Phosphoproteomic Analysis of Feedback Networks in T cell signaling

T 细胞信号传导反馈网络的磷酸化蛋白质组学分析

• 批准号: 9915845
• 负责人: ARTHUR Robert SALOMON
• 金额: $ 40.8万
• 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 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9915845
• 关键词: Address; Antigens; Binding; Biochemical; Biological Assay; Biological Models; Blood; California; Cell Line; Cell physiology; Cells; Cellular Immunity; Cellular Structures; Complex; Computer Analysis; DUSP22 gene; Data; Disease; Equilibrium; Event; Feedback; Funding; Immune response; Immune system; Immunologic Deficiency Syndromes; Immunology; Infection; Insulin-Dependent Diabetes Mellitus; Knowledge; LCP2 gene; Laboratories; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Microbe; Microscopy; Modernization; Molecular; Mus; NR0B2 gene; PLC gamma1; PTPN22 gene; Pathologic; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Play; Process; Protein Tyrosine Kinase; Proteins; Proteomics; Receptor Activation; Receptor Signaling; Regulation; Regulatory Pathway; Research; Rheumatoid Arthritis; Role; SH3 Domains; San Francisco; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Protein; Site; Speed; Stimulus; Structure; Subcellular structure; System; Systemic Lupus Erythematosus; T cell response; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Therapeutic; Therapeutic Intervention; Tyrosine Phosphorylation; Universities; Virus; Wisconsin; adaptive immune response; antigen-specific T cells; combat; design; experimental study; genetic regulatory protein; innovation; insight; member; mouse model; multidisciplinary; mutant; neoplastic cell; new technology; novel; pathogen; phosphoproteomics; preference; protein protein interaction; recruit; response; scaffold; 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 proteomic methods using quantitative mass spectrometry can facilitate the hypothesis-driven characterization of signaling pathways by providing a global view of cellular phosphorylation and protein-protein interactions through a variety of activation states. T cells play a central role in cell-mediated immunity against viruses, a variety of microbes, and cancer. This proposal focuses on the elucidation of the molecular details of the T cell signaling pathway using these new technologies. Lck tyrosine kinase is the central regulator of T cell activation regulated through its phosphorylation state. Lck autophosphorylation at Tyr394 activates the kinase, whereas phosphorylation at Tyr505 inactivates the kinase. Four phosphatases were shown previously to act on Lck Tyr394, but how each one is recruited to Lck and whether other negative regulatory molecules are involved is not understood. The molecular mechanism controlling the proper distribution of Lck between the T cell receptor and downstream signaling nodes such as the SLP76 scaffolded signalosome are not well defined. In the previous funding period, our research team discovered that downstream members of the T cell signaling pathway regulate the phosphorylation of Lck and its substrates. We discovered that the scaffold protein SLP-76 controls both negative and positive feedback loops in T cell receptor signaling at Lck Tyr394. We also discovered that PLCγ1 regulates differential Lck substrate phosphorylation within the TCR and the SLP-76 complex. To gain new insights into the pathways regulating Lck activity and spatial localization, we have assembled a multidisciplinary team to apply novel quantitative proteomic techniques, biochemical methods, and mouse models to provide a detailed view of the network. The central question that we will address in this project is how SLP76 and PLCγ1 set the spatial and temporal equilibrium of Lck activation resulting in appropriate T cell response to antigen. Successful completion of the aims will clarify the identity of the regulatory proteins employed in each feedback loop, define molecular factors controlling the cellular localization of Lck, and define their physiological role.

信号网络对于协调细胞功能以响应刺激至关重要。知识 这些网络结构的研究为理解其病理后果提供了基础 故障并为设计治疗干预措施提供了机会。这些网络的复杂性 信号在细胞中传输的速度使得绘制信号图成为一项艰巨的挑战。这 阐明细胞信号网络结构的典型方法涉及以下迭代过程 创建信号蛋白破坏、结构域突变体和定点突变体，然后进行表征 通过一系列细胞激活测定来分析每个突变体。作为一种补充方法，现代 使用定量质谱的蛋白质组学方法可以促进假设驱动的表征 通过提供细胞磷酸化和蛋白质-蛋白质相互作用的全局视图来研究信号通路 通过各种激活状态。 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。 该提案的重点是利用这些技术阐明 T 细胞信号传导途径的分子细节。 新技术。 Lck 酪氨酸激酶是 T 细胞活化的中央调节因子，通过其调节 磷酸化状态。 Tyr394 处的 Lck 自磷酸化会激活激酶，而 Tyr394 处的磷酸化会激活激酶 Tyr505 使激酶失活。之前有四种磷酸酶被证明可以作用于 Lck Tyr394，但是每种磷酸酶如何作用于 Lck Tyr394？ 其中一个被招募到 Lck 中，并且是否涉及其他负调控分子尚不清楚。这 控制Lck在T细胞受体和下游之间正确分布的分子机制 诸如 SLP76 支架信号体之类的信号节点尚未明确定义。在之前的融资中 期间，我们的研究团队发现T细胞信号通路的下游成员调节 Lck 及其底物的磷酸化。我们发现支架蛋白 SLP-76 控制着 Lck Tyr394 的 T 细胞受体信号传导中的负反馈环和正反馈环。我们还发现PLCγ1 调节 TCR 和 SLP-76 复合物内的差异 Lck 底物磷酸化。 为了获得对调节 Lck 活性和空间定位的途径的新见解，我们组装了 一个多学科团队应用新型定量蛋白质组学技术、生化方法和小鼠 模型提供网络的详细视图。我们在这个项目中要解决的核心问题是 SLP76 和 PLCγ1 如何设置 Lck 激活的空间和时间平衡，从而产生适当的 T 细胞 对抗原的反应。成功完成目标将阐明调节蛋白的身份 在每个反馈环路中使用，定义控制 Lck 细胞定位的分子因素，并定义 它们的生理作用。