Identification of TBK1 complexes required for the phosphorylation of IRF3 and the production of interferon β.
The double-stranded RNA mimetic poly(I:C) and lipopolysaccharide (LPS) activate Toll-like receptors 3 (TLR3) and TLR4, respectively, triggering the activation of TANK (TRAF family member-associated NF-κB activator)-binding kinase 1 (TBK1) complexes, the phosphorylation of interferon regulatory factor 3 (IRF3) and transcription of the interferon β (IFNβ) gene. Here, we demonstrate that the TANK-TBK1 and optineurin (OPTN)-TBK1 complexes control this pathway. The poly(I:C)- or LPS-stimulated phosphorylation of IRF3 at Ser396 and production of IFNβ were greatly reduced in bone marrow-derived macrophages (BMDMs) from TANK knockout (KO) mice crossed to knockin mice expressing the ubiquitin-binding-defective OPTN[D477N] mutant. In contrast, IRF3 phosphorylation and IFNβ production were not reduced significantly in BMDM from OPTN[D477N] knockin mice and only reduced partially in TANK KO BMDM. The TLR3/TLR4-dependent phosphorylation of IRF3 and IFNβ gene transcription were not decreased in macrophages from OPTN[D477N] crossed to mice deficient in IκB kinase ε, a TANK-binding kinase related to TBK1. In contrast with the OPTN-TBK1 complex, TBK1 associated with OPTN[D477N] did not undergo phosphorylation at Ser172 in response to poly(I:C) or LPS, indicating that the interaction of ubiquitin chains with OPTN is required to activate OPTN-TBK1 in BMDM. The phosphorylation of IRF3 and IFNβ production induced by Sendai virus infection were unimpaired in BMDM from TANK KO × OPTN[D477N] mice, suggesting that other/additional TBK1 complexes control the RIG-I-like receptor-dependent production of IFNβ. Finally, we present evidence that, in human HACAT cells, the poly(I:C)-dependent phosphorylation of TBK1 at Ser172 involves a novel TBK1-activating kinase(s).
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Siddharth
S
Bakshi
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Jordan
J
Taylor
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Sam
S
Strickson
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.
Thomas
T
McCartney
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K. p.cohen@dundee.ac.uk.
Philip
P
Cohen
D048868:Adaptor Proteins, Signal Transducing / Q000172:deficiency / Q000235:genetics; D033942:Adaptor Proteins, Vesicular Transport / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D000818:Animals; D018797:Cell Cycle Proteins; D002461:Cell Line, Transformed; D005136:Eye Proteins / Q000235:genetics / Q000276:immunology; D055879:Gene Knock-In Techniques; D006801:Humans; D051550:I-kappa B Kinase / Q000172:deficiency / Q000235:genetics; D050838:Interferon Regulatory Factor-3 / Q000235:genetics* / Q000276:immunology; D016899:Interferon-beta / Q000235:genetics* / Q000276:immunology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D008070:Lipopolysaccharides / Q000494:pharmacology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D026901:Membrane Transport Proteins; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011070:Poly I-C / Q000494:pharmacology; D061251:Primary Cell Culture; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000276:immunology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051196:Toll-Like Receptor 3 / Q000235:genetics* / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000235:genetics* / Q000276:immunology; D000099151:MAP Kinase Kinase Kinase 7
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D018797:Cell Cycle Proteins; D005136:Eye Proteins; D051550:I-kappa B Kinase; D050838:Interferon Regulatory Factor-3; D016899:Interferon-beta; D008070:Lipopolysaccharides; D020930:MAP Kinase Kinase Kinases; D026901:Membrane Transport Proteins; D011070:Poly I-C; D017346:Protein Serine-Threonine Kinases; D034741:RNA, Small Interfering; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D000099151:MAP Kinase Kinase Kinase 7; C494233:Irf3 protein, mouse; C501288:Optn protein, mouse; C470409:TICAM1 protein, human; C519703:TLR3 protein, mouse; C100814:Tank protein, mouse; C493487:Tlr4 protein, mouse; C498588:Tbk1 protein, mouse
IRF3; LPS; TBK1; TLR3; interferon; ubiquitin