Signaling to PIAS1 to Regulate Immune Responses

描述（由申请人提供）：由炎症刺激触发的免疫反应必须受到严格调节。非限制性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激活尚不清楚。NF？B和STAT是两个重要的转录因子家族，它们被多种促炎刺激物激活以诱导基因表达。活化的STAT 1蛋白抑制剂（PIAS 1）通过选择性阻断NF？B和STAT 1与基因启动子连接。我们最近发现了一种新的信号通路，其中促炎刺激激活IKKa介导的PIAS 1磷酸化，以立即抑制炎症基因激活。这项研究的总体目标是表征IKKa-PIAS 1信号通路在免疫应答调节中的作用。具体而言，我们将通过突变分析和激酶测定来表征IKKa介导的PIAS 1 Ser 90磷酸化的分子基础。将使用IKK敲除细胞检查IKK家族其他成员的作用和IKK在介导PIAS 1 Ser 90磷酸化中的特异性。接下来，我们将研究PIAS 1磷酸化如何调节PIAS 1的活性。我们将研究PIAS 1 Ser 90磷酸化在配体诱导的PIAS 1启动子募集中的作用。我们将分析PIAS 1 SAP结构域在PIAS 1的Ser 90磷酸化依赖性启动子募集中的作用。我们将使用Ser 90磷酸模拟突变体测试Ser 90磷酸化是否足以将PIAS 1靶向基因启动子。将通过ChIP试验和突变分析检查PIAS 1的启动子结合区。最后，我们将建立PIAS 1磷酸化缺陷的敲入小鼠模型，以研究PIAS 1磷酸化在病原感染应答中的生理功能。这些研究将探讨一种新的负调节免疫反应的分子信号机制，这将提高我们设计合理的治疗策略来治疗感染性和炎症性疾病的能力。与公共卫生的相关性：由炎症刺激引发的免疫反应必须受到严格的调节。非限制性炎症与免疫紊乱和癌症有关。这项提案是为了研究一种新发现的信号通路，其功能是平衡免疫反应。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.