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Src and interferon
Cells are armed with multiple pattern recognition receptors (PRRs) that sense viral infection. Although PRRs mediate signaling through different adaptor proteins, they mostly converge on activation of the kinase TBK1, which phosphorylates and activates the transcription factor IRF3 to promote expression of genes encoding type I interferons (IFNs). Li et al. found that TBK1, which undergoes serine autophosphorylation to become activated, was also tyrosine-phosphorylated, which was dependent on the kinase Src. Although Src did not directly associate with and phosphorylate TBK1, it was recruited to multiple PRR-associated adaptor-containing complexes that contained TBK1. Furthermore, loss of Src or inhibition of its activity impaired the production of type I IFNs by virally infected macrophages, suggesting that Src acts as a central regulator of the innate immune response to viruses.
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 Tyr179 upon viral infection of RAW264.7 macrophages. Mutation of Tyr179 to alanine resulted in impaired autophosphorylation of TBK1 at Ser172, 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.