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STAM and Hrs interact sequentially with IFN-α Receptor to control spatiotemporal JAK–STAT endosomal activation

Nature Cell Biology volume 25, pages 425–438 (2023)Cite this article

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Abstract

Activation of the JAK–STAT pathway by type I interferons (IFNs) requires clathrin-dependent endocytosis of the IFN-α and -β receptor (IFNAR), indicating a role for endosomal sorting in this process. The molecular machinery that brings the selective activation of IFN-α/β-induced JAK–STAT signalling on endosomes remains unknown. Here we show that the constitutive association of STAM with IFNAR1 and TYK2 kinase at the plasma membrane prevents TYK2 activation by type I IFNs. IFN-α-stimulated IFNAR endocytosis delivers the STAM–IFNAR complex to early endosomes where it interacts with Hrs, thereby relieving TYK2 inhibition by STAM and triggering signalling of IFNAR at the endosome. In contrast, when stimulated by IFN-β, IFNAR signalling occurs independently of Hrs as IFNAR is sorted to a distinct endosomal subdomain. Our results identify the molecular machinery that controls the spatiotemporal activation of IFNAR by IFN-α and establish the central role of endosomal sorting in the differential regulation of JAK–STAT signalling by IFN-α and IFN-β.

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Fig. 1: IFNAR1 interaction with Hrs.
Fig. 2: Interaction of IFNAR1 with Hrs is required for IFN-α-induced JAK–STAT signalling.
Fig. 3: IFNAR1 and IFNAR2 degradations are independent of Hrs.
Fig. 4: The constitutive association of STAM with TYK2 prevents TYK2 activation.
Fig. 5: IFNAR1 constitutively interacts with STAM at the plasma membrane and at the early endosome.
Fig. 6: STAM2A interacts with IFNAR1 and IFNAR2 independently of Hrs.
Fig. 7: IFNAR endocytosis is dispensable for efficient JAK–STAT signalling in cells lacking STAM.
Fig. 8: IFNAR1 interaction with Hrs relieves STAM inhibition of TYK2.

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Data availability

All data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the core facilities and the CurieCoreTech recombinant antibodies platform of Institut Curie, and the staff of the Cell and Tissue Imaging (PICT-IBiSA) and the Nikon Imaging Centre at Institut Curie as well as the members of the French National Research Infrastructure France–BioImaging (ANR10-INBS-04) for their scientific and technical assistance. In particular, we thank F. Waharte and L. Sengmanivong for their help with the FLIM–FRET and fluorescence microscopy experiments. We thank the following people for providing materials or expertise: G. Raposo and P. Benaroch (Institut Curie, Paris), and P. Eid (INSERM, Paris). We thank the Morph-Im core facility (UNamur, Belgium) for Airyscan imaging. This work was supported by institutional grants from the Curie Institute, INSERM, CNRS, and by specific grants from Agence Nationale de la Recherche (ANR NANOSTAT-15-CE11-0025-01 to C.L.) and Marie Curie Actions—Networks for Initial Training (FP7-PEOPLE-2010-ITN to C.L. and D.C.). Funding was also provided by the Deutsche Forschungsgemeinschaft to J. Piehler (NANOSTAT, PI 405/10-1). Fast FLIM imaging was carried out at the NeurImag Imaging core facility, part of the IPNP, INSERM 1266 unit and Université de Paris. Support from Fondation Leducq is acknowledged. N.Z. was supported by a PhD fellowship from Ministère de l’Enseignement Supérieur et de la Recherche and Ligue Nationale contre le Cancer, D.C. by a PhD fellowship from Fondation pour la Recherche Médicale and C.M.B. by a postdoctoral fellowship from Ligue Nationale contre le Cancer. The Lamaze team are members of Labex CelTisPhyBio ANR-10-LBX-0038, part of the IDEX PSL ANR-10-IDEX-0001-02.

Author information

Author notes

  1. These authors contributed equally: Natacha Zanin, Christine Viaris de Lesegno.

Authors and Affiliations

  1. Membrane Mechanics and Dynamics of Intracellular Signaling Laboratory, Institut Curie—Centre de Recherche, PSL Research University, Paris, France

    Natacha Zanin, Christine Viaris de Lesegno, Joanna Podkalicka, Pamela Gonzalez Troncoso, Daniela Chmiest, Cédric M. Blouin & Christophe Lamaze

  2. Institut National de la Santé et de la Recherche Médicale (INSERM), Paris, France

    Natacha Zanin, Christine Viaris de Lesegno, Joanna Podkalicka, Pamela Gonzalez Troncoso, Daniela Chmiest, Cédric M. Blouin & Christophe Lamaze

  3. Centre National de la Recherche Scientifique (CNRS), Paris, France

    Natacha Zanin, Christine Viaris de Lesegno, Joanna Podkalicka, Pamela Gonzalez Troncoso, Daniela Chmiest, Cédric M. Blouin & Christophe Lamaze

  4. Namur Research Institute for Life Sciences (NARILIS), URBC, University of Namur, Namur, Belgium

    Natacha Zanin

  5. Laboratoire Physico–Chimie Curie, Institut Curie, PSL Research University, Sorbonne Université, Paris, France

    Joanna Podkalicka

  6. Laboratory of Cytobiochemistry, Faculty of Biotechnology, University of Wrocław, Wrocław, Poland

    Joanna Podkalicka

  7. Department of Biology and Center for Cellular Nanoanalytics, University of Osnabruck, Osnabruck, Germany

    Thomas Meyer & Jacob Piehler

  8. Membrane Traffic in Healthy and Diseased Brain, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Université de Paris, Paris, France

    Philippe Bun & Lydia Danglot

  9. NeurImag Imaging Facility, Institute of Psychiatry and Neuroscience of Paris (IPNP), INSERM U1266, Université de Paris, Paris, France

    Philippe Bun & Lydia Danglot

  10. Department of Biochemistry, CIIL Biomedical Research Center, University of Lausanne, Epalinges, Switzerland

    Daniela Chmiest

  11. Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK

    Sylvie Urbé

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  1. Natacha Zanin
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Contributions

N.Z., C.M.B., C.V.d.L., J. Podkalicka and T.M. designed the study, performed experiments and analysed results. N.Z., C.M.B., C.V.d.L., J. Piehler and C.L. wrote the manuscript. D.C., P.G.T., P.B., L.D. and S.U. assisted with experiments and provided several reagents. C.L. supervised and directed the research. All authors discussed the results and commented on the manuscript.

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Correspondence to Cédric M. Blouin or Christophe Lamaze.

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Nature Cell Biology thanks Paul Hertzog, Pier Paolo Di Fiore, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 IFN-α-dependent proximity of IFNAR1 and Hrs on endosomes.

a, Immunofluorescent labelling of Hrs, EEA1 and endocytosed IFNAR1 subunits after 10 min IFN-α (upper panels) or IFN-β (lower panels) stimulation in eGFP–Rab5 Q79L-transfected RPE1 cells observed by confocal microscopy with representative plots of co-localization profiles on endosomes. Quantification of Hrs and IFNAR1 co-localization is expressed as the Manders’ coefficient indicating the portion of IFNAR1 pixels containing Hrs pixels. Scale bar, 10 µm. Data represent the mean ± s.e.m., n = 8 cells examined from two independent experiments. Statistical significance was determined using a two-tailed Student’s t-test. ***P < 0,001; NS, not significant. b, Fast FLIM analysis of the IFNAR1–eGFP fluorescence lifetime in segmented endosomes of transfected RPE1 cells (with or without Hrs–mCherry expression) at steady state or following stimulation with IFN-α or IFN-β for 10 or 20 min (one dot is the mean lifetime of all endosomes from one cell); n = 28, 29, 29, 29, 29 and 29 cells, respectively, examined from four independent experiments. Data are the median ± 95% confidence interval. P values were calculated using a one-way ANOVA with the Kruskal–Wallis multiple comparison test. ***P < 0.001; NS, not significant. Source numerical data are provided.

Source data

Extended Data Fig. 2 Effect of Hrs depletion on JAK–STAT signalling and receptor degradation.

a, Nuclear translocation of pSTAT1 from wide-field microscopy imaging (left) and pSTAT1 nucleus/cytosol fluorescence ratio following stimulation with IFN-α, IFN-β or IFN-α2-YNS for 10 min in control RPE1 cells (shCtrl: α = 80, β = 142, α2-YNS = 90) or depleted for Hrs (shHrs: α = 90, β = 90, α2-YNS = 156; right). Data were pooled from two independent experiments. Scale bar, 50 µm. b, Immunoblots of tyrosine phosphorylation levels of IFNAR1 (pIFNAR1) in control (shCtrl) and shHrs RPE1 cells with or without IFN-α stimulation for 10 min, as indicated. c, Immunoblots and quantification of tyrosine phosphorylation levels of JAK1 (pJAK1) in shCtrl and shHrs HeLa S3-GFP cells (top) or S3-mHrs-GFP cells (bottom) with or without IFN-α or IFN-β stimulation for 10 min, as indicated. Control shCtrl + IFN-β was set at 100%. d, Immunofluorescent labelling (left) and quantification (right) of surface and total of IFNAR1 subunits in control or Hrs-depleted RPE1 cells analysed by confocal microscopy (the mean of the control condition was set to 100); n = 16 cells (shCtrl) and n = 28 cells (shHrs) examined from two independent experiments for surface staining and n = 28 cells (shCtrl) and n = 25 cells (shHrs) examined from three independent experiments for total staining. Scale bar, 10 µm. e,f, Immunoblot and quantification of IFNAR1 (e; three independent experiments) and EGFR (f; two independent experiments) degradation in RPE1 cells depleted or not for Hrs following IFN-β or EGF (100 ng ml−1) stimulation, respectively. Mean of the control t = 0 time point was set at 100%. a,d,e, Data are the mean ± s.e.m. Statistical significance was determined using an ANOVA with Dunnett’s multiple comparison test (a,d) or a one-way ANOVA with the Kruskal–Wallis multiple comparison test (e); ****P < 0,0001; NS, not significant. b,c, Data are representative of two independent experiments. Source numerical data and unprocessed blots are provided.

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Extended Data Fig. 3 STAM constitutively interacts with IFNAR1 at the plasma membrane.

a, Immunofluorescent labelling of STAM2A and IFNAR1 subunits at steady state in RPE1 cells observed by confocal microscopy with representative plots of co-localization profiles in the vicinity of the plasma membrane. Scale bars, 10 µm. b, Co-immunoprecipitation of endogenous STAM1 and STAM2 with endogenous IFNAR1 subunit in control or TYK2-depleted RPE1 cells with or without IFN-α or IFN-β treatment for 10 min with. Data are representative of three independent experiments. c, Immunofluorescence imaging (left) of IFNAR1 uptake in control (siRNA scrambled; n = 35) or STAM1 and STAM2-depleted (n = 34) RPE1 cells stimulated for 10 min with IFN-α. Following fixation, the cells were co-labelled for EEA1 and analysed by confocal microscopy. Scale bar, 10 μm. Quantifications (right) of co-localizations are expressed as the Manders’ coefficient, indicating the proportion of IFNAR1 pixels containing EEA1 pixels in cells. Data pooled from three independent experiments and represent the mean ± s.e.m. d, Levels of IFNAR1 surface staining monitored by FACS in control or STAM1 and STAM2-depleted RPE1 cells stimulated with IFN-α or IFN-β for 10 min. The same gating strategy (red polygon, left) was used for all samples. Data are representative of two independent experiments; NS, not significant. Source numerical data and unprocessed blots are provided.

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Extended Data Fig. 4 STAM constitutively interacts with TYK2 and is not required for IFNAR1 endocytosis.

a, RFP-Trap immunoprecipitation of TYK2–mCherry in RPE1 cells expressing eGFP–STAM2A. Control immunoblots for RFP-Trap at steady state (left) without any cells or with cells transfected with either eGFP–STAM2A or eGFP and with either TYK2–mCherry or mCherry. Immunoblots for RFP-Trap (right) on RPE1 cells transfected with TYK2–mCherry and eGFP–STAM2A with or without IFN-α or IFN-β stimulation for 10 min. Data are representative of two independent experiments. b, Immunofluorescence imaging (left) of IFNAR1 uptake in control (siRNA scrambled) or STAM1 and STAM2-depleted RPE1 cells stimulated for 10 min with IFN-α. Following fixation, the cells were co-labelled for EEA1 and analysed by confocal microscopy. Scale bars, 50 µm. Quantifications (right) of co-localizations are expressed as the Manders’ coefficient, indicating the proportion of IFNAR1 pixels containing EEA1 pixels in three independent experiments. c, Levels of IFNAR1 surface staining monitored by FACS in control or STAM1 and STAM2-depleted RPE1 cells stimulated with IFN-α or IFN-β for 10 min. Data are representative of two independent experiments. Statistical significance was determined using a two-way RM ANOVA with Tukey’s multiple comparisons test. d, Immunoblots for TYK2 expression in RPE1 cells with or without siRNA to TYK2 transfection for 72 h (left). PLA experiments monitoring the interaction between total IFNAR1 and STAM2A in control (n = 53) and TYK2 (n = 45)-depleted RPE1 cells pooled from three independent experiments (middle). The corresponding quantifications show the mean ± s.e.m. (right). Statistical significance was determined using an unpaired two-tailed Student’s t-test. NS, not significant. Scale bar, 10 µm. Source numerical data and unprocessed blots are provided.

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Extended Data Fig. 5 Block of clathrin-dependent endocytosis in cells ectopically expressing Hrs at the plasma membrane.

a, Confocal microscopy of endogenous Hrs immunofluorescent labelling in RPE1 cells co-transfected with peGFP-FYVE and with constitutively active RFP-Rab5-CAAX (Q79L) and HA-TC10 (Q75L) to ectopically produce PI3-P at the plasma membrane. Insets: magnified views of the plasma membrane (boxes 1 and 2) and endosomal (box 3) areas. Scale bars, 10 µm. b, RPE1 cells treated with 4 µM ikarugamycin or dimethylsulfoxide (Ctrl) were incubated for 20 min at 4 °C with Alexa Fluor 546 transferrin, anti-IFNAR1 and IFN-α. The cells were then transferred to 37 °C for 10 min to allow endocytosis, fixed, immunostained for EEA1 and observed by confocal microscopy. Inset: magnified views of the plasma membrane areas (top) and the endosomal structures (bottom). Scale bars, 10 µm. Data are representative of three independent experiments.

Extended Data Fig. 6 Blocking of clathrin-dependent endocytosis by dominant-negative Eps15 mutant in cells ectopically expressing Hrs at the plasma membrane.

a, Wide-field microscopy of Alexa Fluor 546 transferrin uptake (10 min) in control Eps15 D3Δ2–eGFP (arrow)-or dominant-negative Eps15 DN–eGFP (arrowhead)-transfected cells. Data are representative of three independent experiments. b, Wide-field microscopy (right) and quantification (left) of pSTAT1 nuclear translocation in RPE1 cells with or without (ø) IFN-α or IFN-γ stimulation for 10 min cells transfected with control Eps15 D3Δ2–eGFP (ø = 24, α = 33, γ = 56), or dominant-negative Eps15 DN–eGFP (ø = 36, α = 28, γ = 35). The nucleus/cytosol fluorescence ratio for pSTAT1 was detremined from the fluorescence imaging. Nuclear translocation of pSTAT1 is inhibited by Eps15 DN–eGFP for IFN-α (white arrow) but not for IFN-γ (white arrowhead). c, Wide-field microscopy (right) and quantification (left) of pSTAT1 nuclear translocation in RPE1 cells with or without IFN-α or IFN-β stimulation for 10 min and co-transfected with control Eps15 D3Δ2–eGFP alone (ø = 162, α = 162, β = 167), RFP-Rab5-CAAX (Q79L) (ø = 158, α = 160, β = 161), dominant-negative Eps15 DN–eGFP alone (ø = 162, α = 167, β = 161) or RFP-Rab5-CAAX (Q79L) (ø = 162, α = 162, β = 161). pSTAT1 nuclear translocation is rescued for IFN-α (white arrowheads) but not for IFN-β (white arrows). The nucleus/cytosol fluorescence ratio for pSTAT1 was determined from the fluorescence imaging. b,c, Data were pooled from three independent experiments and are shown as the median ± 95% confidence interval. Statistical significance was determined using a one-way ANOVA with the Kruskal–Wallis multiple comparison test. ****P < 0.0001; ***P < 0.001; *P < 0.05; NS, not significant. Scale bars, 20 µm. Source numerical data are provided.

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Extended Data Fig. 7 Competition between STAM2A and SOCS-1 for binding to the TYK2–IFNAR1 signalling complex.

Representative live-cell micropatterning experiment with micropatterned receptor HaloTag–mTagBFP–IFNAR1 as the bait protein (cyan), and TYK2–meGFP FERM–SH2 (FS) and full-length (FL) forms (green), STAM2A–mCherry (yellow) and SOCS-1–iRFP (red) as prey proteins. Scale bars, 10 µm. Interactions of TYK2, STAM2A and SOCS-1 with micropatterned IFNAR1 were quantified according to the contrast within micropatterned cells. Cells expressing TYK2-FS/SOCS-1 (n = 10), TYK2-FL/SOCS-1 (n = 15) or TYK2-FL/SOCS-1/STAM2A (n = 20) were analysed for contrast values and visualized in a box-and-whisker plot (representing the median, minima, maxima and the four quartiles); data were pooled from two independent experiments. Statistical significance was determined using a two-sample Kolmogorov–Smirnov tests. **P < 0.01. Source numerical data are provided.

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Zanin, N., Viaris de Lesegno, C., Podkalicka, J. et al. STAM and Hrs interact sequentially with IFN-α Receptor to control spatiotemporal JAK–STAT endosomal activation. Nat Cell Biol 25, 425–438 (2023). https://doi.org/10.1038/s41556-022-01085-6

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