Lck/Hck/Fgr-Mediated Tyrosine Phosphorylation Negatively Regulates TBK1 to Restrain Innate Antiviral Responses

Cytosolic nucleic acid sensing elicits interferon production for primary antiviral defense through cascades controlled by protein ubiquitination and Ser/Thr phosphorylation. Here we show that TBK1, a core kinase of antiviral pathways, is inhibited by tyrosine phosphorylation. The Src family kinases (SFKs) Lck, Hck, and Fgr directly phosphorylate TBK1 at Tyr354/394, to prevent TBK1 dimerization and activation. Accordingly, antiviral sensing and resistance were substantially enhanced in Lck/Hck/Fgr triple knockout cells and ectopic expression of Lck/Hck/Fgr dampened the antiviral defense in cells and zebrafish. Small-molecule inhibitors of SFKs, which are conventional anti-tumor therapeutics, enhanced antiviral responses and protected zebrafish and mice from viral attack. Viral infection induced the expression of Lck/Hck/Fgr through TBK1-mediated mobilization of IRF3, thus constituting a negative feedback loop. These findings unveil the negative regulation of TBK1 via tyrosine phosphorylation and the functional integration of SFKs into innate antiviral immunity. [Display omitted] •TBK1 is dynamically tyrosine-phosphorylated by Src kinases (SFKs) during viral infection•Antiviral responses induce expression of SFKs Lck/Hck/Fgr via IRF3-directed transcription•Lck/Hck/Fgr modify TBK1 at Y394/354 to disrupt TBK1 dimerization and activation•Lck/Hck/Fgr and inhibitors govern antiviral defenses in cells, zebrafish, and mice Liu et al. report the Src family kinases (SFKs) Lck, Hck, and Fgr directly phosphorylate TBK1, a core player in antiviral immunity, to prevent its dimerization and activation, which dampens antiviral defenses in zebrafish and mice. SFKs are induced by antiviral immune signaling, thus constituting a negative feedback loop.