Palmitoylation of Interferon-α (IFN-α) Receptor Subunit IFNAR1 Is Required for the Activation of Stat1 and Stat2 by IFN-α
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.021915
ISSN1083-351X
AutoresJulie Claudinon, Pauline Gonnord, Emilie Beslard, Marta Marchetti, Keith Mitchell, Cédric Boularan, Ludger Johannes, Pierre Eid, Christophe Lamaze,
Tópico(s)Immune Response and Inflammation
ResumoType I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-α-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway. Type I interferons (IFNs) bind IFNAR receptors and activate Jak kinases and Stat transcription factors to stimulate the transcription of genes downstream from IFN-stimulated response elements. In this study, we analyze the role of protein palmitoylation, a reversible post-translational lipid modification, in the functional properties of IFNAR. We report that pharmacological inhibition of protein palmitoylation results in severe defects of IFN receptor endocytosis and signaling. We generated mutants of the IFNAR1 subunit of the type I IFN receptor, in which each or both of the two cysteines present in the cytoplasmic domain are replaced by alanines. We show that cysteine 463 of IFNAR1, the more proximal of the two cytoplasmic cysteines, is palmitoylated. A thorough microscopic and biochemical analysis of the palmitoylation-deficient IFNAR1 mutant revealed that IFNAR1 palmitoylation is not required for receptor endocytosis, intracellular distribution, or stability at the cell surface. However, the lack of IFNAR1 palmitoylation affects selectively the activation of Stat2, which results in a lack of efficient Stat1 activation and nuclear translocation and IFN-α-activated gene transcription. Thus, receptor palmitoylation is a previously undescribed mechanism of regulating signaling activity by type I IFNs in the Jak/Stat pathway. Type I interferons (IFN 7The abbreviations used are: IFNinterferonBRETbioluminescence resonance energy transferISGIFN-stimulated geneISREIFN-stimulated response elementmAbmonoclonal antibodypAbpolyclonal antibodyCHOChinese hamster ovarySNAREsoluble NSF attachment protein receptorSH2Src homology 2YFPyellow fluorescent proteinFACSfluorescence-activated cell sorter. α/β) are potent cellular mediators essential for several key cell functions, including immunomodulatory, antiviral, and antiproliferative activities. These pleiotropic effects occur through the transcriptional regulation of many IFN-stimulated genes (ISGs) (1.Platanias L.C. Nat. Rev. Immunol. 2005; 5: 375-386Crossref PubMed Scopus (2372) Google Scholar). IFN signal transduction relies mainly on the activation of the Janus tyrosine kinase (Jak)/signal-transducing activators of transcription (Stat) pathways, although several other signaling cascades have also been associated with IFN-regulated transcription (2.Kalvakolanu D.V. Pharmacol. Ther. 2003; 100: 1-29Crossref PubMed Scopus (83) Google Scholar, 3.Parmar S. Platanias L.C. Curr. Opin. Oncol. 2003; 15: 431-439Crossref PubMed Scopus (169) Google Scholar). In general, the binding of type I IFNs to the cell surface receptor IFNAR1 and IFNAR2 subunits induces tyrosine phosphorylation in trans of the IFNAR-associated Jak kinases (Tyk2 with IFNAR1 and Jak1 with IFNAR2), which in turn leads to IFNAR tyrosine phosphorylation. Several members of the Stat family can be activated by type I IFNs, and Stat1 and Stat2 are the main downstream effectors of the type I IFN transcriptional response. Upon IFN-α stimulation, cytosolic Stat2 is recruited to the activated IFNAR complex where it becomes tyrosine-phosphorylated by the receptor-associated Jak kinases. Stat2 activation is a key event in IFN-α signaling because it is required for the indirect recruitment, through binding to Stat2, of Stat1 to IFNAR1 and its activation. There is some debate as to whether cytosolic Stat2 is preferentially recruited to IFNAR1 or to IFNAR2 (4.Li X. Leung S. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1997; 17: 2048-2056Crossref PubMed Scopus (165) Google Scholar, 5.Nadeau O.W. Domanski P. Usacheva A. Uddin S. Platanias L.C. Pitha P. Raz R. Levy D. Majchrzak B. Fish E. Colamonici O.R. J. Biol. Chem. 1999; 274: 4045-4052Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 6.Nguyen V.P. Saleh A.Z. Arch A.E. Yan H. Piazza F. Kim J. Krolewski J.J. J. Biol. Chem. 2002; 277: 9713-9721Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 7.Abramovich C. Shulman L.M. 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Samarajiwa S.A. Hertzog P.J. J. Biol. Chem. 2007; 282: 20053-20057Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). interferon bioluminescence resonance energy transfer IFN-stimulated gene IFN-stimulated response element monoclonal antibody polyclonal antibody Chinese hamster ovary soluble NSF attachment protein receptor Src homology 2 yellow fluorescent protein fluorescence-activated cell sorter. Recent data indicate that signal transduction through the Jak/Stat pathway cannot fully account for the diversity and complexity of the biological response elicited by type I IFNs (12.van Boxel-Dezaire A.H. Rani M.R. Stark G.R. Immunity. 2006; 25: 361-372Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar), and that other factors, for example receptor configuration and alternate signaling pathways, have to be considered (13.Baychelier F. Nardeux P.C. Cajean-Feroldi C. Ermonval M. Guymarho J. Tovey M.G. Eid P. Cell. Signal. 2007; 19: 2080-2087Crossref PubMed Scopus (10) Google Scholar). We recently showed that endocytosis plays an important role in the control of IFN-α signaling and biological activity (14.Marchetti M. Monier M.N. Fradagrada A. Mitchell K. Baychelier F. Eid P. Johannes L. Lamaze C. Mol. Biol. Cell. 2006; 17: 2896-2909Crossref PubMed Scopus (87) Google Scholar). Little is known about the potential links between membrane trafficking and the control of the Jak/Stat signaling pathway, and the contribution of IFNAR trafficking to IFN signaling is just beginning to be appreciated (15.Claudinon J. Monier M.N. Lamaze C. Biochimie. 2007; 89: 735-743Crossref PubMed Scopus (27) Google Scholar). To study this poorly investigated aspect of IFN signaling, we examined the role, if any, of receptor palmitoylation. Palmitoylation is a reversible lipid modification involving the specific attachment of a saturated fatty acid chain to cysteines via a thioester bond. Palmitoylation is among the most prevalent post-translational modifications found on the cytoplasmic face of transmembrane proteins. Various functions have been proposed for protein palmitoylation, although the precise mechanisms by which it works remain to be established (16.Charollais J. Van Der Goot F.G. Mol. Membr. Biol. 2009; 26: 55-66Crossref PubMed Scopus (135) Google Scholar, 17.Nadolski M.J. Linder M.E. FEBS J. 2007; 274: 5202-5210Crossref PubMed Scopus (213) Google Scholar, 18.Resh M.D. Sci. STKE 2006. 2006; : re14Google Scholar). Palmitoylation controls the stability of several proteins, including CCR5, yeast SNAREs, the anthrax toxin receptor, and the neutral sphingomyelinase 2, by preventing their ubiquitination and thereby their targeting to lysosomal degradation (19.Abrami L. Leppla S.H. van der Goot F.G. J. Cell Biol. 2006; 172: 309-320Crossref PubMed Scopus (155) Google Scholar, 20.Tani M. Hannun Y.A. J. Biol. Chem. 2007; 282: 10047-10056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21.Valdez-Taubas J. Pelham H. EMBO J. 2005; 24: 2524-2532Crossref PubMed Scopus (158) Google Scholar, 22.Percherancier Y. Planchenault T. Valenzuela-Fernandez A. Virelizier J.L. Arenzana-Seisdedos F. Bachelerie F. J. Biol. Chem. 2001; 276: 31936-31944Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In hematopoietic cells and lymphocytes, palmitoylation regulates signal transduction by promoting the association of the signaling molecules with lipid microdomains at the plasma membrane and by regulating protein-protein interactions (23.Flaumenhaft R. Sim D.S. Hematology. 2005; 10: 511-519Crossref PubMed Scopus (9) Google Scholar). The chemokine receptor CCR5 and Fas are receptors whose palmitoylation is required for the induction of efficient signaling (24.Chakrabandhu K. Hérincs Z. Huault S. Dost B. Peng L. Conchonaud F. Marguet D. He H.T. Hueber A.O. EMBO J. 2007; 26: 209-220Crossref PubMed Scopus (150) Google Scholar, 25.Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Finally, palmitoylation is involved in various trafficking events, including export from the endoplasmic reticulum and the Golgi apparatus, and recycling to the plasma membrane (25.Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 26.Hayashi T. Rumbaugh G. Huganir R.L. Neuron. 2005; 47: 709-723Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 27.Kinlough C.L. McMahan R.J. Poland P.A. Bruns J.B. Harkleroad K.L. Stremple R.J. Kashlan O.B. Weixel K.M. Weisz O.A. Hughey R.P. J. Biol. Chem. 2006; 281: 12112-12122Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We investigated whether palmitoylation contributes to IFNAR1 trafficking and IFN-α-induced signaling. We report that IFNAR1 is palmitoylated on cysteine 463, and although this modification has no major effect on IFNAR1 cellular trafficking, it strongly affects Jak/Stat signaling and the gene transcription induced by IFN-α. Recombinant human IFN-α2b (specific activity of 108 units/mg) from (Biosidus, Argentina) was kindly provided by J. Wietzerbin. Mouse anti-IFNAR1 mAb 34F10 and 64G12 and mouse anti-IFNAR2 mAbs 8F11 and 10E10 were described previously (13.Baychelier F. Nardeux P.C. Cajean-Feroldi C. Ermonval M. Guymarho J. Tovey M.G. Eid P. Cell. Signal. 2007; 19: 2080-2087Crossref PubMed Scopus (10) Google Scholar). Mouse anti-IFNAR1 mAb AA3 and EA12 were the kind gifts from Biogen Inc. (Boston). Rabbit anti-phospho-Stat1 (Tyr-701), anti-phospho-Tyk2 (Tyr-1054/1055), anti-phospho-Jak1 (Tyr-1022/1023), anti-Stat1, and anti-Tyk2 pAb were from Cell Signaling Technology. Rabbit anti-Stat2 and rabbit anti-phospho-Stat2 (Tyr-689) were from Upstate. Rabbit anti-Lamp2, goat anti-EEA1, goat anti-calnexin, and rabbit anti-Rab6 were from Santa Cruz Biotechnology. Biotinylated anti-phosphotyrosine (RC20) was from BD Biosciences. Secondary antibodies were goat Alexa 488-conjugated anti-mouse pAb, goat Cy3-conjugated anti-mouse pAb, goat Cy3-conjugated anti-rabbit pAb, donkey Cy3-conjugated anti-goat pAb, donkey horseradish peroxidase-conjugated anti-mouse pAb, and donkey horseradish peroxidase-conjugated anti-rabbit pAb (Jackson ImmunoResearch). Streptavidin-horseradish peroxidase was from Roche Applied Science. Wild-type human IFNAR1 was expressed in pEFIREShyg derived from pIREShyg as described previously (28.Cajean-Feroldi C. Nosal F. Nardeux P.C. Gallet X. Guymarho J. Baychelier F. Sempé P. Tovey M.G. Escary J.L. Eid P. Biochemistry. 2004; 43: 12498-12512Crossref PubMed Scopus (33) Google Scholar). Mutagenesis of the IFNAR1 receptor chain was performed using the QuickChange site-directed mutagenesis kit (Stratagene, Amsterdam). pcDNA3.1(+)-IFNAR1-YFP was a kind gift from Dr. Jacob Piehler. It was modified by changing the cytomegalovirus promoter by an EF-1α promoter. pRC-CMV-Tyk2-VSV plasmid was a kind gift from Dr. Sandra Pellegrini. Renilla luciferase sequence was amplified from phRluc-C1 plasmid (Clontech) and was inserted in pRC-CMV-Tyk2-VSV plasmid in 5′ of the Tyk2 sequence by PCR. Stat2-GFP and IFNGR1-YFP plasmids were gifts from H. Hauser and J. L. Casanova, respectively. The accuracy of all cDNA was confirmed by DNA sequencing. L929R2 murine fibroblasts stably expressing human IFNAR2 (28.Cajean-Feroldi C. Nosal F. Nardeux P.C. Gallet X. Guymarho J. Baychelier F. Sempé P. Tovey M.G. Escary J.L. Eid P. Biochemistry. 2004; 43: 12498-12512Crossref PubMed Scopus (33) Google Scholar) were transfected by wild-type or mutated forms of human IFNAR1 using FuGENE 6 (Roche Applied Science). Mixed populations of transfected cells were selected under hygromycin selective pressure. The generated cell lines (L929R1R2) were cultured in Dulbecco's modified Eagle's essential medium supplemented with 10% fetal bovine serum, 1% l-glutamine, 1% penicillin/streptomycin, 400 μg/ml hygromycin, and 1.5 mg/ml geneticin. CHO cells were cultured as above except for Dulbecco's modified Eagle's essential medium/F-12 growth medium and without selection antibiotics. Palmitoylation was inhibited by incubating the cells with 200 μm 2-bromopalmitate for 1 h at 37 °C before starting the experiments. Chemical removal of palmitoylation was performed by treating cell extracts with 1 m hydroxylamine, pH 7, for 1 h at room temperature. Protein synthesis was inhibited by a 1-h treatment with 50 μm cycloheximide at 37 °C. All drugs were from Sigma. 40 × 106 cells were detached with phosphate-buffered saline/EDTA 2 mm and lysed 30 min in immunoprecipitation buffer (1% Triton X-100, 50 mm Tris, pH 7.5, 150 mm NaCl, 1 mm EDTA, and a mixture of protease and phosphatase inhibitors (Sigma) with or without 10 mm N-ethylmaleimide). Cell lysates were centrifuged for 30 min at 14,000 × g, and supernatants were incubated overnight at 4 °C on protein G-Sepharose beads (Amersham Biosciences) with 2.5 μg of EA12 mAb for IFNAR1 or 2.5 μg of 8F11 mAb for IFNAR2. After washing the beads, samples were boiled for 5 min before being analyzed by Western blotting using 64G12 mAb for detecting IFNAR1 or 10E10 mAb for detecting IFNAR2. Cells were first starved for 1 h in serum-free medium, then incubated for 4 h at 37 °C in Dulbecco's modified Eagle's essential medium containing 0.2% bovine serum albumin with 200 μCi/ml [9,10-3H]palmitic acid (American Radiolabeled Chemicals), washed, and immunoprecipitated for IFNAR1 or IFNAR2. After fixation (25% isopropyl alcohol, 65% H2O, and 10% acetic acid), gels were incubated for 30 min in enhanced Amplify NAMP100 (GE Healthcare), dried, and exposed for 3 weeks to Hyperfilm MP (GE Healthcare). For analysis of IFNAR1 endocytosis, cells grown on coverslips were incubated on ice with the 34F10 antibody for 30 min. Cells were then incubated at 37 °C for 30 min, washed, fixed using 4% paraformaldehyde, and permeabilized with saponin treatment, and endocytosed antibody-IFNAR1 complexes were revealed with Cy3-conjugated anti-mouse antibody. Cells were imaged with an epifluorescent Leica microscope. For analysis of IFNAR1 intracellular co-localization experiments, cells were first fixed, then permeabilized, and incubated simultaneously with 34F10 and either anti-EEA1, anti-Rab6, anti-Rab11 or anti-Lamp2 antibodies as indicated. Secondary Alexa488-conjugated anti-mouse antibody was used to reveal IFNAR1, and Cy3-conjugated anti-goat antibody was used to reveal EEA1, and Cy3-conjugated anti-rabbit antibody was used to reveal Rab6, Rab11, and Lamp2. Cells were imaged with a confocal Leica microscope. Cells were treated with or without 1000 units/ml IFN-α2b at 37 °C for the indicated times. For biochemical analysis, cells were washed with phosphate-buffered saline at 4 °C and lysed in Lysis Buffer (1% Triton X-100, 50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, and a mixture of proteases and phosphatases inhibitors (Sigma)). After centrifugation for 10 min at 15,000 × g, lysates were resolved on SDS-PAGE and analyzed by Western blot/ECL for activated Tyk2 and Jak1 using anti-pTyk2 and anti-pJak1 or activated Stat1 and Stat2 using anti-pStat1 and anti-pStat2 antibodies. Anti-Jak1 and anti-Stat1 antibodies were used to determine the total amount of Jak1 and Stat1. For immunofluorescent analysis of pStat1 and pStat2 nuclear translocation, cells grown on coverslips were treated with IFN-α2b at 37 °C for the indicated time and fixed with cold methanol at −20 °C for 10 min. pStat1 and pStat2 were then stained by successive incubations with anti-phospho-Stat1, anti-phospho-Stat2, and Cy3-conjugated anti-rabbit antibodies. Cells were detached with phosphate-buffered saline/EDTA 2 mm and stained with AA3 antibody for 40 min in FACS buffer (phosphate-buffered saline supplemented with 3% fetal calf serum and 0.05% sodium azide) on ice. Goat anti-mouse Alexa 488-conjugated antibody (Jackson ImmunoResearch) was used as secondary antibody. Dead cells were excluded by gating on forward/side light scatter. Events corresponding to 2 × 104 gated cells were accumulated per sample. Flow cytometry was performed on a FACSCalibur machine, and data were analyzed by CellQuest software (BD Biosciences). L929R1R2 cells were transfected with an ISG54-luciferase construct kindly provided by S. Pellegrini using Lipofectamine 2000 (Invitrogen). After 48 h, cells were treated or not (control) with 1000 units/ml IFN-α2b for 8 h. Luciferase activity was quantified in cell lysates using a luminometer (Lumat LB9501, Berthold, Wildbald, Germany), and results were reported to the quantity of proteins as quantified with the Bradford method. Results were expressed as fold increase over the basal activity without IFN stimulation. 5 × 106 CHO cells were transiently transfected with 0.1 μg of the DNA construct coding for BRET donor (RLuc-Tyk2) and increasing (0.05–1.5 μg) amounts of the BRET acceptor plasmid (IFNAR1 CC-YFP, IFNAR1 AC-YFP, or IFNGR1-YFP) and 0.1 μg of the DNA construct coding for IFNAR2 using GeneJuice Reagent (Novagen). A total amount of transfected DNA was maintained constant using an appropriate quantity of pcDNA3 (Invitrogen). 48 h after transfection, the luciferase substrate, coelenterazine h (Interchim), was added at a final concentration of 5 μm to 1 × 105 cells. Luminescence and fluorescence were measured simultaneously using the MithrasTM fluorescence-luminescence detector (Berthold). Cells expressing BRET donors alone were used to determine background. Filter sets were 485 ± 10 nm for luciferase emission and 530 ± 12.5 nm for YFP emission. BRET ratios were calculated as described previously (29.Boularan C. Scott M.G. Bourougaa K. Bellal M. Esteve E. Thuret A. Benmerah A. Tramier M. Coppey-Moisan M. Labbé-Jullié C. Fåhraeus R. Marullo S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18061-18066Crossref PubMed Scopus (54) Google Scholar). We recently showed that IFNAR1 uptake at the plasma membrane proceeds through classical clathrin- and dynamin-dependent endocytosis; inhibition of IFNAR1 endocytosis by either small interfering RNA-mediated knockdown of clathrin or inactivation of the GTPase dynamin by the dominant negative mutant K44A inhibits both activation of the Jak/Stat signaling pathway and the antiviral and antiproliferative activities otherwise promoted by IFN-α (14.Marchetti M. Monier M.N. Fradagrada A. Mitchell K. Baychelier F. Eid P. Johannes L. Lamaze C. Mol. Biol. Cell. 2006; 17: 2896-2909Crossref PubMed Scopus (87) Google Scholar). Based on previous studies of the contribution of palmitoylation to the regulation of membrane trafficking (19.Abrami L. Leppla S.H. van der Goot F.G. J. Cell Biol. 2006; 172: 309-320Crossref PubMed Scopus (155) Google Scholar, 30.Alvarez E. Gironès N. Davis R.J. J. Biol. Chem. 1990; 265: 16644-16655Abstract Full Text PDF PubMed Google Scholar) and cell signaling pathways (23.Flaumenhaft R. Sim D.S. Hematology. 2005; 10: 511-519Crossref PubMed Scopus (9) Google Scholar, 24.Chakrabandhu K. Hérincs Z. Huault S. Dost B. Peng L. Conchonaud F. Marguet D. He H.T. Hueber A.O. EMBO J. 2007; 26: 209-220Crossref PubMed Scopus (150) Google Scholar, 25.Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), we tested whether palmitoylation is involved in IFNAR1 endocytosis and IFN-α signaling. We first analyzed the effects of 2-bromopalmitate, a drug that blocks general protein palmitoylation (Fig. 1A) (31.Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). IFNAR1 is rapidly endocytosed, and within 30 min is found in the recycling compartment as shown by co-localization with the small GTPAse Rab11 (data not shown) and as established previously (14.Marchetti M. Monier M.N. Fradagrada A. Mitchell K. Baychelier F. Eid P. Johannes L. Lamaze C. Mol. Biol. Cell. 2006; 17: 2896-2909Crossref PubMed Scopus (87) Google Scholar). However, preincubation of cells with 200 μm 2-bromopalmitate strongly inhibited IFNAR1 endocytosis, and few IFNAR1 subunits were detected in the recycling compartment (Fig. 1A). The inhibition appears to be specific to 2-bromopalmitate because palmitate had no effect; this implicates palmitoylation in IFNAR1 endocytosis. We next tested the effect of 2-bromopalmitate on the Jak/Stat signaling pathway activated by IFN-α because we have shown this process to be dependent on IFNAR1 endocytosis (14.Marchetti M. Monier M.N. Fradagrada A. Mitchell K. Baychelier F. Eid P. Johannes L. Lamaze C. Mol. Biol. Cell. 2006; 17: 2896-2909Crossref PubMed Scopus (87) Google Scholar). Upon IFN-α binding, the Tyk2 and Jak1 kinases associated with IFNAR are activated by tyrosine phosphorylation, resulting in the phosphorylation of tyrosine 701 of Stat1. In L929R1R2 cells, tyrosine phosphorylation of Stat1 (pStat1) was complete after 10 min of stimulation with 1000 units/ml IFN-α (Fig. 1B). Treatment with 2-bromopalmitate inhibited tyrosine phosphorylation of Stat1 in a dose-dependent manner with a maximum effect at 200 μm. We also analyzed later steps of the Jak/Stat signaling pathway by testing for the translocation of pStat1 in the nucleus (Fig. 1C). pStat1 accumulated in the nuclei of control or palmitate-treated cells after 30 min of IFN-α stimulation but not in the nuclei of cells treated with 200 μm 2-bromopalmitate (Fig. 1C). Thus, IFNAR1 endocytosis and IFN-α-dependent Jak/Stat signaling require protein palmitoylation. We analyzed whether the type I IFN receptor subunits IFNAR1 and IFNAR2 were themselves palmitoylated. IFNAR1 and IFNAR2 were immunoprecipitated from lysates of [3H]palmitic acid-labeled L929R1R2 cells. Radiolabeled bands corresponding to both IFNAR1 and IFNAR2 were detected (Fig. 2A), with a stronger signal for IFNAR2. The difference of band intensity was probably due to the higher level of IFNAR2 than IFNAR1 expression in this cell line, as shown on the corresponding whole cell extract (28.Cajean-Feroldi C. Nosal F. Nardeux P.C. Gallet X. Guymarho J. Baychelier F. Sempé P. Tovey M.G. Escary J.L. Eid P. Biochemistry. 2004; 43: 12498-12512Crossref PubMed Scopus (33) Google Scholar). A similar experiment with a Jurkat cell line expressing a FLAG-tagged IFNAR1 showed that IFNAR1 was palmitoylated in this human lymphoid cell line also (data not shown). The two IFN-α receptor subunits are required for IFN-α activity; however, IFNAR1 is essential for IFN-α-induced signaling, and IFNAR2 is more involved in IFN-α binding; we therefore focused our analysis on IFNAR1 (32.Pestka S. Krause C.D. Walter M.R. Immunol. Rev. 2004; 202: 8-32Crossref PubMed Scopus (1299) Google Scholar). Several studies have reported an inducible cycle of palmitoylation and depalmitoylation. For example, stimulation of the β-adrenergic receptor by isoproterenol increases the binding of [3H]palmitic acid reflecting a higher turnover of bound palmitate, whereas it induces the depalmitoylation of the Gα subunits associated with the receptor (33.Mumby S.M. Kleuss C. Gilman A.G. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 2800-2804Crossref PubMed Scopus (222) Google Scholar, 34.Mouillac B. Caron M. Bonin H. Dennis M. Bouvier M. J. Biol. Chem. 1992; 267: 21733-21737Abstract Full Text PDF PubMed Google Scholar). The association of the CD19-CD21-CD81 complex with the activated B cell antigen receptor induces palmitoylation of CD81, thereby enhancing the stability of this association (35.Cherukuri A. Carter R.H. Brooks S. Bornmann W. Finn R. Dowd C.S. Pierce S.K. J. Biol. Chem. 2004; 279: 31973-31982Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Also, 17β-estradiol reduces the palmitoylation of the estradiol receptor in a time- and dose-dependent manner (36.Acconcia F. Ascenzi P. Bocedi A. Spisni E. Tomasi V. Trentalance A. Visca P. Marino M. Mol. Biol. Cell. 2005; 16: 231-237Crossref PubMed Scopus (391) Google Scholar). This prompted us to investigate whether IFN treatment regulates the turnover of palmitate on IFNAR1. Stimulation of the cells with IFN-α from 5 min to 1 h prior to and during radiolabeling with [3H]palmitic acid had no effect on the quantity of palmitate bound to IFNAR1 (Fig. 2B); however, after 2 h of stimulation there was an increase of palmitate incorporation indicating that IFNAR1 palmitoylation turnover can be regulated by IFN-α stimulation. We found that IFNAR1 palmitoylation occurs on cysteine residues via a thioester bond because hydroxylamine, which cleaves the thioester bond, removed the [3H]palmitic acid incorporated in IFNAR1 (Fig. 3B). The cytoplasmic domain of IFNAR1 contains only two cysteines, at positions 463 and 502, that are likely to be palmitoylated. To determine which cysteines are palmitoylated, we mutated each of them to an alanine in single (AC and CA) and double (AA) mutants (Fig. 3A). We transfected L929R2 cells with either the wild-type form of human IFNAR1 or each of the three mutants. Mixed populations of transfected cells were generated, and mutant cell lines expressing levels of cell surface IFNAR1 and IFNAR2 similar to that in cells expressing the wild-type subunits, as determined by flow cytometry, were used for metabolic labeling with [3H]palmitic acid. The CA mutant incorporated as much palmitate as the wild-type CC (Fig. 3B); in contrast, the AC mutant, carrying the cysteine 463 to alanine mutation, did not show any radioactive signal. Similarly, the double mutant AA showed no incorporation of [3H]palmitic acid. Thus, IFNAR1 is palmitoylated on cysteine 463 but not on cysteine 502. This is in agreement with previous reports that transmembrane proteins are mostly palmitoylated on cysteine residues in close proximity to the plasma membrane and near stretches of hydrophobic acids (37.Bijlmakers M.J. Marsh M. Trends Cell Biol. 2003; 13: 32-42Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), an environment corresponding to that of the cysteine 463. Next, we examined the effect of IFNAR1 palmitoylation on the functional properties of IFNAR. For several proteins, a lack of palmitoylation results in various trafficking defects, including reduced export from the Golgi apparatus, accumulation in lysosomes, and defective recycling to the plasma membrane (19.Abrami L. Leppla S.H. van der Goot F.G. J. Cell Biol. 2006; 172: 309-320Crossref PubMed Scopus (155) Google Scholar, 20.Tani M. Hannun Y.A. J. Biol. Chem. 2007; 282: 10047-10056Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 25.Blanpain C. Wittamer V. Vanderwinden J.M. Boom A. Renneboog B. Lee B. Le Poul E. El Asmar L. Govaerts C. Vassart G. Doms R.W. Parmentier M. J. Biol. Chem. 2001; 276: 23795-23804Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 27.Kinlough C.L. McMahan R.J. Poland P.A. Bruns J.B. Harkleroad K.L. Stremple R.J. Kashlan O.B. Weixel K.M. Weisz O.A. Hughey R.P. J. Biol. Chem. 2006; 281: 12112-12122Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Therefore, we investigated whether IFNAR1 palmitoylation regulates any of the steps of IFNAR1 intracellular trafficking. We first tested whether IFNAR1 palmitoylation was required for IFNAR1 uptake from the plasma membrane. Indeed, a lack of protein palmitoylation has been associated with abnormalities in endocy
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