scholarly journals Priming Phosphorylation of TANK-Binding Kinase 1 by IκB Kinase β Is Essential in Toll-Like Receptor 3/4 Signaling

2019 ◽  
Vol 40 (5) ◽  
Author(s):  
Hiroto Abe ◽  
Junko Satoh ◽  
Yutaro Shirasaka ◽  
Amane Kogure ◽  
Hiroki Kato ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Interferon Regulatory Factor ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Double Stranded Rna ◽  
Activate Transcription Factor ◽  
Iκb Kinase ◽  
Phosphorylation Cascade ◽  
Tlr3 Signaling ◽  
Inducible Gene

ABSTRACT TRIF is an essential adaptor for Toll-like receptor 3/4 (TLR3/4) signaling to activate transcription factor interferon regulatory factor 3 (IRF-3). We examined the molecular mechanism of TLR3 signaling and found that TLR3 stimulation by double-stranded RNA (dsRNA) induces phosphorylation of TRIF at Ser210 and is required for IRF-3 recruitment. TANK-binding kinase 1 (TBK1) is known to be responsible for IRF-3 phosphorylation and activation. We found that TBK1 is also responsible for phosphorylation of Ser210 in TRIF. Unexpectedly, we discovered that IκB kinase β (IKKβ) plays an essential role in TLR3/4 signaling using a pharmacological inhibitor and gene deletion. Of note, IKKβ is essential in TLR3/4 but not in retinoic acid-inducible gene I (RIG-I) signaling. Mechanistically, IKKβ transiently associates with and induces the phosphorylation of TBK1 upon TLR3 stimulation. These results suggest a phosphorylation cascade of IKKβ and TBK1, where priming phosphorylation of TBK1 by IKKβ is required to surpass the threshold to induce signaling, thereby activating IRF-3.

scholarly journals The Roles of Two IκB Kinase-related Kinases in Lipopolysaccharide and Double Stranded RNA Signaling and Viral Infection

2004 ◽  
Vol 199 (12) ◽  
pp. 1641-1650 ◽  
Author(s):  
Hiroaki Hemmi ◽  
Osamu Takeuchi ◽  
Shintaro Sato ◽  
Masahiro Yamamoto ◽  
Tsuneyasu Kaisho ◽  
...  
Keyword(s):  
Viral Infection ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Tlr Signaling ◽  
Dramatic Decrease ◽  
Embryonic Fibroblasts ◽  
Double Stranded Rna ◽  
Iκb Kinase ◽  
Inducible Genes ◽  
Rna Signaling

Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-β gene induction. Recently, two IκB kinase (IKK)–related kinases, inducible IκB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-β production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-β and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i–deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-β and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.

scholarly journals Identification of TBK1 complexes required for the phosphorylation of IRF3 and the production of interferon β

2017 ◽  
Vol 474 (7) ◽  
pp. 1163-1174 ◽  
Author(s):  
Siddharth Bakshi ◽  
Jordan Taylor ◽  
Sam Strickson ◽  
Thomas McCartney ◽  
Philip Cohen
Keyword(s):  
Sendai Virus ◽  
Interferon Regulatory Factor ◽  
Poly I:C ◽  
Hacat Cells ◽  
Regulatory Factor ◽  
Interferon Β ◽  
Present Evidence ◽  
Double Stranded Rna ◽  
Iκb Kinase ◽  
Ubiquitin Binding

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).

Interferon regulatory factor-1 gene deletion decreases glomerulonephritis in MRL/lpr mice

2006 ◽  
Vol 36 (5) ◽  
pp. 1296-1308 ◽  
Author(s):  
Christopher M. Reilly ◽  
Selen Olgun ◽  
David Goodwin ◽  
Robert M. Gogal ◽  
Arben Santo ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Interferon Regulatory Factor ◽  
Regulatory Factor ◽  
Interferon Regulatory Factor 1

Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2573-2573
Author(s):  
Daniela Werth ◽  
Anita Bringmann ◽  
Katharina Brauer ◽  
Karin von Schwarzenberg ◽  
Stefanie Held ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Activation ◽  
Nuclear Translocation ◽  
Interferon Regulatory Factor ◽  
Toll Like Receptor ◽  
Regulatory Factor ◽  
Dendritic Cell Activation

Abstract Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam3Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam3Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.

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