title: The tyrosine kinase Src promotes phosphorylation of the kinase TBK1 to facilitate type I interferon production after viral infection.
abstract: 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 Tyr179upon viral infection of RAW264.7 macrophages. Mutation of Tyr179to 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.
authors.affiliation: National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Xuelian
authors.initials: X
authors.lastname: Li
authors.affiliation: National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Mingjin
authors.initials: M
authors.lastname: Yang
authors.affiliation: National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China.
authors.forename: Zhou
authors.initials: Z
authors.lastname: Yu
authors.affiliation: National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai 200433, China.
authors.forename: Songqing
authors.initials: S
authors.lastname: Tang
authors.affiliation: National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.affiliation: National Key Laboratory of Medical Molecular Biology and Department of Immunology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China. chenty@immunol.org caoxt@immunol.org.
authors.forename: Xuetao
authors.identifier: 0000-0001-9677-7647
authors.initials: X
authors.lastname: Cao
authors.affiliation: National Key Laboratory of Medical Immunology and Institute of Immunology, Second Military Medical University, Shanghai 200433, China. chenty@immunol.org caoxt@immunol.org.
authors.forename: Taoyong
authors.identifier: 0000-0002-2507-0027
authors.initials: T
authors.lastname: Chen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000502:physiology; D054884:Host-Pathogen Interactions; D006801:Humans; D007370:Interferon Type I / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism* / Q000821:virology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D014721:Vesicular stomatitis Indiana virus / Q000502:physiology; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D007370:Interferon Type I; C498588:Tbk1 protein, mouse; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases