Signaling to PIAS1 to Regulate Immune Responses

描述(申请人提供)：必须严格控制炎症刺激引发的免疫反应。不受限制的炎症与免疫紊乱有关。细胞外炎症刺激如何向细胞核发出信号以限制炎症基因的激活，目前还知之甚少。核因子？B和STATS是两个重要的转录因子家族，可被多种促炎刺激激活以诱导基因表达。活化的STAT1蛋白抑制因子1(PIAS1)通过选择性地阻断NF？B和STAT1与基因启动子的结合来抑制免疫反应。我们最近发现了一条新的信号通路，在该通路中，促炎刺激激活了IKKA介导的PIAS1的磷酸化，从而立即抑制了炎症基因的激活。这项研究计划的总体目标是描述Ikka-PIAS1信号通路在免疫反应调节中的特征。具体地说，我们将通过突变分析和激酶分析来表征IKKA介导的PIAS1 Ser90磷酸化的分子基础。IKK基因敲除细胞将研究IKK家族其他成员的作用以及IKKS在介导PIAS1 Ser90磷酸化中的特异性。接下来，我们将研究PIAS1的磷酸化如何调节PIAS1的活性。我们将研究PIAS1 Ser90磷酸化在配体诱导的PIAS1启动子招募中的作用。我们将分析PIAS1 SAP结构域在PIAS1的Ser90磷酸化依赖的启动子招募中的作用。我们将使用Ser90磷酸化突变体测试Ser90磷酸化是否足以将PIAS1靶向基因启动子。PIAS1的启动子结合区(S)将通过芯片分析和突变分析进行检测。最后，我们将建立PIAS1磷酸化缺陷敲击小鼠模型，以研究PIAS1磷酸化在病原性感染中的生理功能。这些研究将探索一种新的免疫反应负调控的分子信号机制，这将增强我们设计合理的治疗策略治疗感染性和炎症性疾病的能力。与公共健康相关：必须严格控制炎症刺激引发的免疫反应。不受限制的炎症与免疫紊乱和癌症有关。这项建议是为了研究一种新发现的信号通路，它具有平衡免疫反应的功能。这些研究将提高我们设计治疗免疫性疾病的新治疗策略的能力。

DESCRIPTION (provided by applicant): The immune responses triggered by inflammatory stimuli must be tightly regulated. Unrestricted inflammation is associated with immune disorders. How extracellular inflammatory stimuli signal to the nucleus to restrict inflammatory gene activation is poorly understood. NF?B and STATs are two important families of transcription factors that are activated by a wide variety of pro-inflammatory stimuli to induce gene expression. Protein inhibitor of activated STAT1 (PIAS1) inhibits immune responses by selectively blocking the binding of NF?B and STAT1 to gene promoters. We have recently identified a novel signaling pathway in which pro- inflammatory stimuli activate the IKKa-mediated phosphorylation of PIAS1 for the immediate repression of inflammatory gene activation. The overall goal of this research proposal is to characterize the IKKa-PIAS1 signaling pathway in the regulation of immune responses. Specifically, we will characterize the molecular basis of the IKKa-mediated PIAS1 Ser90 phosphorylation by mutational analysis and kinase assays. The role of other members of the IKK family and the specificity of IKKs in mediating PIAS1 Ser90 phosphorylation will be examined using IKK knockout cells. Next, we will investigate how PIAS1 phosphorylation regulates the activity of PIAS1. We will examine the role of PIAS1 Ser90 phosphorylation in the ligand-induced promoter recruitment of PIAS1. We will analyze the role of PIAS1 SAP domain in the Ser90 phosphorylation-dependent promoter recruitment of PIAS1. We will test if Ser90 phosphorylation is sufficient to target PIAS1 to gene promoters using Ser90 phosphomimic mutants. The promoter binding region(s) of PIAS1 will be examined by ChIP assays and mutational analysis. Finally, we will generate a PIAS1 phosphorylation-defective knockin mouse model to study the physiological function of PIAS1 phosphorylation in response to pathogenic infection. These studies will investigate a novel molecular signaling mechanism for the negative regulation of immune responses, which will enhance our ability to design rational therapeutic strategies for the treatment of infectious and inflammatory diseases. RELEVANCE TO PUBLIC HEALTH: The immune responses triggered by inflammatory stimuli must be tightly regulated. Unrestricted inflammation is associated with immune disorders and cancer. This proposal is to study a newly identified signaling pathway that functions to balance immune responses. These studies will enhance our ability to design novel therapeutic strategies for the treatment of immune diseases.