Phosphoproteomic Analysis of T Cell Activation Pathways

T 细胞激活途径的磷酸化蛋白质组学分析

• 批准号: 8468632
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 35.99万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-15 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8468632
• 关键词: Binding; Biological Assay; Cell Line; Cell membrane; Cell physiology; Cells; Cellular Immunity; Cellular Structures; Classification; Collection; Complex; Critical Pathways; Data; Disease; Equilibrium; Event; Extracellular Signal Regulated Kinases; Foundations; Guanine Nucleotide Exchange Factors; IL2-Inducible T-Cell Kinase; Immune response; Immunologic Deficiency Syndromes; Infection; Insulin-Dependent Diabetes Mellitus; Interleukin-2; Jurkat Cells; Knowledge; Leukocytes; Location; Lymphocyte; MEKs; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Metric; Microbe; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Monitor; Pathway interactions; Phosphatidylinositols; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Positioning Attribute; Process; Production; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Proteomics; Receptor Signaling; Recruitment Activity; Regulation; Relative (related person); Rheumatoid Arthritis; Role; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Site; Speed; Stimulus; Structure; Subcellular structure; Systemic Lupus Erythematosus; T-Cell Activation; T-Cell Activation Pathway; T-Cell Receptor; T-Lymphocyte; Techniques; Test Result; Testing; Therapeutic; Therapeutic Intervention; Transcription Factor AP-1; Tyrosine; Tyrosine Phosphorylation Site; Viral; Virus; base; crosslink; delta protein; design; genetic regulatory protein; human PTPRT protein; inhibitor/antagonist; insight; molecular phenotype; mutant; neoplastic cell; novel; nuclear factors of activated T-cells; pathogen; phospholipase C gamma; protein kinase C-delta; receptor; reconstitution; release of sequestered calcium ion into cytoplasm; response; rho; skeletal; src Homology Region 2 Domain

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, PLCy1, 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 PTPa will be explored with classical molecular approaches.

信号网络对于协调细胞功能以响应刺激是至关重要的。知识 这些网络的结构为理解它们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 而信号在细胞中的传输速度使映射它们成为一项艰巨的挑战。这个 阐明蜂窝信令网络结构的典型方法包括以下迭代过程 产生信号蛋白中断、结构域突变体和定点突变体，然后鉴定 通过一系列的细胞激活分析来确定每个突变体的基因。作为一种补充方法，现代 质谱学中的磷酸蛋白质组学方法可以促进假说驱动的表征 通过各种激活状态提供细胞磷酸化的全局视图的信号通路 或在特定途径蛋白或磷酸化位置被干扰。这一信息提供了合理的基础 用于产生关于信号通路结构的假设。然后，我们通过监控来检验由此产生的假设 破坏网络中特定节点(蛋白质或磷酸化位点)的全球后果。 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。至 对导致T细胞激活的途径有了新的见解，新的磷酸蛋白质组技术 与传统方法相结合，提供T细胞中磷酸化事件网络的详细视图 通过T细胞受体激活的细胞。这种独特方法的前景在初步说明中得到了说明 受体近端蛋白酪氨酸激酶ZAP-70受损的T细胞的磷酸蛋白质组学研究。这个 复制了预期的T细胞信号通路结构，并发现了96个新的磷酸化事件 被发现了。这些新的磷酸化位点位于先前与T相关的蛋白质上 细胞途径以及功能未鉴定的蛋白质。我们现在要检验的假设是，这些小说 通过定量的磷酸蛋白质组学分析，可以将位点放置在通路中的特定位置 途径蛋白LCK、Plcy1、VAV和ERK被破坏的T细胞。特别是，这些东西的放置 与关键途径蛋白SLP76和LAT相关的磷酸化事件将被详细研究 通过结构域和点突变的集合，允许精确放置小说 不同信号通路分支中的磷酸化位点是由这些蛋白启动的。测试 关于Fyn的Zap-70依赖调控的新假说，由磷蛋白质组数据启发 将用经典的分子方法来探索通过PTPA的激酶。