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: 7887159
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 38.86万
• 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/7887159
• 关键词: Binding; Biological Assay; Birds; Cell Line; Cell membrane; Cell physiology; Cells; Cellular Immunity; Cellular Structures; Classification; Collection; Complex; Critical Pathways; Data; Disease; Equilibrium; Event; Extracellular Signal Regulated Kinases; Eye; 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; Lymphocyte-Specific p56LCK Tyrosine Protein Kinase; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Methods; Metric; Microbe; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Modeling; Molecular; Monitor; Pathway interactions; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Positioning Attribute; Process; Production; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Proteomics; Proto-Oncogene Proteins c-fyn; 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; ZAP-70 Gene; base; crosslink; 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; public health relevance; receptor; reconstitution; release of sequestered calcium ion into cytoplasm; response; rho; skeletal; src Homology Region 2 Domain

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.

描述（由申请人提供）：信号网络对于响应刺激的蜂窝函数的编排至关重要。对这些网络结构的了解为理解其故障的病理后果提供了一个基础，并为设计治疗干预提供了机会。这些网络的复杂性以及在单元格中传输信号的速度使它们映射为巨大的挑战。阐明细胞信号网络结构的典型方法涉及迭代的过程，即产生信号蛋白破坏，结构域突变体和位置导向突变体，然后通过一系列细胞激活测定法对每个突变体进行表征。作为一种互补方法，质谱法中的现代磷酸化方法可以通过通过多种激活状态或在特定途径蛋白或磷酸化位点上扰动的各种激活状态或在特定的途径蛋白质上扰动的细胞磷酸化来促进信号传导途径的表征。该信息为生成有关信号通路结构的假设提供了合理的基础。然后，我们通过监视破坏网络中特定特定节点（蛋白质或磷酸化位点）的全局后果来检验产生的假设。 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。为了获得导致T细胞激活的途径的新见解，将新型的磷酸蛋白质组技术与传统方法结合使用，以提供通过T细胞受体激活的T细胞网络网络的详细视图。这种独特的方法的希望在具有干扰受体近端蛋白酪氨酸激酶的T细胞的初步磷酸蛋白质组学研究中说明了。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。这些新型的磷酸化位点既位于先前与T细胞途径以及功能未表征的蛋白质相关的蛋白质上。现在，我们将测试以下假设：这些新颖位点可以通过对途径蛋白LCK，PLC1，VAV和ERK的T细胞进行定量的磷酸蛋白质组学分析，将这些新地点放置在途径内的特定位置。特别是，将通过域和点突变体的集合来详细检查这些磷酸化事件相对于关键途径蛋白SLP76和LAT的放置，从而详细检查了从这些蛋白质引发的不同信号通路分支中新型磷酸化位点的精确放置。通过经典分子方法探索了对FYN激酶对ZAP-70依赖FYN激酶的依赖性调节的新假设的，磷酸蛋白质组学启发的假设的测试。 公共卫生相关性：T细胞信号网络的全面定义是绝对需要的，以了解与建立正常生理学的激活和抑制途径之间的平衡，并破坏这种相互作用，这会导致各种疾病状态，包括免疫缺陷，包括1型糖尿病，型糖尿病，肌lupus lupus erythematosus和Rheythematosus和Rheyumatioid Arthritis。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，并为设计治疗干预提供了机会。在此提案中，我们在质谱中应用现代方法来促进T细胞信号传导途径的假设驱动的表征，通过提供正常和突变细胞激活状态的全局视图。