Phosphatase regulation of NFKappaB activity

描述（由申请人提供）：TNFa和IL-1p是促炎介质，可诱导趋化因子和细胞因子的风暴。它们被认为在多种慢性自身免疫性疾病和传染性疾病中负责延长炎症介质的产生。我们建议研究负责调节对这些原型细胞因子反应的机制。具体来说，我们将定义控制TNF和il -1介导的NF-KB激活所需的磷酸酶。RNAi方法将与nf - kb荧光素酶报告基因一起使用。在SV40大T抗原永生化和原代细胞培养中，已经制备并筛选了500种siRNA结构，用于基础和tnf介导的信号传导。初步数据证明了这一战略的有效性。在我们最初的筛选中鉴定出19个磷酸酶基因。19个基因中有10个先前与NF-KB信号传导无关。该提案列出了三个具体目标。首先是确定参与TNF和IL-1信号传导的磷酸酶。本文详细介绍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信号的研究。每个与NF-KB信号相关的磷酸酶基因的功能将通过过表达得到证实。第二个目标是研究目标1中确定的磷酸酶的直接和间接靶标。我们在信号通路的上方或下方定义了磷酸酶可能起作用的点（kB降解和核易位）。myc标记的磷酸酶组分的共免疫沉淀将用于鉴定NF-KB组分和磷酸酶之间的物理关联。组成活性PP1和PP2A全酶的亚基将被表征。我们还研究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制。第三个目标侧重于磷酸酶调节内源性基因转录的能力。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差异作用。这些研究将扩展到其他磷酸酶基因，我们将研究一系列选定的nf - kb依赖性基因在炎症过程中的转录调控。总之，所提出的实验将提供对磷酸酶在控制nf - kb介导的信号传导中的作用的见解。我们还将评估个体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.