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: 8661694
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 38.21万
• 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 2017-05-07
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661694
• 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.

信号网络对于响应刺激的响应细胞功能的编排至关重要。知识 这些网络的结构提供了理解其病理后果的基础 故障并提供设计治疗干预措施的机会。这些网络的复杂性 信号传输在单元格中的速度使它们映射到它们是巨大的挑战。这 阐明细胞信号网络结构的典型方法涉及一个迭代过程 创建信号蛋白质破坏，域突变体和位置突变体，然后进行表征 每个突变体通过一系列细胞激活测定法。作为一种互补的方法，现代 质谱法中的磷蛋白质组学方法可以促进假设驱动的表征 信号通路通过通过多种激活状态提供细胞磷酸化的全局视图。 或在特定途径蛋白或磷酸化位点受到干扰。此信息提供了合理的基础 用于产生有关信号通路结构的假设。然后，我们通过监视测试结果的假设 破坏网络中特定节点（蛋白质或磷酸化位点）的全球后果。 T细胞在针对病毒，多种微生物和癌症的细胞介导的免疫中起着核心作用。 目前的提议着重于阐明T细胞信号通路的分子细节。到 对导致T细胞激活的途径获得新的见解，新型的磷酸蛋白质组学技术是 结合传统方法，可以详细介绍T中的磷酸化事件网络 通过T细胞受体激活的细胞。初步说明了这种独特方法的承诺 具有破坏受体近端蛋白酪氨酸激酶的T细胞的T细胞的磷酸蛋白质组学研究，ZAP-70。这 重复了预期的T细胞信号通路结构，96个新的磷酸化事件为 发现。这些新型的磷酸化位点均位于先前与T的蛋白质上 细胞途径以及功能未表征的蛋白质。我们现在将检验这些小说的假设 可以通过定量的磷蛋白质组分析将站点放置在途径内的特定位置。 具有破坏途径蛋白LCK，PLCY1，VAV和ERK的T细胞。特别是，这些位置 相对于关键途径蛋白SLP76和LAT的磷酸化事件将进行详细检查 通过域和点突变体的集合，可以精确地放置小说 这些蛋白质引发的不同信号通路分支中的磷酸化位点。测试 关于ZAP-70依赖性调节Fyn的新假设的磷蛋白质组学数据启发的假设 通过经典分子方法将探索通过PTPA的激酶。