Research ArticleCytokines

IFN-γ–inducible antiviral responses require ULK1-mediated activation of MLK3 and ERK5

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Science Signaling  20 Nov 2018:
Vol. 11, Issue 557, eaap9921
DOI: 10.1126/scisignal.aap9921
  • Fig. 1 Putative binding partners of ULK1 after engagement of the IFNGR.

    (A to C) MS/MS analysis of protein-ULK1 complexes from untreated (UT) or IFN-γ–treated KT-1 cells. Venn diagram (A) indicates the number of proteins that interact with endogenous ULK1 under untreated (blue), both (black), and after 10-min IFN-γ treatment (red) conditions. Heatmap analysis (B) is of the putative binding partners of ULK1 identified in untreated, IFN-γ–treated (IFN-γ), and both groups (see also table S1). Ontology analysis (C) is of putative ULK1 interactors identified only after IFN-γ stimulation (see also table S2). Note that red rectangles and asterisks highlight (B) IFN-γ signaling pathway and (C) viral process terms. Data are from one experiment. ncRNA, noncoding RNA; IP, immunoprecipitation.

  • Fig. 2 ULK1 interacts with and phosphorylates MLK3 during engagement of the IFNGR.

    (A and B) Coimmunoprecipitation analysis of ULK1 interaction with MLK3 in KT-1 (A) or U937 (B) cells left untreated or treated with IFN-γ for 10 min, as indicated. Blots are representative of three independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RIgG, rabbit immunoglobulin G. (C) ADP-Glo kinase assay analysis of adenosine diphosphate (ADP) concentration produced by in vitro kinase reaction of recombinant human ULK1 and heat-inactivated MLK3 alone or in combination. Data are means ± SEM of three independent experiments performed in triplicate. RLU, relative luminescence units. (D) Western blot analysis of pMLK3 in lysates from Ulk1/2+/+ and Ulk1/2−/− MEFs treated with IFN-γ for 10 or 30 min, as indicated. Blots (top) are representative of five independent experiments. Quantified data (bottom) are means ± SEM pooled from all experiments. *P < 0.05, ***P < 0.001, and not significant (NS) by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test (C) or two-way ANOVA using Bonferroni correction (D). a.u., arbitrary units.

  • Fig. 3 IFN-γ stimulates MLK3-mediated activation of ERK5, but not ERK1/2 and JNK.

    (A to C) Western blot analysis of pERK5 (A), pERK1/2 (B), and pJNK (C) in lysates from Mlk3+/+ and Mlk3−/− MEFs treated with IFN-γ for 10 or 30 min, as indicated. Blots (left) are representative of three (C) or four (A and B) independent experiments. Quantified data (right) are means ± SEM pooled from all experiments. *P < 0.05, ***P < 0.001, and not significant by two-way ANOVA using Bonferroni’s correction.

  • Fig. 4 ULK1/2 promotes IFN-γ–induced phosphorylation of ERK5 and p90RSK1.

    (A to D) Western blot analysis of pERK5 (A), pERK1/2 (B), pJNK (C), and p-p90RSK1 (D) in lysates from Ulk1/2+/+ and Ulk1/2−/− MEFs treated with IFN-γ for 10 or 30 min, as indicated. Blots (left) are representative of three independent experiments. Quantified data (right) are means ± SEM pooled from all experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and not significant by two-way ANOVA using Bonferroni’s correction.

  • Fig. 5 Ulk1/2 augments IFN-γ–mediated transcription of ISGs.

    (A to E) RNA-seq analysis of transcript expression in Ulk1/2+/+ and Ulk1/2−/− MEFs untreated or treated with mouse IFN-γ for 6 hours, as indicated. MDS plot of all groups (A) and Volcano plot of differentially expressed (DE) genes after IFN-γ treatment in Ulk1/2+/+ versus Ulk1/2−/− MEFs (B). Venn diagram (C) indicates the gene expression overlap existing between differentially expressed genes after IFN-γ treatment in Ulk1/2+/+ (green ellipse) and Ulk1/2−/− (blue ellipse) MEFs. Heatmap analysis (D) of antiviral genes induced in Ulk1/2+/+ MEFs compared to Ulk1/2−/− MEFs by IFN-γ treatment. Box plot (E) of log2 fold change (FC) distribution of IFN-γ–inducible antiviral genes in Ulk1/2+/+ and Ulk1/2−/− MEFs. Data are from four biological replicates per group (see also figs. S2 to S6 and tables S3 to S8). Statistical analyses were performed using Wilcoxon unpaired rank sum test.

  • Fig. 6 ULK1/2 is required for IFN-γ–dependent transcriptional induction of antiviral ISGs.

    (A) Luciferase reporter assay analysis of GAS promoter activity in Ulk1/2+/+ and Ulk1/2−/− cells untreated or treated with IFN-γ for 6 hours. Data are means ± SEM from four independent experiments. (B to D) qRT-PCR analysis of Cxcl10 (B), Oasl2 (C), and Ifit3 (D) mRNA expression in Ulk1/2+/+ and Ulk1/2−/−MEFs untreated or treated with IFN-γ for 6 hours. Data are means ± SEM from three (B) or four (C and D) independent experiments. (E) Western blot analysis of ULK1 in lysate from KT-1 ULK1 KO cells generated by CRISPR-Cas9 genome editing. Blots are representative of three independent experiments. (F to I) qRT-PCR analysis of CXCL10 (F), OAS1 (G), IFIT3 (H), and IRF1 (I) mRNA expression in KT-1 ULK1 WT and KT-1 ULK1 KO cells that were incubated in the presence or absence of IFN-γ for 6 hours. Data are means ± SEM from three (F) or four (G to I) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t test (two-tailed) with Welch’s correction.

  • Fig. 7 Requirement of ULK1/2 for the generation of IFN-γ–dependent antiviral effects.

    (A) Crystal violet viability analysis of EMCV-induced cytopathic effects (CPEs) in Ulk1/2+/+ and Ulk1/2−/− MEFs pretreated with IFN-γ at the indicated doses for 16 hours and subsequently challenged with EMCV for 24 hours. Data are means ± SEM of quadruplicate assays from three independent experiments. (B) Crystal violet viability analysis of EMCV-induced CPEs in human fibrosarcoma 2fTGH cells pretreated for 2 hours with the ULK1 kinase inhibitor MRT68921 (MRT), as indicated, and then exposed to IFN-γ for 16 hours before challenge with EMCV for 24 hours. Data are means ± SEM of quadruplicate assays from three independent experiments. (C) qRT-PCR analysis of Cxcl10 (left) and Ifit3 (right) mRNA expression in Ulk1/2+/+ MEFs were treated with dimethyl sulfoxide (DMSO) (vehicle-control, C), XMD8-92 (XMD), and/or IFN-γ, as indicated. Data are means ± SEM from four independent experiments. (D) Crystal violet viability analysis of EMCV-induced CPEs in human fibrosarcoma 2fTGH cells pretreated for 2 hours with the ERK5 inhibitor XMD8-92, as indicated, and then exposed to IFN-γ for 16 hours before challenge with EMCV for 24 hours. Data are means ± SEM of quadruplicate assays from three independent experiments. (E) Schematic illustration of the potential role of ULK1/2 in IFN-γ signaling. *P < 0.05, **P < 0.01, and ****P < 0.0001 by one-way ANOVA analysis, followed by Tukey’s multiple comparisons test (C) or two-way ANOVA with post hoc t tests (A, B, and D).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/557/eaap9921/DC1

    Fig. S1. ULK1/2 activity is not required for IFN-γ–induced phosphorylation of STAT1.

    Fig. S2. Effects of targeted disruption of Ulk1/2 gene expression on IFN-γ–dependent gene transcription.

    Fig. S3. Genes differentially expressed in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.

    Fig. S4. Genes differentially expressed only in Ulk1/2+/+ MEFs after IFN-γ treatment.

    Fig. S5. Genes differentially expressed only in Ulk1/2−/− MEFs after IFN-γ treatment.

    Fig. S6. GO analysis of IFN-γ–dependent differentially expressed genes.

    Fig. S7. Inhibition of autophagy does not affect transcription of antiviral ISGs.

    Fig. S8. Genomic location for the ULK1 sgRNA in the human ULK1 open reading frame.

    Table S1. GO analysis of putative ULK1 interactors in untreated versus IFN-γ–treated KT-1 cells identified by mass spectrometry analysis.

    Table S2. GO analysis of putative ULK1 interactors identified after IFN-γ stimulation by mass spectrometry analysis.

    Table S3. Genes differentially expressed in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.

    Table S4. Genes differentially expressed only in Ulk1/2+/+ MEFs after IFN-γ treatment.

    Table S5. Genes differentially expressed only in Ulk1/2−/− MEFs after IFN-γ treatment.

    Table S6. GO analysis of differentially expressed genes identified in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.

    Table S7. GO analysis of differentially expressed genes identified only in Ulk1/2+/+ MEFs after IFN-γ treatment.

    Table S8. GO analysis of differentially expressed genes identified only in Ulk1/2−/− MEFs after IFN-γ treatment.

  • The PDF file includes:

    • Fig. S1. ULK1/2 activity is not required for IFN-γ–induced phosphorylation of STAT1.
    • Fig. S2. Effects of targeted disruption of Ulk1/2 gene expression on IFN-γ–dependent gene transcription.
    • Fig. S3. Genes differentially expressed in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.
    • Fig. S4. Genes differentially expressed only in Ulk1/2+/+ MEFs after IFN-γ treatment.
    • Fig. S5. Genes differentially expressed only in Ulk1/2−/− MEFs after IFN-γ treatment.
    • Fig. S6. GO analysis of IFN-γ–dependent differentially expressed genes.
    • Fig. S7. Inhibition of autophagy does not affect transcription of antiviral ISGs.
    • Fig. S8. Genomic location for the ULK1 sgRNA in the human ULK1 open reading frame.
    • Legends for Tables S1 to S8

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). GO analysis of putative ULK1 interactors in untreated versus IFN-γ–treated KT-1 cells identified by mass spectrometry analysis.
    • Table S2 (Microsoft Excel format). GO analysis of putative ULK1 interactors identified after IFN-γ stimulation by mass spectrometry analysis.
    • Table S3 (Microsoft Excel format). Genes differentially expressed in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.
    • Table S4 (Microsoft Excel format). Genes differentially expressed only in Ulk1/2+/+ MEFs after IFN-γ treatment.
    • Table S5 (Microsoft Excel format). Genes differentially expressed only in Ulk1/2−/− MEFs after IFN-γ treatment.
    • Table S6 (Microsoft Excel format). GO analysis of differentially expressed genes identified in both Ulk1/2+/+ and Ulk1/2−/− MEFs after IFN-γ treatment.
    • Table S7 (Microsoft Excel format). GO analysis of differentially expressed genes identified only in Ulk1/2+/+ MEFs after IFN-γ treatment.
    • Table S8 (Microsoft Excel format). GO analysis of differentially expressed genes identified only in Ulk1/2−/− MEFs after IFN-γ treatment.

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