Systematic analysis of the IL ‐17 receptor signalosome reveals a robust regulatory feedback loop
2020; Springer Nature; Volume: 39; Issue: 17 Linguagem: Inglês
10.15252/embj.2019104202
ISSN1460-2075
AutoresHelena Draberova, Sarka Janusova, Daniela Knizkova, Tereza Šemberová, Michaela Přibíková, Andrea Ujevic, Karel Harant, Sofija Knápková, Matouš Hrdinka, Viola Fanfani, Giovanni Stracquadanio, Ales Drobek, Klara Ruppova, Ondřej Štěpánek, Peter Dráber,
Tópico(s)Immune Response and Inflammation
ResumoArticle21 July 2020Open Access Source DataTransparent process Systematic analysis of the IL-17 receptor signalosome reveals a robust regulatory feedback loop Helena Draberova Helena Draberova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Sarka Janusova Sarka Janusova Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Daniela Knizkova Daniela Knizkova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Tereza Semberova Tereza Semberova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Michaela Pribikova Michaela Pribikova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Andrea Ujevic Andrea Ujevic Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Karel Harant Karel Harant Laboratory of Mass Spectrometry, BIOCEV, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Sofija Knapkova Sofija Knapkova Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Matous Hrdinka Matous Hrdinka orcid.org/0000-0002-2981-2825 Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Viola Fanfani Viola Fanfani Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Giovanni Stracquadanio Giovanni Stracquadanio Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ales Drobek Ales Drobek orcid.org/0000-0003-1066-9413 Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Klara Ruppova Klara Ruppova Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Ondrej Stepanek Corresponding Author Ondrej Stepanek [email protected] orcid.org/0000-0002-2735-3311 Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Peter Draber Corresponding Author Peter Draber [email protected] orcid.org/0000-0001-8658-4614 Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Helena Draberova Helena Draberova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Sarka Janusova Sarka Janusova Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Daniela Knizkova Daniela Knizkova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Tereza Semberova Tereza Semberova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Michaela Pribikova Michaela Pribikova Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Andrea Ujevic Andrea Ujevic Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Karel Harant Karel Harant Laboratory of Mass Spectrometry, BIOCEV, Faculty of Science, Charles University, Prague, Czech Republic Search for more papers by this author Sofija Knapkova Sofija Knapkova Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Matous Hrdinka Matous Hrdinka orcid.org/0000-0002-2981-2825 Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic Search for more papers by this author Viola Fanfani Viola Fanfani Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Giovanni Stracquadanio Giovanni Stracquadanio Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ales Drobek Ales Drobek orcid.org/0000-0003-1066-9413 Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Klara Ruppova Klara Ruppova Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Ondrej Stepanek Corresponding Author Ondrej Stepanek [email protected] orcid.org/0000-0002-2735-3311 Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Peter Draber Corresponding Author Peter Draber [email protected] orcid.org/0000-0001-8658-4614 Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic Search for more papers by this author Author Information Helena Draberova1,2,‡, Sarka Janusova2,‡, Daniela Knizkova1,2,‡, Tereza Semberova1,2, Michaela Pribikova1,2, Andrea Ujevic1,2, Karel Harant3, Sofija Knapkova4,5, Matous Hrdinka4,5, Viola Fanfani6, Giovanni Stracquadanio6, Ales Drobek2, Klara Ruppova2, Ondrej Stepanek *,2 and Peter Draber *,1,2 1Laboratory of Immunity & Cell Communication, BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic 2Laboratory of Adaptive Immunity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic 3Laboratory of Mass Spectrometry, BIOCEV, Faculty of Science, Charles University, Prague, Czech Republic 4Department of Haematooncology, University Hospital Ostrava, Ostrava, Czech Republic 5Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic 6Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +42 0241 062155; E-mail: [email protected] *Corresponding author. Tel: +42 0735 208125; E-mail: [email protected] The EMBO Journal (2020)39:e104202https://doi.org/10.15252/embj.2019104202 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract IL-17 mediates immune protection from fungi and bacteria, as well as it promotes autoimmune pathologies. However, the regulation of the signal transduction from the IL-17 receptor (IL-17R) remained elusive. We developed a novel mass spectrometry-based approach to identify components of the IL-17R complex followed by analysis of their roles using reverse genetics. Besides the identification of linear ubiquitin chain assembly complex (LUBAC) as an important signal transducing component of IL-17R, we established that IL-17 signaling is regulated by a robust negative feedback loop mediated by TBK1 and IKKε. These kinases terminate IL-17 signaling by phosphorylating the adaptor ACT1 leading to the release of the essential ubiquitin ligase TRAF6 from the complex. NEMO recruits both kinases to the IL-17R complex, documenting that NEMO has an unprecedented negative function in IL-17 signaling, distinct from its role in NF-κB activation. Our study provides a comprehensive view of the molecular events of the IL-17 signal transduction and its regulation. Synopsis We resolved the hierarchy of IL-17 receptor signalosome formation using a novel mass-spectrometry approach. We identified that LUBAC functions as a new positive regulator of IL-17 signaling, while NEMO-recruited kinases TBK1/IKKε provide very potent inhibitory feedback loop. Recruitment of approximately six Act1 molecules to a triggered IL-17 receptor creates a docking site for trimeric K63-polyubiquitin ligase TRAF6. TRAF6 promotes recruitment of linear ubiquitin chain assembly complex (LUBAC) and combined activity of these two ubiquitin ligases creates docking sites for activators of downstream signaling. NEMO promotes recruitment of TBK1 and IKKε kinases via adaptors TANK and NAP1, independently of its role in NF-κB activation. TBK1 and IKKε phosphorylate ACT1 leading to the release of TRAF6 from the complex, explaining the relatively weak cellular response to IL-17 stimulation. Introduction The interleukin 17 (IL-17) is a major proinflammatory cytokine produced by Th17 cell lineage and several innate immune cell types (Harrington et al, 2005; Park et al, 2005; Cua & Tato, 2010). Studies of mouse models demonstrated that this cytokine is crucial for host defense against opportunistic fungal and bacterial species (Conti et al, 2009; Cho et al, 2010). In accord, patients impaired in IL-17 signaling suffer from chronic mucocutaneous candidiasis (Puel et al, 2011; Conti & Gaffen, 2015). On the other hand, aberrant signaling via IL-17 promotes pathogenesis of several autoimmune disorders, such as psoriasis, atopic dermatitis, rheumatoid arthritis, or multiple sclerosis (Brembilla et al, 2018) and therapeutic antibodies blocking IL-17 or its receptor have been successfully used in clinic to treat severe plaque psoriasis (Bilal et al, 2018; Hawkes et al, 2018). Altogether, IL-17 production and signal transduction must be subjected to a tight control to allow proper immune system response when required, yet preventing autoinflammatory diseases. Interleukin-17 receptor (IL-17R) is composed of two widely expressed subunits IL-17RA and IL-17RC (Toy et al, 2006; Hu et al, 2010). Binding of dimeric IL-17 leads to heterodimerization of the receptor (Ely et al, 2009; Liu et al, 2013; Goepfert et al, 2017) and recruitment of a cytoplasmic protein ACT1 (Chang et al, 2006; Qian et al, 2007). ACT1 was described to enhance the expression of genes encoding proinflammatory cytokines by stabilizing their mRNAs or by activating downstream signaling pathways leading to the activation of their transcription (Li et al, 2019). The gene-activation pathways are dependent on the recruitment of E3 ubiquitin ligases from tumor necrosis factor (TNF) receptor-associated factors (TRAFs) family, most prominently TRAF6. TRAF6 creates non-degradative K63-polyubiquitin linkages which serve as docking sites for a variety of signaling molecules and promote activation of downstream signaling pathways, especially mitogen-activated protein kinase (MAPK) or nuclear factor-κB (NF-κB) and subsequent production of proinflammatory cytokines (Schwandner et al, 2000; Sonder et al, 2011). However, the mechanisms promoting and regulating IL-17 signaling emanating directly from IL-17 receptor are incompletely understood. The IL-17-induced activation of downstream pathways is surprisingly weak in comparison with other proinflammatory cytokines such as IL-1α or TNF, although their receptors all employ the formation of non-degradative polyubiquitin linkages and share multiple proximal signaling proteins (Kupka et al, 2016; Strickson et al, 2017; Li et al, 2019; McGeachy et al, 2019). The molecular basis for these differences is poorly defined. In addition to directly inducing activation of signaling pathways, IL-17 can trigger stabilization of mRNA transcripts via ACT1 and TRAF2/5, which regulate mRNA stability either directly, or by modulating the activity of mRNA binding proteins ARID5A and HuR, splicing factor SF2, and endoribonuclease Regnase-1 (Sun et al, 2011; Herjan et al, 2013, 2018; Somma et al, 2015; Amatya et al, 2018). In this study, we established a novel methodical approach to analyze the assembly of the IL-17 receptor signaling complex (IL-17RSC) via mass spectrometry (MS), which revealed the composition of the complex and its stoichiometry, including a novel signaling mediator, linear ubiquitin chain assembly complex (LUBAC). Importantly, we uncovered a robust negative inhibitory loop mediated by NEMO-recruited kinases TBK1 and IKKε that is specific for the IL-17 pathway, explaining the enigmatic mechanism of a weak signaling response of cells to IL-17 stimulation and showing a unique regulatory role of NEMO in the assembly of IL-17RSC. Results Kinases TBK1 and IKKε are strongly and preferentially activated upon IL-17 stimulation We aimed to resolve the composition of the IL-17 receptor signaling complex formed upon the binding of IL-17 to its receptors. For that purpose, we deployed a strategy for receptor-complex analysis in which cells were stimulated with a recombinant dimeric IL-17 (Fig EV1A–C), followed by the pull-down of the whole signaling complex via the ligand's tandem affinity purification tag (2xStrep-tag and 1xFlag-tag) and MS analysis (Fig 1A). This approach offers the possibility to isolate only ligand-engaged receptors forming membrane-proximal signaling complexes via highly specific tandem affinity purification without the requirement for exogenous expression of tagged proteins in target cells. As a control, the ligand was added after the cell lysis, which did not induce assembly of the signaling complex. The IL-17 stimulation might lead to post-translational modifications of potential contaminants that would change their binding to the beads used for immunoprecipitation. In order to ensure that the identified proteins are bona fide components of the IL-17RSC, we decided for relatively strict definition of background contaminants (as described in Table EV1). Click here to expand this figure. Figure EV1. Isolation of IL-17RSC reveals that TBK1 and IKKε kinases are strongly activated upon IL-17 stimulation The schematic representation of recombinant Strep-Flag-IL-17 (SF-IL-17) construct used in this study. Murine or human IL-17 coding sequence lacking leader peptide was used. The purity and assembly of SF-IL-17 was analyzed by SDS-PAGE followed by Coomassie staining. Samples were either left untreated or reduced with dithiothreitol (DTT) to disrupt the covalent IL-17 dimers. ST2 cells were incubated on ice in the presence or absence of SF-IL-17. Subsequently, the cells were stained with fluorescently labeled anti-Flag antibody and analyzed by FACS. ST2 cells were stimulated with SF-IL-17 (500 ng/ml) for indicated time points or were left unstimulated and IL-17 was added post-lysis. Lysates were subjected to anti-Flag immunoprecitation to isolate IL-17RSC. The samples were analyzed by immunoblotting. HeLa cells were stimulated with IL-17 (500 ng/ml) or TNF (500 ng/ml) for indicated time points and analyzed via immunoblotting. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Kinases TBK1 and IKKε are major components of IL-17RSC Schematic representation of IL-17RSC isolation and analysis. Cells are stimulated with recombinant Strep-Flag-IL-17 (SF-IL-17) (1), which leads to the crosslinking of its two receptor subunits and recruitment of cytoplasmic molecules (2). The whole complex is isolated upon cell lysis via tandem affinity purification of the ligand and analyzed by MS (3). ST2 cells were stimulated for 15 min with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post-lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in five independent experiments are shown. The stoichiometry of IL-17RSC calculated as the ratio between iBAQ intensities of individual IL-17RSC components to iBAQ intensity of IL-17RC. The recruitment of kinases TBK1 and IKKε as compared to related kinases IKKα and IKKβ is significantly enhanced. Mean from five performed MS experiments is shown, the statistical significance was determined using unpaired two-tailed Student's t-test. ST2 cells were stimulated with IL-17 (500 ng/ml), TNF (50 ng/ml), or IL-1α (50 ng/ml) for indicated time points and activation of signaling pathways was analyzed by immunoblotting. A representative of two independent experiments is shown. Source data are available online for this figure. Source Data for Figure 1 [embj2019104202-sup-0008-SDataFig1.rar] Download figure Download PowerPoint The comparison between control and stimulated samples revealed a very specific and highly reproducible set of proteins recruited to IL-17RSC. In contrast to IL-17RA, we detected IL-17RC only in stimulated but not control samples, which reflects that murine IL-17RC binds IL-17 only when it is associated with IL-17RA (Kuestner et al, 2007). Importantly, we identified a number of previously known components of IL-17RSC: core protein ACT1, non-degradative ubiquitin ligases TRAF6 and TRAF2, deubiquitinase A20 and associated adaptors ABIN1 and TAX1BP1, a kinase complex NEMO/IKKα/IKKβ, and homologous kinases TBK1 and IKKε (Amatya et al, 2017). We also identified components of a degradative ubiquitin ligase complex consisting of βTrCP1/2 and Cullin1, which were previously reported to degrade ACT1 upon prolonged stimulation (Shi et al, 2011), although it was not known they are recruited directly to the IL-17RSC. In addition, we identified proteins TANK and NAP1 that have not yet been connected to the IL-17R pathway (Fig 1B and Table EV1). These two adaptors were reported to associate with TBK1 and IKKε (Chau et al, 2008; Helgason et al, 2013) and recruit them to the TNFR1 signaling complex (TNF-RSC) (Lafont et al, 2018). We subsequently calculated the stoichiometry between individual components of the complex using intensity-based absolute quantification (iBAQ) (Schwanhausser et al, 2011). Murine IL-17 binds first strongly to IL-17RA and only subsequently can interact with IL-17RC to form the complex in 1:2:1 stoichiometry between IL-17RA:IL-17:IL-17RC (Ely et al, 2009; Liu et al, 2013; Goepfert et al, 2017). As IL-17RC does not bind directly to IL-17 in the post-lysis control samples, we normalized the iBAQ values of individual proteins to IL-17RC (Fig 1B). Surprisingly, TBK1 and IKKε were among the most abundant components of the complex, largely exceeding the related kinases IKKα and IKKβ that are crucial for NF-κB activation (Fig 1C and Table EV1). We confirmed that both TBK1 and IKKε were recruited and phosphorylated on their activation Ser172 residue (Kishore et al, 2002; Ma et al, 2012) within the IL-17RSC (Fig EV1D). High abundance of TBK1 and IKKε in the IL-17RSC suggested that their activation might be a major signaling event triggered by IL-17 stimulation. Indeed, IL-17 strongly activated TBK1 and IKKε at a comparable or even higher level as the stimulation with strong proinflammatory stimuli TNF or IL-1α (Fig 1D). In a sharp contrast, NF-κB and MAPK signaling pathways were only weakly triggered by IL-17. The same results were obtained in human cell line HeLa (Fig EV1E). Altogether, these data established that IL-17 shows a unique preference for strong activation of TBK1 and IKKε kinases over other signaling events. Kinases TBK1 and IKKε negatively regulate IL-17 signaling Although our data showed that the activation of TBK1 and IKKε is likely the most prominent signaling event upon IL-17 stimulation, the role of TBK1 and IKKε kinases in IL-17 signaling is highly controversial. Ablation of IKKε or TBK1 alone was described to weakly inhibit MAPK signaling and IL-17-mediated stabilization of mRNA (Bulek et al, 2011; Herjan et al, 2018). In accord, stimulation of TBK1 and IKKε DKO cells with IL-17 in the presence of TNF led to markedly decreased transcriptional response, indicating that both kinases are positive regulators of IL-17 signaling responses (Tanaka et al, 2019). In striking contrast, overexpression of either TBK1 or IKKε in cells deficient for both these kinases led to inhibition of IL-17-induced downstream signaling (Qu et al, 2012), indicating that they are in fact negative regulators of IL-17 signaling. In order to resolve the controversial issue concerning the role of these kinases in shaping IL-17 responses, we employed RNA sequencing to analyze the transcription response following IL-17 stimulation in the presence or absence of MRT67307, a highly specific inhibitor of both TBK1 and IKKε (Clark et al, 2011a) (Table EV2 and Fig EV2A). The comparison of unstimulated with IL-17 stimulated cells showed upregulation of 65 genes, most of them being established targets of the IL-17 signaling pathway (Fig 2A). Treatment of cells with MRT67307 alone induced subtle alternations of the transcriptome that were largely non-overlapping with the effects of IL-17 treatment (Fig EV2B and C). However, IL-17-induced pronounced changes in the transcriptional response of cells pretreated with MRT67307 (Fig 2B). Specifically, the inhibition of TBK1 and IKKε augmented the upregulation of almost all IL-17 responsive genes (Fig 2C ans D). Moreover, multiple IL-17 responsive genes (such as Tnf or Cxcl2) reached the significant level of upregulation only when the IL-17 stimulation was performed in the presence of MRT67307 (Fig 2E). Real-time PCR analysis confirmed that inhibition of TBK1 and IKKε markedly enhanced the IL-17-mediated upregulation of selected target genes after 2, 4, and 8 h of stimulation (Fig 2F and EV2D). Altogether, these data demonstrated that TBK1 and IKKε kinase activities lead to the general inhibition of IL-17 transcriptional responses. Click here to expand this figure. Figure EV2. TBK1 and IKKε function as major inhibitors of IL-17-induced signaling and transcriptional response ST2 cells were incubated with or without TBK1/IKKε inhibitor MRT67307 (2 μM) for 30 min and subsequently were left untreated or stimulated for 2 h with IL-17 (500 ng/ml). mRNA was isolated and subjected to RNA sequencing. The principal component analysis from three independent experiments is shown. Analysis of transcriptional response induced by treatment of ST2 cells with TBK1/IKKε inhibitor only. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < −1, −log10 Benjamini–Hochberg adjusted P-value > 2, based on analysis of three independent experiments). Names of several significantly upregulated transcripts are indicated. The Venn diagram representing the number of significantly changed transcripts upon treatment of cells with TBK1/IKKε inhibitor alone or with IL-17 alone. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 μM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 4 or 8 h, and induction of mRNA for selected genes was analyzed by real-time PCR. Mean + SEM from four independent experiments is shown, and statistical significance was determined using unpaired two-tailed Student's t-test. ST2 wild types or TBK1/IKKε DKO cells were stimulated with IL-17 (500 ng/ml) or TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting. ST2 wild types or TBK1/IKKε DKO cells were stimulated with indicated concentration of IL-17 for 15 min and lysates were analyzed by immunoblotting. HeLa cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 μM) were stimulated with IL17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting. HeLla wild types or TBK1/IKKε DKO cells were stimulated with indicated concentration of IL-17 for 15 min and lysates were analyzed by immunoblotting. Data information: Immunoblot results are representative of two (G, H) or three (F) independent experiments. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. TBK1 and IKKε function in redundant manner as inhibitors of IL-17-induced signaling responses A, B. ST2 cells were either left untreated (A) or pretreated with TBK1/IKKε inhibitor MRT67307 (2 μM) for 30 min (B), followed by stimulation with IL-17 (500 ng/ml) for 2 h. Transcription response induced by IL-17 stimulation as compared to unstimulated cells was analyzed by RNA sequencing. In red are transcripts considered to be significantly changed (log2 fold change > 1 or < −1, −log10 Benjamini–Hochberg adjusted P-value > 2, based on the analysis of three independent experiments; tringle is used for transcripts with −log10 adjusted P-value > 100). Names of several significantly upregulated transcripts are indicated. C. The Venn diagram representing the number of significantly changed transcripts upon IL-17 stimulation detected in (A) and (B). D, E. Comparison of IL-17-induced transcriptional response in the presence or absence of TBK1/IKKε inhibitor. (D) Transcripts that are significantly changed in both conditions. (E) Transcripts that pass significance threshold for induction only in the presence of TBK1/IKKε inhibitor. Dashed lines indicate significantly upregulated transcripts (log2 fold change > 1); red lines separate transcripts that are more upregulated upon IL-17 stimulation in the presence of TBK1/IKKε inhibitor as compared to IL-17 alone. F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 μM) were left unstimulated or stimulated with IL-17 (500 ng/ml) for 2 h and induction of mRNA for selected genes was analyzed by real-time PCR. Mean + SEM from five independent experiments is shown, and statistical significance was determined using unpaired two-tailed Student's t-test. G. ST2 cells were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 μM), stimulated with IL17 (500 ng/ml) for indicated time points and analyzed by immunoblotting. H. ST2 wild-type, TBK1 KO, IKKε KO, or cells lacking both kinases (DKO) were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting. I. TBK1/IKKε DKO cells reconstituted with either wild-type or kinase dead mutant (K38A) version of both kinases were stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting. Data information: Immunoblot results are representative of four (G) or three (H, I) independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embj2019104202-sup-0009-SDataFig2.rar] Download figure Download PowerPoint In the next step, we probed the role of TBK1 and IKKε in the IL-17-triggered proximal signaling pathways. The inhibition of TBK1 and IKKε activity dramatically enhanced the activation of NF-κB and MAPKs (Fig 2G). Subsequently, we prepared cells deficient in TBK1, IKKε, or both using CRISPR/Cas9 approach. Ablation of either kinase alone led to weak suppression of responses to IL-17 stimulation. In contrast, deficiency in both kinases led to strikingly enhanced activation of major signaling pathways, demonstrating absolute functional redundancy betwe
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