Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK–STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.
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We thank A. Garcia-Sastre (Icahn School of Medicine at Mount Sinai) for Stat2−/− MEFs, D. Cheresh (Moores UCSD Cancer Center) for MDA-MB-231, G. Stark (Cleveland Clinic) for sharing U-series cell lines, S. Fujita (Ehime University School of Medicine) for KT-1 cells, R. Xiang (The Scripps Research Institute) for WEHI-3B cells, S. Urbe (University of Liverpool) for GFP-fusion STAT2 and USP18 constructs, V. Verkhusha (Albert Einstein College of Medicine) for the mTag-BFP construct, T. Akagi (KAN Research Institute) for providing pCX4-series vectors, S. Kotenko (Rutgers New Jersey Medical School) for the pcDEF-hIFNAR2 DNA construct, D. Baker (Biogen Idec) for supplying recombinant human IFNβ and anti–human IFNAR1 antibody, the staff of Hybrigenics for their contribution, G. Hikade for technical support, and R. Kurre for advice on fluorescence microscopy. This study was supported by NIH R01HL091549 and R01CA177305 to D.-E.Z. and SFB 944 from the DFG to J.P. and J.J.H.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Cell micropatterning for probing the interaction of cytosolic effector proteins at the plasma membranes of living cells.
(a) Binary surface patterning by microcontact printing of poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) (structure in the center), which was either chemically coupled to RGD-peptide (I), (RGD peptide drawn in violet) or coupled to the HaloTag ligand (II), (HaloTag ligand (HTL) drawn in red) (left image). Concept of spatial organization of IFN receptor subunits in the plasma membrane of living cells cultured on the surface of a micropatterned coverslide. IFNAR2 (blue) fused to the HaloTag is captured into HTL-functionalized areas while cell attachment via focal adhesions is mediated by RGD-functionalized areas. Effector proteins such as STAT2 and USP18 are locally recruited to the receptor (right image). (b) HeLa cells transfected with HaloTag-mTagBFP-IFNAR2 (blue channel) and mEGFP (green channel) and TagRFP-T (orange channel) as a negative control. Scale bar: 10 μm. Representative images of 17 cells analyzed in two independent experiments. (c) Constitutive binding of STAT2 and USP18 to micropatterned IFNAR2. HeLa cells transfected with HaloTag-mTagBFP-IFNAR2 (blue channel), STAT2-TagRFP-T (orange channel) and mEGFP-USP18 (green channel). Scale bar: 10 μm. Representative images of 26 cells analyzed in two independent experiments. (d) Intensity profiles of STAT2-TagRFP-T, mEGFP-USP18, TagRFP-T, mEGFP and mTagBFP-IFNAR2 within the yellow ROI depicted in the images projected along the direction of the line pattern. (e) Dynamics of the STAT2 and USP18 interaction with micropatterned IFNAR2 in HeLa cells probed by FRAP and monoexponential fit of the recovery curves (representative of 5 cells analyzed). These time constants describe the exchange kinetics of prey proteins interacting with the immobilized bait and thus are a measure of complex stability, which can be considered rate-limiting because the high expression levels of unbound prey protein ensure fast association.
Stat2-/- MEFs or Stat2-expressing Stat2-/- MEFs were infected with vector control (-) or FLAG-Usp18 (+) retrovirus. These cells were treated with murine IFNβ (100 U/ml) for 0, 6 or 12 hours as indicated. Relative mRNA expression of the indicated IFN-inducible genes (a) Igtp, Ifit1, Gbp1, and Isg15 (b) Irf9, and Cxcl9 were examined by qRT-PCR analysis. Data are presented as mean ± S.D. of two independent experiments. Data are normalized to Gapdh was used as a reference gene.
Supplementary Figure 3 The N- and C-terminal regions of USP18 bind STAT2 and are important for inhibiting the IFN response.
(a) A schematic drawing of USP18 and its deletion mutants used in this study. The ability of a given deletion mutant to interact with STAT2 (+ or - binding) is indicated to the right.. (b) Both N-terminal and C-terminal regions of USP18 interact with STAT2. IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding STAT2-Myc and either FLAG-USP18 or the indicated deletion mutant. (c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding STAT2-FLAG and GFP or USP18 303-312 GFP as indicated. (d) MIP empty vector (-), MIP-FLAG-USP18 (USP18), or MIP-FLAG-USP18 303-312 deletion mutant (USP18∆303-312) expressing 2fTGH cells were treated with IFNα (1000 U/ml) for 15minutes. Cell lysates were analyzed by Western blotting with indicated antibodies. (e) MIP vector, USP18, or USP18 303-312 aa deletion mutant expressing KT-1 cells were treated with IFNα (1000 U/ml) for 30 minutes. Cell lysates were analyzed by Western blotting with indicated antibodies.
Supplementary Figure 4 The N- and C-terminal regions of USP18 bind IFNAR2 and are important for inhibiting the IFN response.
(a) A schematic drawing of USP18 and its deletion mutants used in this study. The ability of a given deletion mutant to interact with IFNAR2 (+ or - binding) is indicated to the right. (b) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GST-IFNAR2 ICD and mock, or FLAG-USP18 and its deletion mutants. (c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding IFNAR2-FLAG and GFP or USP18 36-51 GFP. (d) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GST-IFNAR2 ICD and mock, or FLAG-USP18 36-242aa, or FLAG-USP18 51-242aa. (e) 293T cells were co-transfected with plasmids encoding FLAG-STAT1 and FLAG-USP18 or its mutants as indicated. Twenty-four hours after transfection, cells were treated with IFNα (1000 U/ml) for 15minutes. Cell lysates were analyzed with indicated antibodies.
Supplementary Figure 5 Role of STAT2 in the recruitment of USP18 to IFNAR2, as probed by cell micropatterning.
(a) mEGFP-USP18 binding to micropatterned full-length HaloTag-IFNAR2 in HeLa cells. Cartoon of the assays (left) and total internal reflection fluorescence microscopy (TIRFM) image (right). The contrast within the highlighted area is shown below the TIRFM image. The red channel corresponds to fluorescent ATTO655IFNα2 for staining of IFNAR2. Representative image of 15-20 cells analyzed in two independent experiments. Scale bar: 10 μm. (b) mEGFP-USP18 (left) and JAK1-mEGFP (right) binding to micropatterned IFNAR2 truncated after residue 346, i.e. downstream of the JAK1 binding site. Representative images of 21 cells analyzed in two independent experiments. Scale bar: 10 μm. (c) USP18-mEGFP binding to micropatterned IFNAR2 truncated after residue 375. Representative images of 16 cells analyzed in two independent experiments. Scale bar: 10 μm. (d) Cell micropatterning assays with fragments of the intracellular IFNAR2 domain (aa 265-515) for probing binding of STAT2-TagRFP and mEGFP-USP18, respectively. The fragments used as bait are depicted at the top. (e) Contrast observed for STAT2 and USP18 binding to micropatterned IFNAR2 or IFNAR2 fragments fused to a transmembrane domain (TMD) as depicted in a and d, respectively. STAT2 and USP18 were either separately expressed (blue boxes) or co-expressed (orange and green boxes, respectively) in Hela cells. For statistical evaluation, 14-29 cells were analyzed in two independent experiments. n indicates number of cells used in each experiment. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P ˂ 0.001, n.s. not significant. (f) Representative TIRFM images of 29 cells analyzed in two independent experiments obtained for a fragment comprising the minimum constitutive STAT2 binding site of IFNAR2. Scale bar: 10 μm.
(a) FITC-labeled IFNα was titrated in order to determine the optimal staining concentration for cell-binding assays. Mean fluorescence intensity (MFI) of FITC-labeled IFNα, at the indicated concentrations, bound to the surface of 2fTGH cells was analyzed by flow cytometry. For all additional experiments FITC-labeled IFNα was used at a saturating concentration of 20 nM. (b) Relative amount of FITC-labeled IFNα bound to the surface of the indicated cell lines was examined by flow cytometry. Data are normalized to the mean fluorescence intensity of 2fTGH cells. Data are presented as mean ± S.E.M. for 3 independent experiments. ****P < 0.0001. (c) FITC-labeled IFNα retains an equivalent biological effect to unlabeled IFNα. 2fTGH cells were treated with 20 nM IFNα or FITC-labeled IFNα for the indicated time and cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (d) Interaction between USP18 and STAT2 WT or STAT2 Y690F. IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after transfection with STAT2-Myc or STAT2 Y690F-Myc and mock or FLAG-USP18.
(a) Identification of STAT2 mutations that disrupt STAT2-IRF9 interaction. IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after transfection with FLAG-IRF9 and STAT2 CC/DB Myc and its mutants as indicated. (b) Cells indicated in Figure 7B were treated with IFNα (1000 U/ml) for 12 hours and then expression of IFIT1 was analyzed by q-PCR. Data represents mean ± S.D. for 2 independent experiments. (c) MIP or MIP-STAT2 CC/DB 3A expressing THP-1 cells were treated with IFNβ (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for 3 independently generated stable cell lines. *P < 0.05. (d) THP-1 and KT-1 cells were treated with TAT or USP18 aa 302-313 TAT peptide. Two hours after peptide treatment, IFNα (1000 U/ml) was added for 12 hours and then expression of GBP1 was analyzed by q-PCR. Data represents mean ± S.D. for 2 independent experiments.
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Arimoto, Ki., Löchte, S., Stoner, S. et al. STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling. Nat Struct Mol Biol 24, 279–289 (2017). https://doi.org/10.1038/nsmb.3378
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