STING recruits TBK1 and IRF3

In dsDNA-stimulated human and mouse cells TBK1 has been shown to move to cytoplasmic punctate structures, where it associates with STING to induce IRF3 activation (Ishikawa et al. 2009, Saitoh et al. 2009, Sun et al. 2009, Tanaka & Chen 2012). Co-immunoprecipitation assays in HEK 293T cells expressing HA-tagged STING and Flag-tagged TBK1 showed that TBK1 directly interacts with STING. Moreover, glutathione S-transferase (GST) pull-down assays showed that recruitment of TBK1 by STING was enhanced upon c-di-GMP binding (Ouyang et al. 2012).<p>STING was reported to mediate TBK1-dependent activation of transcription factor IRF3 (Zhong B et al. 2008, Tanaka and Chen 2012). Both TBK1 and IRF3 can directly interact with STING through its C-terminal region (Tanaka & Chen 2012). A construct of human STING containing only 39 amino acid residues of its C-terminus (341 to 379) was sufficient to activate IRF3 in cytosolic extracts of HeLa cells. Further mutagenesis studies showed, that two residues, Ser366 and Leu374, within the C-terminal tail of STING were required for IRF3 binding and phosphorylation, but were dispensable for TBK1 binding and activation (Tanaka & Chen 2012). Thus, STING is thought to function as a scaffold to recruit cytosolic TBK1 and IRF3, which results in TBK1-dependent phosphorylation of IRF3. Importantly, though both STING monomers and dimers can bind TBK1, only STING dimers activates Type I IFN (Ouyang et al. 2012). The nucleotide binding domain and leucine-rich repeat-containing (NLR) protein NLRC3 interacts with STING and TBK1, reducing STING-TBK1 association and reduces the trafficking of STING to the perinuclear region, leading to decreased activation of innate immune cytokines (Zhang et al. 2014).

在dsDNA刺激的人和小鼠细胞中，TBK1已被证实转移至细胞质中的点状结构，并与STING结合以诱导IRF3激活（Ishikawa等，2009年，Saitoh等，2009年，Sun等，2009年，Tanaka & Chen，2012年）。在表达HA标记的STING和Flag标记的TBK1的HEK 293T细胞中进行的共免疫沉淀实验表明，TBK1与STING直接相互作用。此外，谷胱甘肽S-转移酶（GST）拉下实验显示，在c-di-GMP结合后，STING对TBK1的募集作用增强（Ouyang等，2012年）。据报道，STING介导TBK1依赖性转录因子IRF3的激活（Zhong B等，2008年，Tanaka和Chen，2012年）。TBK1和IRF3均可通过其C端区域直接与STING相互作用（Tanaka和Chen，2012年）。仅包含人类STINGC端39个氨基酸残基（341至379）的构建体足以在HeLa细胞的细胞质提取物中激活IRF3。进一步的突变研究显示，STING C端尾部的两个残基，Ser366和Leu374，对于IRF3的结合和磷酸化是必需的，但对于TBK1的结合和激活则是非必需的（Tanaka和Chen，2012年）。因此，STING被认为作为一种支架，以募集细胞质中的TBK1和IRF3，从而导致TBK1依赖性的IRF3磷酸化。值得注意的是，尽管STING单体和二聚体均可与TBK1结合，但只有STING二聚体能够激活I型干扰素（Ouyang等，2012年）。核苷酸结合结构域和富含亮氨酸重复序列（NLR）蛋白NLRC3与STING和TBK1相互作用，减少STING-TBK1的结合，并降低STING向核周区域的转运，从而导致固有免疫细胞因子活化的减少（Zhang等，2014年）。