Li Xuelian, Yang Mingjin, Yu Zhou, Tang Songqing, Wang Lei, Cao Xuetao, Chen Taoyong
National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai 200433, China.
National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China.
Sci Signal. 2017 Jan 3;10(460):eaae0435. doi: 10.1126/scisignal.aae0435.
Various pattern recognition receptors (PRRs) are activated in response to viral infection to stimulate the production of type I interferons (IFNs). However, central to the responses of all of these receptors is their activation of the kinase TBK1, which stimulates transcription by IFN regulatory factor 3 (IRF3). We investigated the mechanism by which the kinase activity of TBK1 is stimulated in response to viral infection. We found that the tyrosine kinase Src promoted the phosphorylation of TBK1 on Tyr upon viral infection of RAW264.7 macrophages. Mutation of Tyr to alanine resulted in impaired autophosphorylation of TBK1 at Ser, which is required for TBK1 activation. The TBK1 Y179A mutant failed to rescue type I IFN production by virally infected RAW264.7 macrophages deficient in TBK1. Pharmacological inhibition of Src with AZD0530 and clustered regularly interspaced short palindromic repeats/Cas9-mediated knockout of Src demonstrated that Src was critical for activating the TBK1-IRF3 pathway and stimulating type I IFN production. However, Src did not directly bind to recombinant TBK1 in vitro but instead bound to the proline-X-X-proline motifs within key PRR adaptor proteins, such as TRIF, MAVS, and STING, which formed complexes with TBK1 after PRR engagement. Together, our data suggest that Src is the major tyrosine kinase that primes TBK1 for autophosphorylation and activation, thus providing mechanistic insights into the regulation of TBK1 activity by various PRRs as part of the innate antiviral response.
多种模式识别受体（PRR）在病毒感染时被激活，以刺激I型干扰素（IFN）的产生。然而，所有这些受体反应的核心是它们对激酶TBK1的激活，TBK1通过干扰素调节因子3（IRF3）刺激转录。我们研究了病毒感染时TBK1激酶活性被刺激的机制。我们发现，酪氨酸激酶Src在RAW264.7巨噬细胞受到病毒感染时促进了TBK1酪氨酸位点的磷酸化。将酪氨酸突变为丙氨酸导致TBK1丝氨酸位点的自磷酸化受损，而这是TBK1激活所必需的。TBK1 Y179A突变体无法挽救缺乏TBK1的RAW264.7巨噬细胞被病毒感染后I型干扰素的产生。用AZD0530对Src进行药理学抑制以及通过成簇规律间隔短回文重复序列/Cas9介导敲除Src表明，Src对于激活TBK1-IRF3通路和刺激I型干扰素的产生至关重要。然而，Src在体外并不直接与重组TBK1结合，而是与关键PRR衔接蛋白（如TRIF、MAVS和STING）中的脯氨酸-X-X-脯氨酸基序结合，这些衔接蛋白在PRR参与后与TBK1形成复合物。总之，我们的数据表明，Src是引发TBK1自磷酸化和激活的主要酪氨酸激酶，从而为作为先天抗病毒反应一部分的各种PRR对TBK1活性的调节提供了机制性见解。
