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• W2041355362 abstract "The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR. The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR. The thrombopoietin receptor (TpoR) 1The abbreviations used are: TpoR, thrombopoietin receptor; CIS, cytokine-inducible Src homology 2-containing protein; Endo-H, endoglycosidase H; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorter; γc, common gamma subunit; GFP, green fluorescent protein; HA, hemagglutinin; IFN, interferon; IL, interleukin; IRES, internal ribosome entry site; JAK, Janus kinase; Luc, luciferase; MAP, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SRE, serum response element; STAT, signal transducers and activators of transcription; TK, thymidine kinase; Tyk, tyrosine kinase; EpoR, erythropoietin receptor. 1The abbreviations used are: TpoR, thrombopoietin receptor; CIS, cytokine-inducible Src homology 2-containing protein; Endo-H, endoglycosidase H; ERK, extracellular signal-regulated kinase; FACS, fluorescence-activated cell sorter; γc, common gamma subunit; GFP, green fluorescent protein; HA, hemagglutinin; IFN, interferon; IL, interleukin; IRES, internal ribosome entry site; JAK, Janus kinase; Luc, luciferase; MAP, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; SRE, serum response element; STAT, signal transducers and activators of transcription; TK, thymidine kinase; Tyk, tyrosine kinase; EpoR, erythropoietin receptor.is a member of the cytokine receptor superfamily that regulates hematopoietic stem cell renewal (1Solar G.P. Kerr W.G. Zeigler F.C. Hess D. Donahue C. de Sauvage F.J. Eaton D.L. Blood. 1998; 92: 4-10Crossref PubMed Google Scholar), megakaryocyte differentiation, and platelet formation (2de Sauvage F.J. Hass P.E. Spencer S.D. Malloy B.E. Gurney A.L. Spencer S.A. Darbonne W.C. Henzel W.J. Wong S.C. Kuang W.J. Nature. 1994; 369: 533-538Crossref PubMed Scopus (1212) Google Scholar, 3Kaushansky K. Lok S. Holly R.D. Broudy V.C. Lin N. Bailey M.C. Forstrom J.W. Buddle M.M. Oort P.J. Hagen F.S. Nature. 1994; 369: 568-571Crossref PubMed Scopus (917) Google Scholar). Downstream signaling mediated by the TpoR is dependent on two cytoplasmic Janus tyrosine kinases, JAK2 and Tyk2 (4Drachman J.G. Griffin J.D. Kaushansky K. J. Biol. Chem. 1995; 270: 4979-4982Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 5Ezumi Y. Takayama H. Okuma M. FEBS Lett. 1995; 374: 48-52Crossref PubMed Scopus (134) Google Scholar, 6Rodriguez-Linares B. Watson S.P. Biochem. J. 1996; 316: 93-98Crossref PubMed Scopus (61) Google Scholar, 7Sattler M. Durstin M.A. Frank D.A. Okuda K. Kaushansky K. Salgia R. Griffin J.D. Exp. Hematol. 1995; 23: 1040-1048PubMed Google Scholar, 8Drachman J.G. Kaushansky K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2350-2355Crossref PubMed Scopus (137) Google Scholar), with JAK2 being the main JAK required for TpoR effects (9Tortolani P.J. Johnston J.A. Bacon C.M. McVicar D.W. Shimosaka A. Linnekin D. Longo D.L. O'Shea J.J. Blood. 1995; 85: 3444-3451Crossref PubMed Google Scholar, 10Drachman J.G. Millett K.M. Kaushansky K. J. Biol. Chem. 1999; 274: 13480-13484Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Ligand binding triggers activation and phosphorylation of JAKs and of the cytoplasmic domain of the TpoR, providing docking sites for the Src homology 2 domains of many signaling proteins, such as the signal transducers and activators of transcription 1, 3, and 5 (STAT1, STAT3, and STAT5, respectively), Shc, SHIP, Grb2, SOS, Vav, Cbl, and phosphatidylinositol 3-kinase (8Drachman J.G. Kaushansky K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2350-2355Crossref PubMed Scopus (137) Google Scholar, 11Miyakawa Y. Oda A. Druker B.J. Kato T. Miyazaki H. Handa M. Ikeda Y. Blood. 1995; 86: 23-27Crossref PubMed Google Scholar, 12Miyakawa Y. Oda A. Druker B.J. Ozaki K. Handa M. Ohashi H. Ikeda Y. Blood. 1997; 89: 2789-2798Crossref PubMed Google Scholar, 13Oda A. Miyakawa Y. Druker B.J. Ishida A. Ozaki K. Ohashi H. Wakui M. Handa M. Watanabe K. Okamoto S. Ikeda Y. Blood. 1996; 88: 4304-4313Crossref PubMed Google Scholar, 14Onishi M. Mui A.L. Morikawa Y. Cho L. Kinoshita S. Nolan G.P. Gorman D.M. Miyajima A. Kitamura T. Blood. 1996; 88: 1399-1406Crossref PubMed Google Scholar, 15Morita H. Tahara T. Matsumoto A. Kato T. Miyazaki H. Ohashi H. FEBS Lett. 1996; 395: 228-234Crossref PubMed Scopus (60) Google Scholar, 16Nagata Y. Todokoro K. FEBS Lett. 1995; 377: 497-501Crossref PubMed Scopus (63) Google Scholar). Recently it has been found that JAK proteins may play important roles in regulating the cellular localization and traffic of their cognate receptors. In the case of the EpoR and oncostatin M receptor, expression of their cognate JAKs, JAK2 and JAK1, was found important for receptor cell surface localization, with enhanced endoplasmic reticulum to Golgi maturation. The overall cellular levels of these receptors were not changed by the presence or absence of JAKs (17Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 18Radtke S. Hermanns H.M. Haan C. Schmitz-Van De Leur H. Gascan H. Heinrich P.C. Behrmann I. J. Biol. Chem. 2002; 277: 11297-11305Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Sequences encompassing the N-terminal domain JH7-JH6 regions of JAKs were required for these effects. Early on after the discovery of the JAK-STAT pathway, the IFNAR1 subunit of the type I IFN receptor complex was shown to require the expression of Tyk2 for stability at the cell surface (19Gauzzi M.C. Barbieri G. Richter M.F. Uze G. Ling L. Fellous M. Pellegrini S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11839-11844Crossref PubMed Scopus (104) Google Scholar). This effect was mediated by sequences in the JH7-JH6 region of Tyk2 (20Richter M.F. Dumenil G. Uze G. Fellous M. Pellegrini S. J. Biol. Chem. 1998; 273: 24723-24729Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Tyk2 expression leads to enhanced protein levels of IFNAR1 by preventing endosomal targeting (21Ragimbeau J. Dondi E. Alcover A. Eid P. Uze G. Pellegrini S. EMBO J. 2003; 22: 537-547Crossref PubMed Scopus (163) Google Scholar). JAK3 was found to promote cell surface localization of the common gamma (γc) subunit of the IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptor complexes (22Hofmann S.R. Lam A.Q. Frank S. Zhou Y.J. Ramos H.L. Kanno Y. Agnello D. Youle R.J. O'Shea J.J. Mol. Cell. Biol. 2004; 24: 5039-5049Crossref PubMed Scopus (43) Google Scholar). Furthermore, overexpression of JAK1, but not of JAK2, Tyk2, or JAK3, leads to enhanced cell surface localization of IL-9Rα and IL-2Rβ, whereas JAK3 and not the other JAKs promotes enhanced cell surface localization of the γc. 2Y. Royer, P. J. Courtoy, and S. N. Constantinescu, unpublished observations. 2Y. Royer, P. J. Courtoy, and S. N. Constantinescu, unpublished observations.In these cases, it was the FERM domain of JAKs and not the kinase or pseudokinase domain that was required for mediating enhanced cell surface localization of receptors. Because the endogenous JAK1 was reported to be exclusively localized at the plasma membrane via interactions with cytokine receptors (23Behrmann I. Smyczek T. Heinrich P.C. Schmitz-Van de Leur H. Komyod W. Giese B. Muller-Newen G. Haan S. Haan C. J. Biol. Chem. 2004; 279: 35486-35493Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), it is now considered that receptor-JAK complexes function as one unit, equivalent with receptor tyrosine kinases. Furthermore, the plasma membrane localization of JAK3 depends on coexpressed γc (22Hofmann S.R. Lam A.Q. Frank S. Zhou Y.J. Ramos H.L. Kanno Y. Agnello D. Youle R.J. O'Shea J.J. Mol. Cell. Biol. 2004; 24: 5039-5049Crossref PubMed Scopus (43) Google Scholar). Taken together these data indicate that JAKs may fulfill important roles other than triggering downstream signaling by cytokine receptors. The traffic of the TpoR to and from the cell surface is special among cytokine receptors for three reasons. First, TpoR was found to recycle to the membrane in hematopoietic cells after activation and withdrawal of ligand (24Dahlen D.D. Broudy V.C. Drachman J.G. Blood. 2003; 102: 102-108Crossref PubMed Scopus (46) Google Scholar). Second, clearance of circulating Tpo may occur via binding, internalization of the ligand-receptor complex, and degradation of Tpo by platelets, which express high affinity receptors for Tpo (25Fielder P.J. Gurney A.L. Stefanich E. Marian M. Moore M.W. Carver-Moore K. de Sauvage F.J. Blood. 1996; 87: 2154-2161Crossref PubMed Google Scholar, 26Fielder P.J. Hass P. Nagel M. Stefanich E. Widmer R. Bennett G.L. Keller G.A. de Sauvage F.J. Eaton D. Blood. 1997; 89: 2782-2788Crossref PubMed Google Scholar, 27Broudy V.C. Lin N.L. Sabath D.F. Papayannopoulou T. Kaushansky K. Blood. 1997; 89: 1896-1904Crossref PubMed Google Scholar, 28Li J. Xia Y. Kuter D.J. Br. J. Haematol. 1999; 106: 345-356Crossref PubMed Scopus (135) Google Scholar). TpoR apparently is not recycled in platelets, but its recycling in myeloid progenitors and hematopoietic cell lines may be relevant for Tpo functions in early hematopoiesis. Third and most interestingly, TpoR traffic, maturation, glycosylation, and stability were found to be altered in myeloproliferative diseases such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis (29Moliterno A.R. Hankins W.D. Spivak J.L. N. Engl. J. Med. 1998; 338: 572-580Crossref PubMed Scopus (257) Google Scholar, 30Moliterno A.R. Spivak J.L. Blood. 1999; 94: 2555-2561Crossref PubMed Google Scholar). Whether this defective maturation is linked to the pathogenesis of these diseases or is a sign of stress hematopoiesis (31Cohen-Solal K. Vitrat N. Titeux M. Vainchenker W. Wendling F. Blood. 1999; 93: 2859-2866Crossref PubMed Google Scholar) is not known, but all available evidence indicates that the study of TpoR traffic may reveal novel regulation mechanisms. Thus, we investigated whether the two JAKs can affect TpoR traffic and metabolism. Here we show that JAK2 and Tyk2, but not JAK1 or JAK3, strongly promote cell surface localization of the TpoR by stimulating recycling and enhancing the protein stability of the mature, Golgi-processed form of the TpoR. Expression of JAK2 or Tyk2 did not change the internalization kinetics of the TpoR, whereas it promoted recycling. Under cycloheximide treatment, which blocks protein synthesis, inhibitors of proteasome degradation prolonged the half-life of the mature TpoR band in the absence of overexpressed JAK2 or Tyk2, suggesting that a fraction of the mature TpoR is normally degraded via the proteasome. Confocal microscopy studies suggest that JAK2 and Tyk2 prevent degradation of an intracellular pool of TpoR, which partially colocalizes with recycling transferrin. By site-directed mutagenesis, we show that an intact FERM domain of JAK2 or of Tyk2 is required for this effect and that receptor sequences encompassing Box1 and Box2 are likely to make the first contact with JAK FERM domains. Although a hydrophobic motif preceding Box1 is not required for JAK2-dependent traffic effects, this motif is crucial, as for the homologous EpoR, for switching on JAK kinase activity upon ligand binding to the TpoR extracellular domain. We discuss the significance of our data for the mechanisms of signaling, down-modulation, and recycling of the TpoR. cDNA Constructs—Receptors were tagged after the cleavage site of the signal sequence as described (32Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (321) Google Scholar). The HA-tagged mouse TpoR and the HA-tagged human γc were subcloned into the pMX-IRES-GFP bicistronic retroviral vector upstream of the IRES as described previously (33Liu X. Constantinescu S.N. Sun Y. Bogan J.S. Hirsch D. Weinberg R.A. Lodish H.F. Anal. Biochem. 2000; 280: 20-28Crossref PubMed Scopus (122) Google Scholar). The human IL-9Rα and the human JAK3 were subcloned into the pREX-IRES-CD4 vector, and the murine JAK1 and the human Tyk2 were subcloned into the pREX-IRES-CD2 vector. Furthermore, we used the previously described HA-tagged EpoR, wild type JAK2, and kinase-inactive JAK2 cloned in the same vectors (17Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). cDNAs coding for the mutated HA-tagged TpoR were generated by PCR using native Pfu polymerase (Stratagene) and overlap extension primers. cDNAs coding for the mutated JAKs were generated using the QuikChange Site-directed Mutagenesis Kit (Stratagene). We constructed two distinct clones for each mutant cDNA. Cells—Ba/F3 cells are IL-3-dependent mouse pro-B cells (34Palacios R. Steinmetz M. Cell. 1985; 41: 727-734Abstract Full Text PDF PubMed Scopus (583) Google Scholar) cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum, antibiotics, and a 5% supernatant of the WEHI-3B cell line as a source of IL-3. γ2A cells are JAK2-deficient human fibrosarcoma cells (35Kohlhuber F. Rogers N.C. Watling D. Feng J. Guschin D. Briscoe J. Witthuhn B.A. Kotenko S.V. Pestka S. Stark G.R. Ihle J.N. Kerr I.M. Mol. Cell. Biol. 1997; 17: 695-706Crossref PubMed Scopus (175) Google Scholar, 36Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (315) Google Scholar) and were cultured in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum, antibiotics, and 400 μg/ml G418. For confocal microscopy studies we used a derivative of HeLa cells, the HeLa Tet-Off cells (BD Clontech) that contain an integrated copy of the pTet-Off regulator plasmid (37Gossen M. Bujard H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5547-5551Crossref PubMed Scopus (4232) Google Scholar). These cells are highly transfectable with ExGen500 (MBI Fermentas) and can be used for tetracycline-dependent expression of genes of interest. BOSC cells are 293-derived ecotropic packaging cells (38Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar). Generation of Stable Cell Lines—High titer replicative-defective retroviral supernatants were generated by calcium phosphate transient transfection of BOSC packaging cells with the bicistronic vectors encoding the different constructs, as described previously (39Constantinescu S.N. Huang L.J. Nam H. Lodish H.F. Mol. Cell. 2001; 7: 377-385Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Viruses have been used to transduce Ba/F3 cells by centrifugation in the presence of 4 μg/ml Polybrene (Sigma). The efficiency of infection was usually around 40-50%. Populations of cells expressing the marker above a predetermined level (top 10%) were isolated by a fluorescence-activated cell sorter (FACS). Surface Expression of HA-TpoR and HA-EpoR—Surface expression of receptors was measured in Ba/F3 cells by flow cytometry using 10 μg/ml monoclonal anti-HA antibody (HA.11, Covance) and 5 μg/ml R-phycoerythrin-conjugated donkey F(ab′)2 anti-mouse IgG secondary antibody (Jackson ImmunoResearch), as described previously (17Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). The monoclonal HA.11 antibody was iodinated with Na125I to a specific activity of 15 Curies/g using IODO-GEN-precoated tubes (Pierce) following the recommendations of the manufacturer. 125I-Anti-HA binding was used to measure the cell surface localization of the TpoR. The cells were incubated for 2 h at 4 °C with 2 μg/ml 125I-anti-HA in the presence or absence of 200 μg/ml cold anti-HA and then separated from unbound antibodies by a centrifugation through a cushion of serum. Bound and unbound antibody fractions were measured using a gamma counter. All measurements were done in triplicate. Immunoprecipitation, Endoglycosidase H (Endo-H) Digestion, and Immunoblotting—Ba/F3 cells were lysed in Nonidet P-40 buffer with sodium orthovanadate, sodium fluoride, phenylmethanesulfonyl fluoride, and Complete protease inhibitor mixture (Roche Applied Science), as described previously (40Seubert N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). The lysates were then incubated with antibodies against the HA tag (monoclonal HA.11). Immune complexes were recovered by binding to protein G-agarose beads (Invitrogen). Proteins bound to the beads were then eluted with 0.5% SDS and 1% β-mercaptoethanol and digested or not with Endo-H (New England BioLabs) for 1 h at 37 °C. Proteins were separated on 10% SDS-PAGE followed by immunoblotting with anti-HA antibody. Ba/F3 cells stably transduced were lysed directly in Laemmli buffer. Proteins were separated on 10% SDS-polyacrylamide gels, and Western blot analysis was performed with anti-JAK1 (Upstate Biotechnology), anti-JAK2 (C-20, Santa Cruz), anti-JAK3 (C-21, Santa Cruz), anti-Tyk2 (C-20, Santa Cruz), anti-CIS (N-19, Santa Cruz), anti-HA, or anti-β-actin (Sigma) antibodies. To investigate the phosphorylation of the TpoR signaling partners, the cells were starved in RPMI medium supplemented with 0.5% bovine serum albumin (Roche Applied Science) for 4 h at 37 °C and were then stimulated with 10 ng/ml Tpo for 15 min. Phosphorylation was verified with specific anti-phosphorylated antibodies against JAK2 Tyr1007-Tyr1008, Tyk2 Tyr1054-Tyr1055, STAT1 Tyr701, STAT3 Tyr705, and STAT5 Tyr694 (all from Cell Signaling Technology). Bound primary antibodies were detected with secondary antibodies coupled to the horseradish peroxidase and with the enhanced chemoluminescence system (Amersham Biosciences). Measurements of TpoR Half-life, Degradation, and Recycling—Stably transduced Ba/F3 cells were incubated at 37 °C with 20 μg/ml cycloheximide for different periods of time to block protein synthesis. Control cells were kept nontreated to have 100% cell surface localization of the TpoR and γc at the same moment. Analysis of the surface pool of the TpoR or γc was performed by flow cytometry (HA.11) on the living cells because both receptors were tagged with HA at their N terminus. Three independent experiments were performed. An aliquot of each cell line was taken at each time point to verify the receptor expression by Western blot. For measuring the contribution of proteasome or lysosome-mediated degradation of the TpoR, stably transduced Ba/F3 cells were incubated at 37 °C for 3-20 h with 20 μg/ml cycloheximide and proteasome inhibitors MG132 (10 μm) or lactacystin (10 μm) (41Lee D.H. Goldberg A.L. Trends Cell Biol. 1998; 8: 397-403Abstract Full Text Full Text PDF PubMed Scopus (1238) Google Scholar) or with 20 μg/ml cycloheximide and lysosome inhibitors chloroquine (200 μm) (42Wibo M. Poole B. J. Cell Biol. 1974; 63: 430-440Crossref PubMed Scopus (425) Google Scholar) or leupeptin (200 μm) (43Kirschke H. Langner J. Wiederanders B. Ansorge S. Bohley P. Broghammer U. Acta Biol. Med. Ger. 1976; 35: 285-299PubMed Google Scholar, 44Kirschke H. Langner J. Wiederanders B. Ansorge S. Bohley P. Eur. J. Biochem. 1977; 74: 293-301Crossref PubMed Scopus (285) Google Scholar). Cells were then lysed and analyzed by Western blotting with antibodies against HA for the relative levels of the mature and immature TpoR. Internalization Measurements—Ba/F3 cells expressing the HA-tagged TpoR or γc along with various JAKs were labeled for 2 h at 4 °C with 2 μg/ml 125I-anti-HA in the presence or absence of 200 μg/ml cold anti-HA. After three washes, the cells were incubated at 37 °C for different periods of time. Cell surface radioactivity was removed by acid wash for 1 h at 4 °C (250 mm sodium acetate, 150 mm NaCl, pH 0.5), and acid-resistant radioactivity was intracellular. The mean efficiency of the acid wash was ∼75%. Viability of Ba/F3 cells in this acidic buffer was verified at room temperature for 2 h. Separation between the cells and the supernatant was performed by centrifugation through a cushion of serum. Radioactivity was measured using a gamma counter. Counts that could not be stripped by the acid wash in the absence of internalization were subtracted from the overall counts. All measurements were done in triplicate, and three independent experiments were performed. Confocal Microscopy—Transferrin-Alexa 488 (Molecular Probes) was absorbed and recycled by the cells for 25 min at 37 °C and then washed. Adherence of Ba/F3 cells was achieved by spinning the cells softly on poly-l-lysine-treated coverslips. Cells were then fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.05% saponin (Sigma). Quenching of the fluorescence and blocking of the nonspecific labeling were realized by the addition of 50 mm glycine at all steps and 100 μg/ml goat γ-globulins during the 30-min blocking step. The cells were stained with monoclonal anti-HA followed by a goat anti-mouse IgG linked to Alexa Fluor 568 (Molecular Probes). The cells were treated with methanol/acetone at -20 °C to remove the GFP and mounted with ProLong Antifade (Molecular Probes). The complete procedure was performed at room temperature. For the LAMP1 staining, the cells were incubated with anti-HA followed by a goat anti-mouse IgG linked to Alexa Fluor 647 (Molecular Probes) and anti-mouse LAMP1 (1D4B, Developmental Studies Hybridoma Bank) followed by a goat anti-rat IgG linked to Alexa Fluor 568. Image acquisitions were made on a Bio-Rad MRC-1024 confocal laser scanning imaging system associated with the Lasersharp 2000 (Bio-Rad) acquisition software. For the LAMP1 staining, similar experiments were performed with HeLa cells that were transiently transfected using ExGen500 with the cDNA coding for the HA-tagged TpoR or γc. The cells were incubated with rabbit polyclonal HA.11 followed by a goat anti-rabbit IgG linked to Alexa Fluor 568 and anti-human LAMP1 (H4A3, Developmental Studies Hybridoma Bank) followed by a goat anti-mouse IgG linked to Alexa Fluor 647. Assay for Tpo-dependent Proliferation—Ba/F3 cells expressing wild type TpoR or mutated TpoR were washed three times in RPMI 1640 medium to remove IL-3 completely. Washed cells were plated at 100,000 cells/well in a 24-well plate with 1 ng/ml Tpo in RPMI supplemented with 10% fetal bovine serum. Proliferation was measured after 3, 5, and 7 days using a Coulter Counter Z1. All experiments were done in triplicate. Dual Luciferase Assays—STAT1 and STAT3 transcriptional activation was assessed by measuring luciferase production in γ2A cells transfected with the pGRR5-Luc, which contains five copies of a GRR sequence (32Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (321) Google Scholar). MAP kinase activation was assessed with the pSRE-Luc construct (45Treisman R. EMBO J. 1995; 14: 4905-4913Crossref PubMed Scopus (346) Google Scholar). As an internal control, we used the pRL-TK vector (Promega) containing the Renilla luciferase gene under the control of the thymidine kinase promoter. γ2A cells were transfected with Lipofectamine 2000 (Invitrogen) for 8 h and then stimulated or not with 50 ng/ml Tpo. The cells were lysed 16 h after transfection, and luciferase assays were performed in triplicate using the dual luciferase reporter assay kit (Promega). Effect of JAK Proteins on Cell Surface Levels of TpoR—To study the effect of the different Janus kinases on the cell surface expression of the TpoR, we constructed four different Ba/F3 cell lines overexpressing JAK1, JAK2, JAK3, or Tyk2, as we have described previously for JAK2 (40Seubert N. Royer Y. Staerk J. Kubatzky K.F. Moucadel V. Krishnakumar S. Smith S.O. Constantinescu S.N. Mol. Cell. 2003; 12: 1239-1250Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This was achieved by retroviral transduction using bicistronic retroviral vectors, which code for the gene of interest separated from a marker (GFP, CD4, or CD2) by an IRES (33Liu X. Constantinescu S.N. Sun Y. Bogan J.S. Hirsch D. Weinberg R.A. Lodish H.F. Anal. Biochem. 2000; 280: 20-28Crossref PubMed Scopus (122) Google Scholar). The cells were sorted for high marker expression (CD4 or CD2), and the JAK protein levels were measured by Western blot (Fig. 1A). Compared with parental Ba/F3 cells, the transduced cells overexpressed 7-10-fold higher levels of JAKs. Parental Ba/F3 cells and the JAK-expressing cells were then transduced by a TpoR expressing an HA tag at the N terminus. Surface levels of TpoR were measured by FACS analysis using monoclonal HA.11 antibody. Fig. 1B shows that JAK2 and Tyk2 increased 3-4 times the TpoR cell surface levels, whereas JAK3 had no influence on the surface pool of TpoR. Although JAK2 and Tyk2 are the Janus kinases activated by TpoR, JAK1 was also found in a complex with the TpoR and other signaling molecules (i.e. SH-PTP2) (46Mu S.X. Xia M. Elliott G. Bogenberger J. Swift S. Bennett L. Lappinga D.L. Hecht R. Lee R. Saris C.J. Blood. 1995; 86: 4532-4543Crossref PubMed Google Scholar), although Tpo binding to the TpoR does not activate JAK1. In Fig. 1B we show that overexpression of JAK1 increases the cell surface levels of TpoR weakly, but this effect is much less significant than the effect of JAK2 or Tyk2. In all cases, the increases in cell surface receptor levels were detected for similar GFP levels, which indicate similar expression of the bicistronic constructs coding for the receptors in the absence or presence of JAK overexpression. Determination of the Structural Requirements of JAK2 and Tyk2 for Enhancing TpoR Cell Surface Levels—Next, we investigated whether these increases were linked to the kinase activity of JAK2. Wild type JAK2 and JAK2 K882D (kinase-inactive JAK2) were transduced in IL-9R-expressing Ba/F3 cells proliferating in 50 units/ml IL-9. The use of IL-9 allowed us to avoid the negative selection against JAK2 K882D observed in IL-3-proliferating Ba/F3 because IL-3 signaling requires JAK2 activity. Cell surface TpoR levels were enhanced in the presence of mutated JAK2 K882D, but not to the level induced by the wild type JAK2 (Fig. 1C). This is in opposition to the EpoR, where JAK2 K882D was able to promote the receptor cell surface expression to at least the same extent as the wild type JAK2 (Fig. 1C and Ref. 17Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar) and where JAK2 activity was shown to be required for targeting the receptor for degradation (47Walrafen P. Verdier F. Kadri Z. Chretien S. Lacombe C. Mayeux P. Blood. 2005; 105: 600-608Crossref PubMed Scopus (133) Google Scholar). For the TpoR, the kinase activity of the JAK2 JH1 domain could be involved in regulating receptor cell surface levels. Because the positive effects of JAKs on cytokine receptor cell surface levels involved the N terminus FERM domains of JAKs (17Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 18Radtke S. Hermanns H.M. Haan C. Schmitz-Van De Leur H. Gascan H. Heinrich P.C. Behrmann I. J. Biol. Chem. 2002; 277: 11297-11305Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20Richter M.F. Dumenil G. Uze G. Fellous M. Pellegrini S. J. Biol. Chem. 1998; 273: 24723-24729Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we also tested whether this was true for the JAK2/Tyk2-TpoR interaction. We introduced mutations in the FERM domain of JAK2 and Tyk2 by replacing a conserved tyrosine residue by an alanine, singly (Tyr → Ala for JAK1, JAK2, and" @default.
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• W2041355362 date "2005-07-01" @default.
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• W2041355362 title "Janus Kinases Affect Thrombopoietin Receptor Cell Surface Localization and Stability" @default.
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• W2041355362 doi "https://doi.org/10.1074/jbc.m501376200" @default.
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