Functional Characterization of Suppressor of IKKepsilon

描述（由申请方提供）：病原体攻毒引起先天免疫系统的立即反应。多种先天性免疫信号传导途径会聚以激活TANK结合激酶1（TBK 1），一种普遍表达的激酶。TBK 1的底物已被证明是协调宿主防御的关键参与者，决定了下游先天免疫反应的形状。我们定义了一种新的TBK 1底物，IKK抑制因子（SIKE）。SIKE最初被鉴定为TBK 1抑制剂。在我们定义SIKE介导的TBK 1抑制的研究中，我们发现SIKE被TBK 1磷酸化，并且TBK 1：SIKE相互作用由SIKE磷酸化调节。相对于其他TBK 1底物，SIKE作为TBK 1的纳摩尔混合型抑制剂。虽然SIKE作为高亲和力底物影响TBK 1活性，但SIKE的主要功能仍不清楚。为了解决这一知识差距，我们研究了SIKE的本地化模式和互动网络。大体定位研究表明，GFP-SIKE是胞质的，聚集在细胞骨架结构，和致密的胞质颗粒。串联MS/MS数据从免疫沉淀的SIKE复合物显示，SIKE相互作用的细胞骨架，翻译机器，和mRNA转运蛋白与核糖核蛋白复合物（RNP）。RNP在病毒感染后形成，以减少用于病毒复制的翻译机器的可用性。这种关系表明，RNP形成宿主的抗病毒防御的一部分，但先天免疫信号传导和这些RNA介导的防御之间的联系是不明确的。基于我们的初步数据，我们假设，SIKE功能作为一个桥梁之间的先天免疫反应和转录后的mRNA池在胞质RNP介导的快速宿主反应的翻译水平。在目标1中，我们将在上皮细胞中建立SIKE相互作用，该相互作用形成对入侵病原体的防御屏障， RNP形成可能代表一种关键的防御机制，吞噬免疫细胞负责隔离/去除病原体，其中快速的细胞骨架重排和细胞迁移是基本功能。我们将使用聚肌苷酸-聚胞苷酸形式的双链RNA评估病原体特异性SIKE相互作用，该双链RNA模拟病毒感染的关键方面，是RNP和S.鼠伤寒沙门氏菌，其被吞噬细胞摄取并激活TBK 1依赖性途径。在目标2中，我们将研究SIKE的磷酸化状态对细胞类型和病原体刺激的影响。我们将通过功能测定，包括细胞迁移、吞噬作用、含病原体空泡的形成以及RNP的形成和分解，研究SIKE在细胞周边与细胞骨架相关的功能与其在胞质颗粒中的作用。这些确定SIKE在桥接TBK 1介导的先天免疫应答和RNA颗粒中的功能的研究将为TBK 1如何塑造我们的先天免疫防御提供关键的新见解，并为建立长期研究TBK 1介导的先天免疫应答的功能，调节和病毒颠覆奠定基础。

DESCRIPTION (provided by applicant): Pathogen challenge elicits an immediate response by the innate immune system. Multiple innate immune signaling pathways converge to activate TANK binding kinase 1 (TBK1), a ubiquitously expressed kinase. Substrates of TBK1 have proven to be critical players in coordinating host defenses, dictating the shape of the downstream innate immune response. We have defined a new TBK1 substrate, Suppressor of IKKϵ (SIKE). SIKE was first identified as a TBK1 inhibitor. In our studies to define SIKE-mediated TBK1 inhibition, we discovered that SIKE was phosphorylated by TBK1 and the TBK1:SIKE interaction was modulated by SIKE phosphorylation. With respect to other TBK1 substrates, SIKE acted as a nanomolar, mixed-type inhibitor of TBK1. Although SIKE influenced TBK1 activity as a high affinity substrate, SIKE's primary function remains unknown. To address this gap in knowledge, we examined SIKE's localization pattern and interaction network. Gross localization studies indicated that GFP-SIKE was cytosolic, accumulated in cytoskeletal structures, and dense cytosolic granules. Tandem MS/MS data from immunoprecipitated SIKE complexes revealed that SIKE interacted with the cytoskeleton, translation machinery, and mRNA transport proteins associated with ribonucleoprotein complexes (RNPs). RNPs are formed following viral infection to reduce the availability of translation machinery for viral replication. This relationship suggests that RNPs form part of the host's antiviral defenses, yet the linkage between innate immune signaling and these RNA-mediated defenses is poorly defined. Based upon our preliminary data, we hypothesize that SIKE functions as a bridge between the innate immune response and post-transcriptional mRNA pools in cytosolic RNPs to mediate a rapid host response at the level of translation. In Aim 1, we will establish SIKE interactions in epithelial cells that form a defensive barrier to invading pathogen and where rapid RNP formation may represent a key defense mechanism, and phagocytic immune cells responsible for sequestering/removing pathogen where rapid cytoskeletal rearrangement and cell migration are essential functions. We will assess pathogen-specific SIKE interactions using dsRNA in the form of polyinosinic-polycytidylic acid, which mimics key aspects of viral infection, an inducer of RNPs, and S. typhimurium, which is taken up by phagocytic cells and activates TBK1-dependent pathways. In Aim 2, we will examine the impact of SIKE's phosphorylation state on its localization with respect to cell-type and pathogen stimuli. We will investigate SIKE' function at the cell periphery in association with the cytoskeleton versus its role in cytosolic granules through functional assays including cell migration, phagocytosis, formation of pathogen containing vacuoles, and RNP formation and resolution. These studies to determine SIKE's function in bridging TBK1-mediated innate immune responses and RNA granules will provide critical new insight into how TBK1 shapes our innate immune defenses and establish a foundation for building long-term studies on the function, regulation and viral subversion of TBK1-mediated innate immune responses.