FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

(A) His-3xFLAG-FAT10 (FLAG-FAT10), HA-tagged MK3, HA-MK3 TT/EE (constitutively active mutant), or HA-MK3 TT/AA (inactive mutant), were transiently overexpressed in HEK293 cells for 24 h, followed by lysis and immunoprecipitation (IP) with a monoclonal phosphoserine-reactive antibody. Subsequently, an immunoblot (IB) was performed using the antibodies indicated. Where indicated, cells were stimulated with TNF/IFNγ for 24 h before harvesting and lysis. Moreover, cells were starved for 24 h (0.3% FCS DMEM) followed by TPA treatment (30 min) before lysis, where indicated. Calf intestinal alkaline phosphatase phosphatase was added to the lysates used in lanes 3 and 5, 2 h before performing the immunoprecipitation. (B) HEK293 cells were transiently transfected with an expression plasmid for FLAG-tagged FAT10 and where indicated, additionally treated with TNF/IFNγ for 24 h. Endogenous FAT10 expression was induced by treating HEK293 cells with TNF/IFNγ for 24 h. Where indicated, lysates were incubated with 400 U of λ phosphatase for 30 min at 30°C, before the immunoprecipitation was performed. Subsequently, an immunoprecipitation against FAT10 was performed using a monoclonal FAT10-reactive antibody (clone 4F1, [Aichem et al, 2010]) coupled to protein A sepharose, followed by Phos-tag/SDS–PAGE and IB analysis with the antibodies indicated. γ-tubulin was used as loading control. (C) Cells were prepared as in (A) and treated as specified, followed by FLAG-IP, Phos-tag/SDS–PAGE, and IB analysis with the indicated antibodies. One representative example out of three independent experiments with similar outcomes is shown.

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(A) HEK293 cells were transiently transfected with a His-3xFLAG-FAT10 (FLAG-FAT10) expression construct and stimulated for 24 h with TNF. Lysates were subjected to immunoprecipitation using FLAG-reactive antibodies, coupled to sepharose beads, and subsequently analyzed by Phos-tag/SDS–PAGE/IB analysis. Where indicated, cells were pretreated before TNF stimulation with the displayed inhibitors for a total of 3 h (10 µM each). (B) HEK293 cells were transiently transfected with expression plasmids for the different kinases. Cells were harvested, lysed, and subjected to immunoprecipitation using anti-FLAG or anti-HA antibodies, coupled to sepharose beads. Subsequently, the immunoprecipitated kinases were incubated with recombinant FAT10 (rFAT10) and an in vitro reaction was performed in the kinase buffer. The phosphorylation status of FAT10 was analyzed by Phos-tag/SDS–PAGE and IB. Asterisks mark unspecific background bands. (C) FLAG-FAT10 and the indicated kinases were transiently overexpressed in HEK293 cells followed by TNF stimulation. After 24 h, cells were lysed and subjected to immunoprecipitation against the FLAG-tag, combined with Phos-tag/SDS–PAGE and IB analysis. (D) Recombinant FAT10 (rFAT10) was incubated with recombinant kinases IKKβ, IKKε or MK3 for 45 min at 30°C. Subsequently, proteins were separated on a Phos-tag/SDS–PAGE followed by immunoblot analysis using the antibodies indicated. (E) HEK293 cells were prepared as described in (A). Where specified, cells were pretreated with the inhibitors indicated (10 µM each) for a total of 3 h before stimulation with TNF. One representative example out of three independent experiments with the same outcomes is shown.

Source data are available for this figure.

(A) FAT10 mRNA levels were analyzed and quantified by real-time PCR in A549 cells 24 h after TNF/IFNγ treatment or 24 h after IAV infection. (B) A549 cells were infected with IAV (MOI:1) for 0, 6, 24 or 48 h, as indicated. Cell lysates were subjected to SDS–PAGE combined with immunoblot analysis with the antibodies indicated. (C) Immunoblot showing endogenous FAT10 expression in A549 cells treated for 24 h with TNF/IFNγ, or infected for 24 h with IAV. FAT10 was immunoprecipitated with a monoclonal FAT10-reactive antibody (clone 4F1) and an immunoblot was performed with the antibodies indicated. (D) A549 cells stably expressing His-3xFLAG-FAT10 (FLAG-FAT10) were lysed after 24 h of TNF stimulation or after 24 h of IAV infection. The lysates were subjected to immunoprecipitation using FLAG-reactive antibodies coupled to sepharose beads. Subsequently, a Phos-tag/SDS–PAGE and immunoblot analysis with the indicated antibodies was performed. Asterisk marks an unspecific background band. (E) Recombinant 6His-SUMO-FAT10 was purified via Ni-NTA and left bound to the beads. 400 units λ phosphatase was added and incubated twice for 30 min at 30°C. Beads were extensively washed and FAT10 was eluted by treatment with the ULP1 enzyme to receive untagged and non-phosphorylated FAT10. Lysates from untreated or IAV-infected A549 cells were prepared and incubated with the purified FAT10, as indicated, for 30 min at 30°C. Proteins were separated on a Phos-tag/SDS–PAGE and analyzed by immunoblotting with a FAT10-reactive, polyclonal antibody. (F) A549 cells stably expressing FLAG-FAT10 were infected with IAV for 24 h. Cell lysates were subjected to a FLAG-immunoprecipitation and analyzed by Phos-tag/SDS–PAGE, followed by immunoblotting using the antibodies indicated. Where indicated, cells were pretreated with the indicated inhibitors (5 mM IKKβ inhibitor, 10 μM IKKε inhibitor) for 3 h, before IAV infection. For each panel, one representative example out of three independent experiments with similar outcomes is shown.

Source data are available for this figure.

(A) A549 cells stably expressing WT, phosphorylation-deficient (A) or phospho-mimetic (E) versions of FLAG-FAT10 were stimulated with TNF, as indicated. 1 d later, cells lysates were prepared and subjected to a FLAG-immunoprecipitation and a Phos-tag/SDS–PAGE and immunoblot analysis with the indicated antibodies. (B) Quantification of the ECL signals from three independent experiments as shown in (A). Values were normalized to the respective FLAG-FAT10 (WT, -A, or -E) expression in the lysate (load). The value of WT FLAG-FAT10–expressing cells was set to 100% and all other values were calculated accordingly. (C) HEK293 cells were transiently transfected with constructs expressing the indicated forms of FLAG-FAT10. 1 d later, cells were lysed followed by FLAG-immunoprecipitation, SDS–PAGE, and immunoblot analysis with the indicated antibodies. (D) HEK293 cells were transiently transfected with constructs expressing the indicated forms of FLAG-FAT10 followed by a cycloheximide (CHX) chase over 5 h. Where indicated, cells were additionally treated for 6 h with MG132 (10 µM). The immunoblot was performed with the indicated antibodies. GAPDH was used as loading control. (E) Quantification of the ECL signals from three independent experiments as shown in (D). Levels were normalized to the respective levels of the housekeeping gene GAPDH. Values at 0 h were set to unity and the other values were calculated accordingly. (F) A549 cells were transiently transfected with constructs expressing the specified forms of FLAG-FAT10 (green) and the cellular localization of FAT10 was assessed by confocal microscopy. Nuclei were stained with DAPI. Scale bars represent 20 μm. One representative example out of three independent experiments is shown.

Source data are available for this figure.

(A) A549 WT and stably transfected A549 FLAG-FAT10, A549 FLAG-FAT10 A, and A549 FLAG-FAT10 E cell lines were infected with influenza A virus (MOI:1). After 24 h, the supernatants were collected and an IFN-β ELISA was performed. (B) The experiment was performed as in (A) and the supernatants were collected 0, 6, 24, and 48 h after influenza A virus infection. (C) A549 cells were transiently transfected twice (at 0 and 24 h) with the indicated FLAG-FAT10 expression plasmids. After 48 h, cells were infected with vesicular stomatitis virus-GFP (MOI:0.1) for 1 h. After 24 h, the supernatants were collected and the concentration of IFN-β in the supernatant was measured by ELISA. (D) The cell pellets from (C) were lysed and protein expression was analyzed by immunoblotting using the antibodies indicated. Data in (A, B, C) are mean ± SEM (n = 3); P < 0.05 (t test); ns: not significant; Asterisks in (D) mark unspecific background signals.

Source data are available for this figure.

(A) A549 WT and stably transduced A549 FLAG-FAT10, A549 FLAG-FAT10 A and A549 FLAG-FAT10 E expressing cells were lysed 24 h after Poly (I:C) transfection. The lysates were subjected to SDS–PAGE and immunoblot analysis with the antibodies indicated. (B) Quantification of the RIG-I expression levels calculated from the immunoblot shown in (A), normalized to GAPDH. (C) HEK293 cells were transiently transfected with expression plasmids for HA-OTUB1, FLAG-FAT10 WT, -A, and -E variants, as indicated. After 24 h, cells were harvested and lysed. Subsequently, an immunoprecipitation against the FLAG tag was performed, followed by SDS–PAGE and immunoblot analysis with the antibodies indicated. γ-tubulin served as loading control. (D) HEK293 cells were transiently transfected with expression plasmids for HA-ubiquitin, Y2-OTUB1, and the different FLAG-FAT10 variants, as indicated. After 24 h, cells were harvested and lysed. Subsequently, endogenous TRAF3 was immunoprecipitated with a monoclonal TRAF3-reactive antibody followed by SDS–PAGE and immunoblot analysis with the indicated antibodies. γ-tubulin served as loading control. (E) Non-transfected A549 WT and A549 OTUB1 KO cells (WT) (black bars), and transiently with FLAG-FAT10 wt (light blue) or FLAG-FAT10 E (dark blue) expression plasmids transduced A549 and A549 OTUB1 KO cells were additionally transfected with Poly (I:C) for 24 h. The supernatant was subjected to IFN-β ELISA. Levels were normalized to the levels of the respective non-transfected sample. Data are mean ± SEM (n = 3). (F) A549 WT and OTUB1 KO cells were left untreated (black bars) or were transiently transduced with expression plasmids for FLAG-FAT10 WT (light blue) or the FLAG-FAT10 E mutant (dark blue). After 48 h, cells were infected with Influenza A virus (MOI:1) for 1 h. After 24 h, the supernatants were collected and the concentration of IFN-β in the supernatants was measured by ELISA. (G) Endogenous FAT10 expression was induced by treating A549 WT, FAT10 KO, OTUB1 KO, and FAT10 KO/OTUB1 KO cells with TNF/IFNγ for 24 h. Then, cells were transfected with Poly (I:C) and stimulated at the same time with TNF/IFNγ for the next 24 h. The supernatants were collected and the concentration of IFN-β in the supernatants was measured by ELISA. Data are mean ± SEM (n = 3). (H) Endogenous FAT10 expression was induced by treating A549 WT, FAT10 knockout (KO), OTUB1 KO, and FAT10 KO/OTUB1 KO cells with TNF/IFNγ for 24 h. Subsequently, cells were infected with influenza A virus (MOI:1) for 1 h. Cells were again stimulated with TNF/IFNγ for the next 24 h after which the supernatant was collected and the concentration of IFN-β in the supernatant was measured by ELISA. Data are mean ± SEM (n = 5). P < 0.05 (t test), ns: not significant.

Source data are available for this figure.