Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon β
Polyubiquitin Binding to Optineurin Is Required for Optimal Activation of TANK-binding Kinase 1 and Production of Interferon β
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DOI：
10.1074/jbc.m111.267567
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发表时间：
2011-10-14
影响因子：
4.8
通讯作者：
Cohen, Philip
中科院分区：
文献类型：
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作者：
Gleason, Catherine E.;Ordureau, Alban;Cohen, Philip
TANK 结合激酶 (TBK1) 对于响应脂多糖 (LPS) 和双链 RNA 的干扰素 (IFN) β 基因转录至关重要，但 TBK1 激活的分子机制尚不完全清楚。此前，我们确定了 NF-κ B 必需调节剂 (NEMO) 相关的多聚泛素结合蛋白 optineurin (OPTN) 作为 TBK1 的新型结合伴侣。为了确定 OPTN 的泛素结合功能是否参与调节 TBK1 和 IFN β 的产生，我们培育了一只小鼠，其中野生型 optineurin 被多聚泛素结合缺陷突变体 OPTND477N/D477N 取代。在这项研究中，我们发现 OPTND477N/D477N 小鼠的骨髓源性巨噬细胞 (BMDM) 中 LPS 或聚 (I:C) 诱导的 TBK1 活性显着降低。与此一致的是，干扰素调节因子 3 (IRF3) 的磷酸化以及干扰素β mRNA 的产生和分泌均减少。用 LPS 刺激 BMDM 会引发 OPTN 的磷酸化，磷酸酶处理可逆转这种磷酸化，并可通过经典 I kappa B 激酶 (IKK α/β) 和 IKK 相关激酶 (TBK1/IKK epsilon) 的药理抑制来预防。相比之下，LPS 刺激的 OPTN(D477N) 磷酸化在 OPTND477N/D477N 小鼠的 BMDM 中显着降低，并且单独抑制经典 IKK 可以阻止磷酸化，这进一步证明泛素与 OPTN 的结合有助于 LPS 诱导的 TBK1 激活。在体外，TBK1 和 IKK 分别优先在 Ser-177 和 Ser-513 处磷酸化 OPTN。总之，我们的结果表明 OPTN 与响应 LPS 和 Poly(I:C) 形成的多泛素化物种结合，增强 TBK1 的激活，这是 IRF3 最佳磷酸化和 IFN beta 产生所需的。
TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) beta gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-kappa B essential modulator (NEMO)-related polyubiquitin-binding protein, optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFN beta production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTND477N/D477N. In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTND477N/D477N mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFN beta mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical I kappa B kinases (IKK alpha/beta) and the IKK-related kinases (TBK1/IKK epsilon). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTND477N/D477N mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKK beta phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquity-lated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFN beta.