Phosphatase regulation of NFKappaB activity

磷酸酶调节 NFKappaB 活性

• 批准号: 7841727
• 负责人: MARTIN E DORF
• 金额: $ 36.71万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7841727
• 关键词: Adaptor Signaling Protein; Autoimmune Process; Cells; Chronic; Co-Immunoprecipitations; Communicable Diseases; Complex; Data; Effectiveness; Elements; Enzymes; Epitopes; Genes; Genetic Transcription; Goals; Holoenzymes; Immune; Individual; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Response; Interleukin-1; Large T Antigen; Libraries; Luciferases; Mediating; Mediator of activation protein; Methodology; NF-kappa B; Nuclear Translocation; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Positioning Attribute; Primary Cell Cultures; Process; Production; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; RNA Interference; Recruitment Activity; Regulation; Reporter; Role; Series; Signal Pathway; Signal Transduction; Simian virus 40; Site; Small Interfering RNA; Specificity; Staging; TNF gene; TNFRSF1B gene; TRAF2 gene; Tissues; Transcript; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Update; chemokine; computerized data processing; cytokine; insight; p65; promoter; prototype; receptor; research study; response

DESCRIPTION (provided by applicant): TNFa and IL-1p are proinflammatory mediators that induce a storm of chemokines and cytokines. They are thought to be responsible for prolonging production of inflammatory mediators in multiple chronic autoimmune and infectious diseases. We propose to investigate the mechanisms responsible for regulating responses to these prototype cytokines. Specifically, we will define the phosphatase requirements for controlling TNF and IL-1-mediated NF-KB activation. An RNAi approach will be used with a NF-KB-luciferase reporter. 500 siRNA constructs have been prepared and screened for basal and TNF-mediated signaling in SV40 large T antigen immortalized and primary cell cultures. Preliminary data demonstrate the effectiveness of this strategy. 19 phosphatase genes were identified in our initial screens. 10 of 19 genes were not previously associated with NF-KB signaling. The proposal lists three specific Aims. The first is to identify phosphatases involved in TNF and IL-1 signaling. This Aim details the methodology required to validate our initial findings, and applies our RNAi strategy to the study of IL-1 signaling. The function of each phosphatase gene associated with NF-KB signaling will be confirmed by over-expression. The second Aim investigates the direct and indirect targets for the phosphatases identified in Aim #1. We define points (kB degradation and nuclear translocation) in the signaling pathway above or below which the phosphatases are likely to operate. Co-immunoprecipitation of myc-tagged phosphatase components will be used to identify physical associations among NF-KB components and phosphatases. The subunits comprising the active PP1 and PP2A holoenzymes will be characterized. We also examine dephosphorylation of suspected target proteins, including TRAF2 and the p65 subunit of NF-KB. We then detail the mechanisms of action for a few phosphatases. The third Aim focuses on the ability of phosphatases to regulate transcription of endogenous genes. Preliminary data demonstrate the differential effects of phosphatase genes on regulation of chemokine and cytokine transcription. These studies will be extended to additional phosphatase genes, and we will examine transcriptional regulation of a selected series of NF-KB-dependent genes involved in the inflammatory process. In summary, the proposed experiments will provide insights into the role of phosphatases in controlling NF-KB-mediated signaling. We will also evaluate the impact of individual genes in controlling transcription of endogenous inflammatory mediators.

描述（由申请人提供）：TNFA和IL-1P是促进介质的促介质，可诱导趋化因子和细胞因子的风暴。他们被认为是导致多种慢性自身免疫和传染病中延长炎症介体的产生的原因。我们建议研究负责调节这些原型细胞因子反应的机制。具体而言，我们将定义控制TNF和IL-1介导的NF-KB激活的磷酸酶需求。 RNAi方法将与NF-KB-荧光素酶报告基因一起使用。已经制备了500个siRNA构建体，并筛选了SV40大型T抗原永生和原代细胞培养的基础和TNF介导的信号传导。初步数据证明了该策略的有效性。在我们的初始筛选中鉴定了19个磷酸酶基因。 19个基因中的10个以前与NF-KB信号传导无关。该提案列出了三个具体目标。首先是鉴定涉及TNF和IL-1信号传导的磷酸酶。此目的详细介绍了验证我们的初始发现所需的方法，并将我们的RNAi策略应用于IL-1信号的研究。与NF-KB信号相关的每个磷酸酶基因的功能将通过过表达确认。第二个目标研究了目标1中鉴定的磷酸酶的直接和间接靶标。我们在磷酸酶可能运行的信号传导途径中定义点（KB降解和核转运）。 MYC标记的磷酸酶成分的共免疫沉淀将用于鉴定NF-KB成分和磷酸酶之间的物理关联。将表征包含活性PP1和PP2A全酶的亚基。我们还检查了可疑靶蛋白的去磷酸化，包括TRAF2和NF-KB的p65亚基。然后，我们详细介绍了几种磷酸酶的作用机理。第三个目标侧重于磷酸酶调节内源基因转录的能力。初步数据证明了磷酸酶基因对趋化因子和细胞因子转录的调节的不同影响。这些研究将扩展到其他磷酸酶基因，我们将检查涉及炎症过程的一系列NF-KB依赖基因的转录调节。总而言之，提出的实验将提供有关磷酸酶在控制NF-KB介导的信号传导中的作用的见解。我们还将评估单个基因在控制内源性炎症介质转录方面的影响。