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Acute Lymphoblastic Leukemia-associated JAK1 Mutants Activate the Janus Kinase/STAT Pathway via Interleukin-9 Receptor α Homodimers

2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês

10.1074/jbc.m807531200

ISSN

1083-351X

Autores

Tekla Hornakova, Judith Staerk, Yohan Royer, Elisabetta Flex, Marco Tartaglia, Stefan N. Constantinescu, Laurent Knoops, Jean‐Christophe Renauld,

Tópico(s)

Lymphoma Diagnosis and Treatment

Resumo

Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Rα, γc, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Rα to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, STAT activation was partially inhibited by expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by γc, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rβ. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants. Activating mutations in JAK1 have been reported in acute lymphoblastic leukemias, but little is known about the mechanisms involved in their constitutive activation. Here, we studied the ability of JAK1 V658F and A634D to activate the Janus kinase (JAK)/STAT pathway upon ectopic expression in HEK293 cells alone or together with the other components of the interleukin-9 receptor complex (IL-9Rα, γc, and JAK3). Expression of JAK1 mutants alone failed to trigger STAT activation, but co-expression of the IL-9Rα chain promoted JAK1 mutant phosphorylation and STAT activation. Mutation of the FERM domain of JAK1, which is critical for cytokine receptor association, or of the single tyrosine of IL-9Rα involved in STAT recruitment abolished this activity, indicating that JAK1 mutants need to associate with a functional IL-9Rα to activate STAT factors. Several lines of evidence indicated that IL-9Rα homodimerization was involved in this process. IL-9Rα variants with mutations of the JAK-interacting BOX1 region not only failed to promote JAK1 activation but also acted as dominant negative forms reverting the effect of wild-type IL-9Rα. Coimmunoprecipitation experiments also showed the formation of IL-9Rα homodimers. Interestingly, STAT activation was partially inhibited by expression of γc, suggesting that overlapping residues are involved in IL-9Rα homodimerization and IL-9Rα/γc heterodimerization. Co-expression of wild-type JAK3 partially reverted the inhibition by γc, indicating that JAK3 cooperates with JAK1 mutants within the IL-9 receptor complex. Similar results were observed with IL-2Rβ. Taken together, our results show that IL-9Rα and IL-2Rβ homodimers efficiently mediate constitutive activation of ALL-associated JAK1 mutants. Janus kinases (JAKs) 5The abbreviations used are: JAK, Janus kinase; IL, interleukin; IL-9Rα, IL-9 receptor complex; EPOR, erythropoietin receptor; TPOR, thrombopoietin receptor; ALL, acute lymphoblastic leukemia; T-ALL, T cell ALL; STAT, signal transducers and activators of transcription; HA, hemagglutinin; FACS, fluorescence-activated cell sorter; WT, wild type. represent a family of four non-receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that is associated with cytokine receptors of no intrinsic kinase activity (1Schindler C. Levy D.E. Decker T. J. Biol. Chem. 2007; 282: 20059-20063Abstract Full Text Full Text PDF PubMed Scopus (958) Google Scholar). During the last few years several acquired JAK mutations have been identified in different malignancies. These mutations led to a gain of kinase function and are tumorigenic. The best example is the JAK2 V617F mutation associated with myeloproliferative neoplasms (2James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2917) Google Scholar, 3Kralovics R. Passamonti F. Buser A.S. Teo S.S. Tiedt R. Passweg J.R. Tichelli A. Cazzola M. Skoda R.C. N. Engl. J. Med. 2005; 352: 1779-1790Crossref PubMed Scopus (2972) Google Scholar, 4Levine R.L. Wadleigh M. Cools J. Ebert B.L. Wernig G. Huntly B.J. Boggon T.J. Wlodarska I. Clark J.J. Moore S. Adelsperger J. Koo S. Lee J.C. Gabriel S. Mercher T. D'Andrea A. Frohling S. Dohner K. Marynen P. Vandenberghe P. Mesa R.A. Tefferi A. Griffin J.D. Eck M.J. Sellers W.R. Meyerson M. Golub T.R. Lee S.J. Gilliland D.G. Cancer Cell. 2005; 7: 387-397Abstract Full Text Full Text PDF PubMed Scopus (2462) Google Scholar, 5Baxter E.J. Scott L.M. Campbell P.J. East C. Fourouclas N. Swanton S. Vassiliou G.S. Bench A.J. Boyd E.M. Curtin N. Scott M.A. Erber W.N. Green A.R. Lancet. 2005; 365: 1054-1061Abstract Full Text Full Text PDF PubMed Scopus (2300) Google Scholar). JAK2 V617F retains its ability to interact with cytokine receptors (6Vainchenker W. Constantinescu S.N. Hematology Am. Soc. Hematol. Educ. Program. 2005; 2005: 195-200Crossref Scopus (91) Google Scholar), and an intact FERM domain, which mediates recruitment to cytokine receptors, is required for inducing transformation of hematopoietic cells (7Wernig G. Gonneville J.R. Crowley B.J. Rodrigues M.S. Reddy M.M. Hudon H.E. Walz C. Reiter A. Podar K. Royer Y. Constantinescu S.N. Tomasson M.H. Griffin J.D. Gilliland D.G. Sattler M. Blood. 2008; 111: 3751-3759Crossref PubMed Scopus (109) Google Scholar). At physiological levels of expression, JAK2 V617F needs to be associated to JAK2 binding homodimeric type I cytokine receptors such as the erythropoietin receptor (EPOR) or the thrombopoietin receptor (TPOR) to allow constitutive signaling (8Lu X. Levine R. Tong W. Wernig G. Pikman Y. Zarnegar S. Gilliland D.G. Lodish H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18962-18967Crossref PubMed Scopus (258) Google Scholar, 9Lu X. Huang L.J. Lodish H.F. J. Biol. Chem. 2008; 283: 5258-5266Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Because EpoR is a preformed dimer in the absence of ligand (10Constantinescu S.N. Keren T. Socolovsky M. Nam H. Henis Y.I. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4379-4384Crossref PubMed Scopus (215) Google Scholar), a model was proposed where dimerization of JAK2 V617F via interactions with a preformed EpoR dimer promotes signaling by JAK2 V617F (8Lu X. Levine R. Tong W. Wernig G. Pikman Y. Zarnegar S. Gilliland D.G. Lodish H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18962-18967Crossref PubMed Scopus (258) Google Scholar). Constitutive and increased erythropoietin or thrombopoietin signaling provide a mechanism for the erythocytosis and thrombocytosis observed in these disorders (11Staerk J. Kallin A. Demoulin J.B. Vainchenker W. Constantinescu S.N. J. Biol. Chem. 2005; 280: 41893-41899Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The A572V mutation in JAK3 has later been identified in patients with acute megakaryoblastic leukemia (12Walters D.K. Mercher T. Gu T.L. O'Hare T. Tyner J.W. Loriaux M. Goss V.L. Lee K.A. Eide C.A. Wong M.J. Stoffregen E.P. McGreevey L. Nardone J. Moore S.A. Crispino J. Boggon T.J. Heinrich M.C. Deininger M.W. Polakiewicz R.D. Gilliland D.G. Druker B.J. Cancer Cell. 2006; 10: 65-75Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Recently, mutations in JAK1, such as A634D, R724H, R879C (13Flex E. Petrangeli V. Stella L. Chiaretti S. Hornakova T. Knoops L. Ariola C. Fodale V. Clappier E. Paoloni F. Martinelli S. Fragale A. Sanchez M. Tavolaro S. Messina M. Cazzaniga G. Camera A. Pizzolo G. Tornesello A. Vignetti M. Battistini A. Cave H. Gelb B.D. Renauld J.C. Biondi A. Constantinescu S.N. Foa R. Tartaglia M. J. Exp. Med. 2008; 205: 751-758Crossref PubMed Scopus (267) Google Scholar), and the V658F mutation (14Jeong E.G. Kim M.S. Nam H.K. Min C.K. Lee S. Chung Y.J. Yoo N.J. Lee S.H. Clin. Cancer Res. 2008; 14: 3716-3721Crossref PubMed Scopus (156) Google Scholar) have been identified in adult B and T cell-acute lymphoblastic leukemia (ALL). These mutations allow for constitutive JAK1 activation when overexpressed in JAK1-deficient cell lines (11Staerk J. Kallin A. Demoulin J.B. Vainchenker W. Constantinescu S.N. J. Biol. Chem. 2005; 280: 41893-41899Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 13Flex E. Petrangeli V. Stella L. Chiaretti S. Hornakova T. Knoops L. Ariola C. Fodale V. Clappier E. Paoloni F. Martinelli S. Fragale A. Sanchez M. Tavolaro S. Messina M. Cazzaniga G. Camera A. Pizzolo G. Tornesello A. Vignetti M. Battistini A. Cave H. Gelb B.D. Renauld J.C. Biondi A. Constantinescu S.N. Foa R. Tartaglia M. J. Exp. Med. 2008; 205: 751-758Crossref PubMed Scopus (267) Google Scholar), as was shown for JAK2 V617F in JAK2-deficient cell lines (2James C. Ugo V. Le Couedic J.P. Staerk J. Delhommeau F. Lacout C. Garcon L. Raslova H. Berger R. Bennaceur-Griscelli A. Villeval J.L. Constantinescu S.N. Casadevall N. Vainchenker W. Nature. 2005; 434: 1144-1148Crossref PubMed Scopus (2917) Google Scholar). Moreover, these A634D and R724H mutants induce the autonomous growth of the cytokine-dependent Ba/F3 cell line, whereas the A634D and R879C mutants protect the murine ALL cell line BW5147 from dexamethasone-induced apoptosis, indicating that they represent gain of function mutations. However, the potential role of JAK1 binding receptors, which are all heterodimeric, in the mechanism of mutant JAK1-induced constitutive signaling has never been studied. IL-9 is a multifunctional TH2 cytokine that was shown to be involved in T cell tumorigenesis in mouse and in humans (15Renauld J.C. van der Lugt N. Vink A. van Roon M. Godfraind C. Warnier G. Merz H. Feller A. Berns A. Van Snick J. Oncogene. 1994; 9: 1327-1332PubMed Google Scholar, 16Merz H. Houssiau F.A. Orscheschek K. Renauld J.C. Fliedner A. Herin M. Noel H. Kadin M. Mueller-Hermelink H.K. Van Snick J. Feller A.C. Blood. 1991; 78: 1311-1317Crossref PubMed Google Scholar, 17Fischer M. Bijman M. Molin D. Cormont F. Uyttenhove C. van Snick J. Sundstrom C. Enblad G. Nilsson G. Leukemia. 2003; 17: 2513-2516Crossref PubMed Scopus (54) Google Scholar, 18Kelleher K. Bean K. Clark S.C. Leung W.Y. Yang-Feng T.L. Chen J.W. Lin P.F. Luo W. Yang Y.C. Blood. 1991; 77: 1436-1441Crossref PubMed Google Scholar). Moreover, in vitro dysregulation of the IL-9 response is associated with autonomous cell growth and malignant transformation of lymphoid cells, leading to the constitutive activation of JAK-STAT pathway (19Uyttenhove C. Druez C. Renauld J.C. Herin M. Noel H. Van Snick J. J. Exp. Med. 1991; 173: 519-522Crossref PubMed Scopus (47) Google Scholar, 20Demoulin J.B. Uyttenhove C. Lejeune D. Mui A. Groner B. Renauld J.C. Cancer Res. 2000; 60: 3971-3977PubMed Google Scholar, 21Knoops L. Renauld J.C. Growth Factors. 2004; 22: 207-215Crossref PubMed Scopus (106) Google Scholar). Its activities are mediated via a heterodimeric receptor complex formed by the IL-9Rα chain (IL-9Rα), which associates with JAK1, and the IL-2Rγ chain, also called γc (common γ chain), which associates with JAK3. γc is in addition involved in IL-2, -4, -7, -15, and -21 signaling, a family of cytokines involved in lymphocyte development and/or activation. IL-9Rα is sufficient to confer high affinity cytokine binding, but formation of the heterodimeric complex with γc is needed for signal transduction (21Knoops L. Renauld J.C. Growth Factors. 2004; 22: 207-215Crossref PubMed Scopus (106) Google Scholar). Upon IL-9 binding, JAK1 and JAK3 are cross-activated, and IL-9Rα is phosphorylated on a single tyrosine (Tyr-116). This phosphorylated tyrosine is the only docking site for STAT1, -3, and -5, the STATS activated by IL-9 (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar). In this paper, in order to study the potential interactions between ALL-associated JAK1 mutants and the different components of IL-9 receptor complex, we co-expressed these different proteins in HEK293 cells, which lack IL-9Rα, γc, and JAK3. Our data show that JAK1 mutants alone fail to activate STAT transcriptional factors but that this process/activation is promoted by IL-9Rα homodimerization in the absence of γc and JAK3. Cells and Cell Culture-HEK293 human embryonic kidney cells and COS-7 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. These cells do not express endogenous IL-9Rα, γc, and JAK3. Human fibrosarcoma U4C cells (JAK1-deficient) and γ2A cells (JAK2-deficient) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Recombinant human IL-9 was produced in the baculovirus system in our laboratory and was purified as previously described (23Druez C. Coulie P. Uyttenhove C. Van Snick J. J. Immunol. 1990; 145: 2494-2499PubMed Google Scholar). Recombinant human IL-2 was provided by Chiron. Plasmid Constructions-V658F and A634D mouse JAK1 mutants were described previously (11Staerk J. Kallin A. Demoulin J.B. Vainchenker W. Constantinescu S.N. J. Biol. Chem. 2005; 280: 41893-41899Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 13Flex E. Petrangeli V. Stella L. Chiaretti S. Hornakova T. Knoops L. Ariola C. Fodale V. Clappier E. Paoloni F. Martinelli S. Fragale A. Sanchez M. Tavolaro S. Messina M. Cazzaniga G. Camera A. Pizzolo G. Tornesello A. Vignetti M. Battistini A. Cave H. Gelb B.D. Renauld J.C. Biondi A. Constantinescu S.N. Foa R. Tartaglia M. J. Exp. Med. 2008; 205: 751-758Crossref PubMed Scopus (267) Google Scholar). Double JAK1 mutants V658F/Y107A and A634D/Y107A were generated using QuikChange XL II site-directed mutagenesis kit (Stratagene, La Jolla, CA). The Y107A mutation was previously described to abolish the binding of JAK1 to cytokine receptors (24Haan C. Is'harc H. Hermanns H.M. Schmitz-Van De Leur H. Kerr I.M. Heinrich P.C. Grotzinger J. Behrmann I. J. Biol. Chem. 2001; 276: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Both wild-type and mutated murine JAK1 cDNAs were subcloned into the pMX-GFP biscistronic retroviral vector upstream of the internal ribosome entry site (IRES) (11Staerk J. Kallin A. Demoulin J.B. Vainchenker W. Constantinescu S.N. J. Biol. Chem. 2005; 280: 41893-41899Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 13Flex E. Petrangeli V. Stella L. Chiaretti S. Hornakova T. Knoops L. Ariola C. Fodale V. Clappier E. Paoloni F. Martinelli S. Fragale A. Sanchez M. Tavolaro S. Messina M. Cazzaniga G. Camera A. Pizzolo G. Tornesello A. Vignetti M. Battistini A. Cave H. Gelb B.D. Renauld J.C. Biondi A. Constantinescu S.N. Foa R. Tartaglia M. J. Exp. Med. 2008; 205: 751-758Crossref PubMed Scopus (267) Google Scholar). Human IL-9Rα and IL-9Rα Y116F were generated as previously described (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar) and were subcloned into the pREX-IRES-CD4 vector (25Liu 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) or into the pEFBos-puro vector (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar). The ability of murine JAK1 to transduce the signal from human IL-9Rα has been previously described (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar, 26Renauld J.C. Druez C. Kermouni A. Houssiau F. Uyttenhove C. Van Roost E. Van Snick J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5690-5694Crossref PubMed Scopus (119) Google Scholar). The IL-9Rα BOX1 mutant was generated by mutation of two proline residues to two serine residues in the BOX1 motif (PXPtoSXS) of wild-type IL-9Rα using the GeneEditor in vitro site-directed mutagenesis system (Promega, Madison, WI). Clones obtained were sequenced using DYEnamic ET Dye Terminator kit (Amersham Biosciences). The IL-9Rα IC34 construct was generated as previously described (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar). Myc and HA tags were introduced after the signal sequence cleavage site in the cDNA coding for human IL-9Rα as described (10Constantinescu S.N. Keren T. Socolovsky M. Nam H. Henis Y.I. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4379-4384Crossref PubMed Scopus (215) Google Scholar). The IL-2Rβ and IL-2Rγ (γc) were cloned to pREX-IRES-CD4 vector (25Liu 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). Dual Luciferase Assay-STAT1, STAT3, and STAT5 transcriptional activity was assessed by measurements of luciferase expression in HEK293, COS-7, U4C, and γ2A cells upon transient transfection of appropriate cDNA constructs and luciferase reporter vectors, namely pGL3-pap1-luc, pLHRE-luc, or pGRR5-luc. Another reporter plasmid, Renilla luciferase (pRLTk, Promega), was co-transfected as an internal transfection control. STAT5-mediated transcription was evaluated with the pLHRE-luc reporter gene constructs harboring tandem copies of the STAT5-inducible lactogenic hormone response element (LHRE) of the rat β-casein gene promoter, inserted upstream a luciferase gene (27Gerland K. Bataille-Simoneau N. Basle M. Fourcin M. Gascan H. Mercier L. Mol. Cell. Endocrinol. 2000; 168: 1-9Crossref PubMed Scopus (21) Google Scholar). STAT3-mediated transcription was evaluated with pGL3-pap1-luc plasmid containing the luciferase gene under the control of the STAT3-inducible rat Pap1 (pancreatitis-associated protein-1) promoter (28Peelman F. Iserentant H. De Smet A.S. Vandekerckhove J. Zabeau L. Tavernier J. J. Biol. Chem. 2006; 281: 15496-15504Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). STAT1, STAT3, and STAT5-mediated transcription was evaluated using the pGRR5-luc construct (provided by Dr. P. Brennan, Imperial Cancer Research Fund, London, UK) that contains five copies of the STAT-binding site of the FcγRI gene inserted upstream from a luciferase gene controlled by the thymidine kinase promoter. Transient transfection of HEK293, COS-7, U4C, and γ2A cells by Lipofectamine (Invitrogen) was previously described (29Dumoutier L. Van Roost E. Colau D. Renauld J.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10144-10149Crossref PubMed Scopus (324) Google Scholar). Briefly, cells were seeded in 24-well plates at 2 × 105 cells/well 1 day before transfection. Transfection was carried out according to the manufacturer's recommendations using 250 ng of the appropriate constructs, 500 ng of pGL3-pap1 or pLHRE reporter plasmids, 50 ng of pRLTk control plasmid, and empty vector to normalize total transfected DNA to 1.5 μg. 4 h after transfection cells were stimulated or left unstimulated for 20 h. 24 h after transfection cells were lysed by 150 μl of Passive Lysis Buffer 1× supplied by Promega. Luciferase assays were performed using the dual luciferase reporter assay kit (Promega). Western Blots-For Western blot analysis, 106 HEK293 cells were seeded in 6-well plates 1 day before transient transfection. In brief, HEK293 cells were transfected with 3.75 μg of different constructs depending on the experimental settings using a standard calcium phosphate transfection procedure (Promega). The day after transfection cells were collected and lysed in 250 μl of Laemmli buffer (Bio-Rad) using a 20-gauge syringe. Cellular lysates were boiled for 3 min before loading 20 μlon 12% precast Tris-glycine gels (Invitrogen) and electrophoretically transferred to nitrocellulose membranes (Hybond-C; Amersham Biosciences). After blocking the membranes in 5% milk/Tris-buffered saline-Tween (TBS-Tween), immunoblotting was performed by overnight incubations at 4 °C with primary antibodies in 0.1% TBS-Tween, using the dilution recommended by the manufacturer. After 3 × 5 min of washing in 0.1% TBS-Tween, the membranes were incubated with secondary anti-rabbit-horseradish peroxidase (HRP) or anti-mouse-HRP antibodies from Cell Signaling Technology (Beverly, MA), diluted 1/5000 in 5% phosphate-buffered saline-TBS-Tween. After 3 × 5 min of washing, a SuperSignal West Pico detection kit (Pierce) and Kodak Biomax Light Film were used for detection. The following phosphospecific antibodies were used: anti-pY1001/1002 JAK1 and anti-pY705 Stat3 (Cell Signaling Technology). Blots were re-probed with anti-JAK1, anti-STAT3 (Cell Signaling Technology), and anti-β-actin (Sigma) antibodies as control. Immunoprecipitation-For immunoprecipitation, 6 × 106 HEK293 cells were seeded in 100-mm plates 1 day before transfection. The day after, cells were co-transfected using a standard calcium phosphate transfection procedure (Promega) with the 7 μg of Myc-tagged IL-9Rα together with 7 μg of HA-tagged IL-9Rα or HA-tagged EpoR. The day after transfection, cells were collected and lysed in 1 ml of ice-cold modified radioimmune precipitation lysis buffer (1% Nonidet P-40, 0.25% deoxycholate, 0.1% SDS, 50 mm Tris (pH 8), 300 mm NaCl, 1 mm EDTA, 2 mm Na3VO4, 1 mm NaF, and complete protease inhibitor mixture tablet (Roche Applied Science) used according to the manufacturer's recommendations) (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar) using a 20-gauge syringe and kept 30 min on ice. Clarified lysates were immunoprecipitated with 2 μg of anti-c-Myc antibodies (Santa Cruz) and 20 μl of protein-G-agarose (Roche Applied Science). Immunoprecipitates were boiled, subjected to Tris-glycine gel electrophoresis, and electrotransferred onto nitrocellulose membranes followed by immunoblotting with anti-HA1.1 (Covance) and anti-c-Myc antibody (Santa Cruz). Washing and detection were performed as described above. FACS Analysis of IL-9Rα Cell Surface Expression-The cell surface expression of the different IL-9Rα constructs was verified by FACS on an aliquot of the cells used for the luciferase assay 1 day after transient transfection. FACS analysis was performed on a BD Biosciences FACSCalibur™ flow cytometer after staining cells with biotinylated anti-human IL-9Rα antibody (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar) followed with phycoerythrin-conjugated streptavidin (BD Biosciences) As a control, an aliquot of the transfected cells was stained only with phycoerythrin-conjugated streptavidin (BD Biosciences) antibody. Reverse Transcription and Quantitative PCR-Total RNA was extracted from the aliquot of the HEK293 cells used for the luciferase assay 1 day after transient transfection. Roughly 5 × 105 cells was used to extract RNA using the TriPure isolation reagent (Roche Applied Science) according to the manufacturer's instructions. Reverse transcription was performed on 1 μg of total RNA with an oligo-(dT) primer (Roche Applied Science) and Moloney murine leukemia virus reverse transcription (Invitrogen). Quantitative PCR reactions were performed using primer sets corresponding to murine JAK1 or human JAK1 with qPCR™ Mastermix for SYBR® Green I (Eurogentec). A series of dilutions of the respective PCR product subcloned into pCR2.1-TOPO vector (Invitrogen) was used a standard for quantification. The sequences of the species-specific JAK1 primers (final concentration, 300 nm) were: mJAK1, 5′-GGAGTGCAGTATCTCTCCTCTCT-3′ (forward) and 5′-CCATGCCCAGGCACTCATTTTCA-3′ (reverse); hJAK1, 5′-TCTTGGAATCCAGTGGAGGCATAAA-3′ (forward) and 5′-CACTCTTCCCGGATCTTGTTTTTCT-3′ (reverse). Samples were first heated for 2 min at 50 °C then 10 min at 95 °C. cDNA was amplified by 40 cycles of a two-step PCR program at 95 °C for 15 s and 60 °C for 1 min. Melting point analysis was carried out by heating the amplicon from 60 to 95 °C. ALL-associated JAK1 Mutants (V658F and A634D) Induce Constitutive STAT Activation in the Presence of IL-9Rα-ALL-associated JAK1 mutants induce constitutive signaling after ectopic expression in Ba/F3 or BW5147 hematopoietic cells (13Flex E. Petrangeli V. Stella L. Chiaretti S. Hornakova T. Knoops L. Ariola C. Fodale V. Clappier E. Paoloni F. Martinelli S. Fragale A. Sanchez M. Tavolaro S. Messina M. Cazzaniga G. Camera A. Pizzolo G. Tornesello A. Vignetti M. Battistini A. Cave H. Gelb B.D. Renauld J.C. Biondi A. Constantinescu S.N. Foa R. Tartaglia M. J. Exp. Med. 2008; 205: 751-758Crossref PubMed Scopus (267) Google Scholar). These cells presumably express many receptor complexes that could bind the mutant JAKs and promote constitutive signaling. To study the role of such receptors, we co-expressed components of the IL-9 receptor complex together with JAK1 mutants in HEK293 cells, which are deficient for γc, IL-9Rα, and JAK3. Expression of two ALL-associated JAK1 mutants alone (V658F and A634D) did not induce a significant increase in the level of STAT3 transcriptional activity as assessed by a luciferase assay (Fig. 1). However, co-expression of IL-9Rα with JAK1 mutants (but not WT JAK1) in the absence of JAK3 and γc increased STAT3 activation. Similar results were obtained for STAT5 activation using pLHRE-luc reporter and with the pGRR5-luc reporter, which also responds to STAT1 (data not shown). Stimulation with IL-9 had no effect on IL-9Rα-mediated activation of STAT3 by JAK1 mutants, which is in line with the fact that, in the absence of JAK3 and γc, IL-9 receptor signaling complex cannot support IL-9-induced signal transduction. To prove that mutant JAK1-induced STAT3 activation was dependent on the presence of a functional IL-9Rα, we co-expressed the defective IL-9Rα Y116F, in which the only STAT-recruiting tyrosine residue Tyr-116 was mutated to phenylalanine. We previously showed that this mutation abolishes STAT activation by IL-9 (22Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (168) Google Scholar). As expected, co-expression of the defective IL-9Rα Y116F did not promote mutant JAK1-induced constitutive STAT3 signaling (Fig. 1), indicating that recruitment of STATs by the IL-9Rα phosphotyrosine was necessary for mutant JAK1-induced constitutive signaling. These results contrast with a previous report showing that expression of JAK1 V658F in JAK1-deficient U4C fibrosarcoma cells allows for constitutive STAT3 activation (11Staerk J. Kallin A. Demoulin J.B. Vainchenker W. Constantinescu S.N. J. Biol. Chem. 2005; 280: 41893-41899Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). To address the hypothesis that this apparent discrepancy was due to the lack of expression of endogenous JAK1 in U4C cells, we measured STAT3 activation in JAK1-deficient U4C or JAK2-deficient γ2A fibrosarcoma cells transfected with wild-type or V658F JAK1. In U4C cells expression of JAK1 V658F alone allowed for constitutive STAT3 activation (8-fold increased luciferase production, as compared with wild-type JAK1). However, co-expression of IL-9Rα chain induced a further 3-fold increase in STAT3 activation (supplemental Fig. 1A). By contrast, in γ2A cells expression of JAK1 V658F only marginally increased (1.6-fold) STAT3 transcriptional activity unless the IL-9Rα chain was co-expressed (supplemental Fig. 1B). To confirm that JAK1 V658F constitutive activity in U4C cells results from the absence of expression of wild-type JAK1, we cotransfected both isoforms into U4C cells. Co-expression of increasing amounts of wild-type JAK1 together with the JAK1 V658F significantly decreased STAT3 activation (supplemental Fig. 1C). Y107A Mutation in the FERM Domain of JAK1 Mutants Abolishes Constitutive STAT3 Activation-To prove that the JAK1 mutants need to associate to IL-9Rα to constitutively activate STAT3, we mutated tyrosine residue Tyr-107 to alanine in the FERM domain of JAK1 V658F and A634D. Several residues conserved within the FERM domain of JAKs are crucial for cytokine receptor binding (30Saharinen P. Takaluoma K. Silvennoinen O. Mol. Cell. Biol. 2000; 20: 3387-3395Crossref PubMed Scopus (284) Google Scholar), and the Y107A mutation within the FERM domain of JAK1 has been shown to abolish JAK1 binding to the gp130 receptor chain (24Haan C. Is'harc H. Hermanns H.M. Schmitz-Van De Leur H. Kerr I.M. Heinrich P.C. Grotzinger J. Behrmann I. J. Biol. Chem. 2001; 276: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). As shown in Fig. 2 for two different cell lines (HEK293 and COS-7 cells), the Y107A mutation also abolished the capacity of JAK1 mutants to activate STAT3 in the presence of IL-9Rα. These results indicate that an intact FERM domain of JA

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