Phosphatase regulation of NFKappaB activity

描述(申请人提供)：TNFa和IL-1p是促炎介质，可诱导趋化因子和细胞因子的风暴。它们被认为是在多种慢性自身免疫性和传染性疾病中延长炎性介质产生的原因。我们建议调查负责调节对这些原型细胞因子的反应的机制。具体地说，我们将定义控制肿瘤坏死因子和白介素1介导的核因子-KB激活所需的磷酸酶。RNAi方法将与NF-KB-荧光素酶报告程序一起使用。在SV40大T抗原永生化和原代细胞培养中，已经制备和筛选了500个siRNA结构物，用于基础和肿瘤坏死因子介导的信号转导。初步数据证明了这一策略的有效性。在我们最初的筛选中，共鉴定出19个磷酸酶基因。19个基因中有10个以前没有与核因子-KB信号相关。该提案列出了三个具体目标。第一个是确定参与肿瘤坏死因子和白介素1信号转导的磷酸酶。这个目标详细说明了验证我们最初发现所需的方法学，并将我们的RNAi策略应用于IL-1信号转导的研究。与核因子-KB信号相关的每个磷酸酶基因的功能将通过过度表达得到证实。第二个目标是研究AIM#1中确定的磷酸酶的直接和间接靶点。我们在信号通路中定义了磷酸酶可能在其上方或下方发挥作用的点(kB降解和核转位)。Myc标记的磷酸酶组分的免疫共沉淀将用于鉴定核因子-KB组分和磷酸酶之间的物理联系。将对由活性PP1和PP2A全酶组成的亚基进行表征。我们还检查了可疑目标蛋白的去磷酸化，包括TRAF2和NF-KB的p65亚单位。然后，我们详细介绍了几种磷酸酶的作用机制。第三个目标集中在磷酸酶调节内源基因转录的能力上。初步数据表明，磷酸酶基因在调节趋化因子和细胞因子转录方面的不同作用。这些研究将扩展到更多的磷酸酶基因，我们将检查参与炎症过程的一系列依赖于核因子-KB的基因的转录调控。总之，拟议的实验将提供对磷酸酶在控制核因子-KB介导的信号转导中的作用的深入了解。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。

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.