The tyrosine kinase Src promotes phosphorylation of the kinase TBK1 to facilitate type I interferon production after viral infection

, 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.