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

基本信息

批准号：
10132943
负责人：
ARTHUR Robert SALOMON
金额：
$ 40.81万
依托单位：
BROWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-15 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10132943
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 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

项目摘要

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在单元格中传输的速度使它们成为巨大的挑战。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。作为一种完整的方法，现代使用定量质谱法的蛋白质组学方法可以促进假设驱动的表征信号通路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的免疫培养基中起着核心作用。该提案的重点是使用这些提案阐明T细胞信号通路的分子细节新技术。 LCK酪氨酸激酶是通过其T细胞激活的中心调节剂。磷酸化状态。 Tyr394处的LCK自磷酸化激活了激酶，而在 Tyr505使激酶失活。先前显示了四个磷酸酶在LCK Tyr394上作用，但是每种磷酸酶一个招募到LCK，是否涉及其他负调节分子。这控制LCK在T细胞接收器和下游之间正确分布的分子机制信号节点（例如SLP76脚手架信号体）的定义不当。在以前的资金中时期，我们的研究小组发现，T细胞信号通路的下游成员调节 LCK及其底物的磷酸化。我们发现脚手架蛋白SLP-76都控制 LCK Tyr394处T细胞接收器信号传导中的负反馈回路。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。为了获得调节LCK活动和空间定位的途径的新见解，我们已经组装了多学科团队应用新颖的定量蛋白质组学技术，生化方法和鼠标提供网络详细视图的模型。我们将在这个项目中解决的主要问题是 SLP76和PLCγ1如何设置LCK激活的空间和临时等效物，从而导致适当的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回路中使用，定义控制LCK细胞定位的分子因素，并定义他们的身体角色。