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: 8079748
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 42.21万
• 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 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079748
• 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; 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）：信号网络对于协调细胞功能以响应刺激至关重要。了解这些网络的结构为理解其功能障碍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。这些网络的复杂性以及信号在小区中传输的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涉及创建信号蛋白破坏、结构域突变体和定点突变体的迭代过程，然后通过一系列细胞激活测定来表征每个突变体。作为一种补充方法，质谱中的现代磷酸化蛋白质组学方法可以通过各种激活状态或特定途径蛋白质或磷酸化位点的扰动提供细胞磷酸化的全局视图，从而促进信号途径的假设驱动表征。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，我们通过监测破坏网络中特定节点（蛋白质或磷酸化位点）的全局后果来测试由此产生的假设。 T 细胞在针对病毒、多种微生物和癌症的细胞介导免疫中发挥着核心作用。本提案的重点是阐明 T 细胞信号传导途径的分子细节。为了获得对导致 T 细胞激活的途径的新见解，将新型磷酸化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 细胞中磷酸化事件网络的详细视图。这种独特方法的前景在对具有破坏的受体近端蛋白酪氨酸激酶 Zap-70 的 T 细胞进行的初步磷酸化蛋白质组学研究中得到了证明。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。这些新的磷酸化位点既位于先前与 T 细胞通路相关的蛋白质上，也位于功能上未表征的蛋白质上。现在，我们将通过对具有破坏的途径蛋白 LCK、PLC1、VAV 和 ERK 的 T 细胞进行定量磷酸蛋白质组学分析，来测试这些新位点可以放置在途径内的特定位置的假设。特别是，将通过一系列结构域和点突变体详细检查这些磷酸化事件相对于关键通路蛋白 SLP76 和 LAT 的位置，从而允许在从这些蛋白启动的不同信号通路分支内精确放置新的磷酸化位点。将通过经典分子方法探索新假设的磷酸化蛋白质组学数据启发的关于 Fyn 激酶通过 PTP 的 Zap-70 依赖性调节的假设的测试。 公共健康相关性：T细胞信号网络的全面定义对于理解激活和抑制途径之间的平衡是绝对必要的，激活和抑制途径结合起来建立正常的生理学，以及这种相互作用的破坏，从而导致各种疾病状态，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。了解这些网络的细胞内结构为理解其功能障碍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。在本提案中，我们应用现代质谱方法，通过提供正常和突变细胞激活状态的全局视图，促进 T 细胞信号通路的假设驱动表征。