Phosphoproteomic Analysis of T Cell Activation Pathways

描述(由申请人提供)：信号网络对于协调细胞功能以响应刺激是至关重要的。对这些网络结构的了解为理解其故障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。这些网络的复杂性和信号在小区中的传输速度使得映射它们成为一项艰巨的挑战。阐明细胞信号网络结构的典型方法包括产生信号蛋白中断、结构域突变和定点突变的迭代过程，然后通过一系列细胞激活分析来表征每个突变。作为一种补充方法，现代的磷酸蛋白质组学方法在质谱学中可以通过提供细胞通过各种激活状态或在特定途径蛋白或磷酸化部位被扰动的磷酸化的全局视图来促进信号通路的假说驱动的表征。这些信息为产生关于信号通路结构的假说提供了合理的基础。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)的全球后果来测试由此产生的假设。T细胞在对抗病毒、各种微生物和癌症的细胞免疫中起着核心作用。目前的建议侧重于阐明T细胞信号通路的分子细节。为了获得对导致T细胞激活的途径的新的见解，新的磷酸蛋白质组学技术与传统方法相结合，以提供通过T细胞受体激活的T细胞中的磷酸化事件网络的详细视图。这种独特的方法的前景在T细胞的初步磷酸蛋白质组学研究中得到了说明，该T细胞的受体近端蛋白酪氨酸激酶ZAP-70被破坏。复制了预期的T细胞信号通路结构，发现了96个新的磷酸化事件。这些新的磷酸化位点既位于先前与T细胞途径相关的蛋白质上，也位于功能未鉴定的蛋白质上。现在，我们将通过对带有中断的途径蛋白LCK、Plc1、VAV和ERK的T细胞进行定量的磷酸蛋白质组学分析，来检验这些新的位置可以被放置在途径中的特定位置的假设。特别是，这些磷酸化事件相对于关键途径蛋白SLP76和LAT的放置将通过一系列结构域和点突变进行详细研究，从而允许在由这些蛋白质启动的不同信号通路分支中精确定位新的磷酸化位点。测试一个新的假设，磷蛋白质组数据启发的假说，ZAP-70依赖调节Fyn激酶通过PTP将探索与经典的分子方法。 与公共健康相关：全面定义T细胞信号网络是绝对必要的，以了解激活和抑制通路之间的平衡，这些通路结合在一起建立正常的生理，以及这种相互作用的破坏导致各种疾病状态，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。对这些网络的细胞内结构的了解为理解其故障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。在这项建议中，我们应用现代的质谱学方法，通过提供正常和突变细胞的激活状态的全局视图，促进T细胞信号通路的假说驱动的表征。

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.