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: 8277233
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 38.35万
• 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/8277233
• 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细胞功能的协调至关重要。知识 这些网络的结构为理解其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 并且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。的 用于阐明蜂窝信令网络结构的典型方法包括迭代过程 产生信号蛋白破坏、结构域突变体和定点突变体， 通过一系列的细胞活化试验来检测每一个突变体。作为一种补充方法，现代 质谱中的磷酸化蛋白质组学方法可以促进假设驱动的表征， 通过提供通过各种激活状态的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。这些信息提供了一个合理的基础 来产生关于信号通路结构的假说。然后，我们通过监测来测试由此产生的假设。 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。到 获得新的见解的途径，导致T细胞活化，新的磷酸蛋白质组学技术， 结合传统的方法，提供了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。这种独特的方法的承诺是说明在初步 具有破坏的受体近端蛋白酪氨酸激酶Zap-70的T细胞的磷酸蛋白质组学研究。的 预期的T细胞信号通路结构被复制，96个新的磷酸化事件被 发现了这些新的磷酸化位点位于以前与T 细胞通路以及功能上未表征的蛋白质。我们现在将测试这些新的假设， 位点可以通过磷酸化蛋白质组学的定量分析， 具有破坏的途径蛋白LCK、PLC γ 1、VAV和ERK的T细胞。特别是，这些 将详细检查与关键途径蛋白SLP 76和LAT相关的磷酸化事件 通过一系列的结构域和点突变体，允许小说的精确定位， 这些蛋白质起始的不同信号通路分支内的磷酸化位点。的测试 一个新的假设，磷酸化蛋白质组学数据启发的关于Zap-70依赖性调节Fyn的假说 通过PTPa的激酶将探索与经典的分子方法。