Abstract
TANK-binding kinase (TBK1) is essential for transcription of the interferon (IFN) β gene in response to lipopolysaccharide (LPS) and double-stranded RNA, but the molecular mechanisms that underlie the activation of TBK1 are incompletely understood. Previously, we identified the NF-κB essential modulator (NEMO)-related polyubiquitin-binding protein, optineurin (OPTN), as a novel binding partner of TBK1. To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFNβ production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTN(D477N/D477N). In this study, we found that LPS or poly(I:C)-induced TBK1 activity was significantly reduced in bone marrow-derived macrophage (BMDM) from OPTN(D477N/D477N) mice. Consistent with this, the phosphorylation of IFN regulatory factor 3 (IRF3) and the production of IFNβ mRNA and secretion were reduced. Stimulation of BMDMs with LPS triggered the phosphorylation of OPTN, which was reversed by phosphatase treatment and prevented by pharmacological inhibition of both the canonical IκB kinases (IKKα/β) and the IKK-related kinases (TBK1/IKKε). In contrast, LPS-stimulated phosphorylation of OPTN(D477N) was markedly reduced in BMDMs from OPTN(D477N/D477N) mice, and inhibition of the canonical IKKs alone prevented phosphorylation, providing further evidence that ubiquitin binding to OPTN contributes to LPS-induced TBK1 activation. TBK1 and IKKβ phosphorylated OPTN preferentially at Ser-177 and Ser-513, respectively, in vitro. In conclusion, our results suggest that OPTN binds to polyubiquitylated species formed in response to LPS and poly(I:C), enhancing the activation of TBK1 that is required for optimal phosphorylation of IRF3 and production of IFNβ.
Highlights
Optineurin is a polyubiquitin-binding protein of unknown function
Recent studies have established an essential role for ubiquitin-binding adaptor proteins such as NF-B essential modulator (NEMO) and ABIN1 in modulating pro-inflammatory cytokine production via regulation of key kinases, such as IKK␣/ and TAK1, much less is known regarding the contribution of K63pUb chains and ubiquitin-binding adaptor proteins in the regulation of kinases, such as TANK-binding kinase 1 (TBK1), that are critical for induction of type 1 IFNs
We have found that TBK1 activity is reduced significantly in response to activation of TLR4 (LPS) and TLR3 ((poly)I:C) in bone marrow-derived macrophage (BMDM) that express the polyubiquitin-binding defective mutant of OPTN
Summary
Optineurin is a polyubiquitin-binding protein of unknown function. Result: Macrophages from mice expressing a polyubiquitin-binding defective mutant of optineurin show reduced activation of TANK-binding kinase 1 (TBK1) and reduced production of interferon . To determine whether the ubiquitin-binding function of OPTN is involved in regulating TBK1 and IFN production, we generated a mouse in which wild-type optineurin was replaced by the polyubiquitin binding-defective mutant, OPTND477N/D477N. The importance of polyubiquitin binding to NEMO in regulating innate immunity has been established by the discovery of human mutations (e.g. the NEMO(D311N) mutation) [13] that abrogate binding to polyubiquitin [9, 11] and prevent activation of the canonical IKK complex or NF-B-dependent gene transcription [14] by inflammatory stimuli. The recognition of viral and bacterial components, such as dsRNA and LPS, is mediated by host pattern recognition receptors, including TLR3, TLR4, and the cytosolic RNA and DNA receptors [25,26,27] When engaged, these receptors stimulate the phosphorylation of IRF3, which is catalyzed by TBK1, and the related IB kinase family member IKK⑀. By utilizing a genetic approach, our findings describe an in vivo role for OPTN and demonstrate the importance of polyubiquitin-binding to OPTN for optimal activation of TBK1 and IFN production
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