed by characterization of each mutant through a battery of cellular activation assays. As a complementary approach, modern phosphoproteomic methods in mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation through a variety of activation states or perturbed at specific pathway proteins or phosphorylation sites. This information provides a rational basis for generating hypotheses about signaling pathway structure. We then test resulting hypotheses by monitoring the global consequences of disrupting specific nodes (proteins or phosphorylation sites) in the network. T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. The present proposal focuses on the elucidation of the molecular details of the T cell signaling pathway. To gain new insights into the pathways leading to T cell activation, novel phosphoproteomic techniques are combined with traditional methods to provide a detailed view of the network of phosphorylation events in T cells activated through the T cell receptor. The promise of this unique approach is illustrated in preliminary phosphoproteomic studies of T cells with a disrupted receptor proximal protein tyrosine kinase, Zap-70. The expected T cell signaling pathway structure was replicated and 96 novel phosphorylation events were discovered. These novel phosphorylation sites are located both on proteins previously associated with the T cell pathway as well as functionally uncharacterized proteins. We will now test the hypothesis that these novel sites can be placed in specific locations within the pathway through quantitative phosphoproteomic analysis of T cells with disrupted pathway proteins LCK, PLC1, VAV, and ERK. In particular, the placement of these phosphorylation events relative to the critical pathway protein SLP76 and LAT will be examined in detail through a collection of domain and point mutants, allowing for the precise placement of the novel phosphorylation sites within different signaling pathway branches initiated from these proteins. Testing of a newly postulated, phosphoproteomic data-inspired hypothesis about the Zap-70 dependent regulation of Fyn kinase through PTP will be explored with classical molecular approaches. PUBLIC HEALTH RELEVANCE: A comprehensive definition of the T cell signaling network is absolutely required to understand the balance between activating and inhibitory pathways that combine to establish normal physiology and the disruption of this interplay that leads to a variety of disease states including immunodeficiency, Type 1 diabetes mellitus, systemic lupus erythematosus, and rheumatoid arthritis. Knowledge of the intracellular structure of these networks provides a basis for understanding the pathological consequences of their malfunction and offers opportunities for designing therapeutic interventions. In this proposal we apply modern methods in mass spectrometry to facilitate the hypothesis-driven characterization of the T cell signaling pathway by providing a global view of the activation state of normal and mutant cells.