Phosphatase regulation of NFKappaB activity

磷酸酶调节 NFKappaB 活性

• 批准号: 7316242
• 负责人: MARTIN E DORF
• 金额: $ 36.37万
• 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/7316242
• 关键词: Adaptor Signaling Protein; Autoimmune Process; Cells; Chronic; Co-Immunoprecipitations; Communicable Diseases; Complex; Data; Effectiveness; Enzymes; Epitopes; Genes; Genetic Transcription; Goals; Holoenzymes; Immune; Individual; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1; Large T Antigen; Libraries; Luciferases; Mediating; Mediator of activation protein; Methodology; Molecular Target; NF-kappa B; Nuclear Translocation; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Primary Cell Cultures; Process; Production; Protein Biosynthesis; 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; TNF gene; TNF receptor-associated factor 2; TRAF2 gene; Thinking; Tissues; Transcript; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Update; chemokine; computerized data processing; cytokine; human TNF protein; insight; p65; 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）：TNF α和IL-1 β是诱导趋化因子和细胞因子风暴的促炎介质。他们被认为是负责延长生产的炎症介质在多种慢性自身免疫性和感染性疾病。我们建议调查的机制负责调节这些原型细胞因子的反应。具体来说，我们将确定控制TNF和IL-1介导的NF-κ B活化的磷酸酶要求。RNAi方法将与NF-κ B-荧光素酶报告基因一起使用。已经制备了500个siRNA构建体，并在SV 40大T抗原永生化和原代细胞培养物中筛选了基础和TNF介导的信号传导。初步数据证明了这一战略的有效性。在我们的初步筛选中鉴定了19个磷酸酶基因。19个基因中有10个先前与NF-κ B信号传导无关。该提案列出了三个具体目标。第一个是鉴定参与TNF和IL-1信号传导的磷酸酶。该目标详细说明了验证我们的初步发现所需的方法，并将我们的RNAi策略应用于IL-1信号转导的研究。将通过过表达证实与NF-κ B信号传导相关的每个磷酸酶基因的功能。第二个目标研究目标#1中鉴定的磷酸酶的直接和间接靶标。我们定义点（kB降解和核转位）在信号通路的磷酸酶可能会操作的上方或下方。myc标记的磷酸酶组分的免疫共沉淀将用于鉴定NF-κ B组分和磷酸酶之间的物理关联。将表征包含活性PP 1和PP 2A全酶的亚基。我们还研究了疑似靶蛋白的去磷酸化，包括TRAF 2和NF-κ B的p65亚基。然后，我们详细介绍了一些磷酸酶的作用机制。第三个目标集中于磷酸酶调节内源基因转录的能力。初步的数据表明，不同的磷酸酶基因对趋化因子和细胞因子转录的调节作用。这些研究将扩展到其他磷酸酶基因，我们将检查一系列选定的NF-κ B依赖性基因参与炎症过程的转录调控。总之，所提出的实验将提供对磷酸酶在控制NF-κ B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中的影响。