The Role of Disulfide-linked Dimerization in Interleukin-3 Receptor Signaling and Biological Activity
2000; Elsevier BV; Volume: 275; Issue: 7 Linguagem: Inglês
10.1074/jbc.275.7.5124
ISSN1083-351X
AutoresFei Le, Frank C. Stomski, Joanna M. Woodcock, Angel F. López, Thomas J. Gonda,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoCysteine residues 86 and 91 of the β subunit of the human interleukin (hIL)-3 receptor (hβc) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 → Ala (Mc4) and Cys-91 → Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3Rα and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type hβc exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type hβc, despite failing to undergo IL-3-stimulated hβc tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and hβc tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements. Cysteine residues 86 and 91 of the β subunit of the human interleukin (hIL)-3 receptor (hβc) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 → Ala (Mc4) and Cys-91 → Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3Rα and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type hβc exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type hβc, despite failing to undergo IL-3-stimulated hβc tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and hβc tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements. granulocyte-macrophage colony-stimulating factor interleukin 3 and interleukin 5 GM-CSF receptor α chain human common β subunit of the GM-CSF, IL-3 and IL-5 receptors human βc wild type phosphatidylinositide-3′-OH Dulbecco's modified Eagle's medium fetal calf serum phosphate-buffered saline fluorescence-activated cell sorter mitogen-activated protein electrophoretic mobility shift analysis signal transducer and activator of transcription The receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF),1 IL-3 and IL-5, are members of the hematopoietin receptor superfamily, often termed the cytokine receptor family. The high affinity GM-CSF·IL-3·IL-5 receptor complexes are composed of specific α chains that bind GM-CSF·IL-3·IL-5 with low affinity (1.Gearing D.P. King J.A. Gough N.M. Nicola N.A. EMBO J. 1989; 8: 3667-3676Crossref PubMed Scopus (523) Google Scholar, 2.Kitamura T. Sato N. Arai K. Miyajima A. Cell. 1991; 66: 1165-1174Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 3.Tavernier J. Devos R. Cornelis S. Tuypens T. Van der Heyden J. Fiers W. Plaetinck G. Cell. 1991; 66: 1175-1184Abstract Full Text PDF PubMed Scopus (494) Google Scholar) and a common β chain (βc) that converts the binding to high affinity by forming a heterodimer with the α subunit (4.Hayashida K. Kitamura T. Gorman D.M. Arai K. Yokota T. Miyajima A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9655-9659Crossref PubMed Scopus (519) Google Scholar). The oligomerization of α and β subunits mediated by ligand binding is thought to initiate signal transduction. It is generally accepted that whereas the cytoplasmic domains of both α and β subunits are required for receptor activation, in some cases dimerization of the cytoplasmic domain of the β subunit alone is sufficient. This was demonstrated by a study in which a chimeric receptor consisting of the extracellular domain of the erythropoietin receptor and the intracellular domain of βc or βIL-3was constructed. Erythropoietin induced proliferation signals in Ba/F3 cells through the chimeric receptors, indicating that the homodimerization of the β subunit is sufficient for receptor activation (5.Sakamaki K. Wang H.M. Miyajima I. Kitamura T. Todokoro K. Harada N. Miyajima A. J. Biol. Chem. 1993; 268: 15833-15839Abstract Full Text PDF PubMed Google Scholar). Likewise, a mutant GM-CSF receptor α chain (GM-CSFRα), in which the cytoplasmic domain was replaced with that of the βc, formed a high affinity receptor with the normal βc and transduced proliferative signals, again indicating the importance of the dimerization of the β cytoplasmic domain (6.Muto A. Watanabe S. Miyajima A. Yokota T. Arai K. Biochem. Biophys. Res. Commun. 1995; 208: 368-375Crossref PubMed Scopus (29) Google Scholar). In contrast, the βc mutant in which the cytoplasmic domain was substituted with that of GM-CSFRα forms a high affinity GM-CSFR with the GM-CSFRα but was unable to induce a proliferation signal, indicating that the dimerization of the GM-CSFRα cytoplasmic domain was not sufficient for signaling (6.Muto A. Watanabe S. Miyajima A. Yokota T. Arai K. Biochem. Biophys. Res. Commun. 1995; 208: 368-375Crossref PubMed Scopus (29) Google Scholar). Despite the lack of an intrinsic tyrosine kinase in the receptors, GM-CSF, IL-3, and IL-5 induce rapid tyrosine phosphorylation of various cellular proteins, including the β subunits themselves. It is now known that cytoplasmic JAK family tyrosine kinases associate with the β subunit via conserved membrane proximal regions known as “box-1” and “box-2” (7.Watanabe S. Itoh T. Arai K. J. Allergy Clin. Immunol. 1996; 98: 183-191Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 8.Quelle F.W. Sato N. Witthuhn B.A. Inhorn R.C. Eder M. Miyajima A. Griffin J.D. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4335-4341Crossref PubMed Google Scholar). The activated JAK kinases phosphorylate tyrosine residues of many signaling proteins, among which are the latent cytoplasmic transcription factors known as signal transducer and activator of transcription (STAT). The activation of JAKs and/or STATs is important for many if not all activities of cytokine receptors (7.Watanabe S. Itoh T. Arai K. J. Allergy Clin. Immunol. 1996; 98: 183-191Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 9.Sakai I. Kraft A.S. J. Biol. Chem. 1997; 272: 12350-12358Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 10.Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1265) Google Scholar). The Ras-Raf-MAP kinase pathway is another major signaling pathway activated in response to GM-CSF and IL-3 (11.Sato N. Sakamaki K. Terada N. Arai K. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (330) Google Scholar, 12.Okuda K. Sanghera J.S. Pelech S.L. Kanakura Y. Hallek M. Griffin J.D. Druker B.J. Blood. 1992; 79: 2880-2887Crossref PubMed Google Scholar, 13.Welham M.J. Duronio V. Sanghera J.S. Pelech S.L. Schrader J.W. J. Immunol. 1992; 149: 1683-1693PubMed Google Scholar). It has been demonstrated that a membrane-distal region of βc is required for activation of the Ras-Raf-MAP kinase pathway (11.Sato N. Sakamaki K. Terada N. Arai K. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (330) Google Scholar). Circumstantial evidence implies that JAK2 is also required for activation of this pathway by IL-3 and GM-CSF. Deletion of the box-1 region in βc (which is believed to be the site of JAK2 association with βc) or expression of a dominant negative form of JAK2 prevented phosphorylation of molecules involved in Ras activation (Shc and SHP2) (7.Watanabe S. Itoh T. Arai K. J. Allergy Clin. Immunol. 1996; 98: 183-191Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Moreover, these same manipulations also blocked IL-3/GM-CSF-induced activation of the c-fos promoter (7.Watanabe S. Itoh T. Arai K. J. Allergy Clin. Immunol. 1996; 98: 183-191Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 14.Watanabe S. Kubota H. Sakamoto K.M. Arai K. Blood. 1997; 89: 1197-1206Crossref PubMed Google Scholar), which is also blocked by dominant negative Ras (14.Watanabe S. Kubota H. Sakamoto K.M. Arai K. Blood. 1997; 89: 1197-1206Crossref PubMed Google Scholar). This pathway may also be important for cell survival (15.Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5031) Google Scholar, 16.Kinoshita T. Yokota T. Arai K. Miyajima A. EMBO J. 1995; 14: 266-275Crossref PubMed Scopus (306) Google Scholar, 17.Kinoshita T. Shirouzu M. Kamiya A. Hashimoto K. Yokoyama S. Miyajima A. Oncogene. 1997; 15: 619-627Crossref PubMed Scopus (103) Google Scholar) (see also “Discussion”). Signaling pathways that promote cell survival also include the phosphatidylinositide-3′-OH kinase (PI3K)-AKT pathway. The AKT kinase is a general mediator of cytokine-induced survival and has been shown to suppress the apoptotic death of a number of cell types induced by a variety of stimuli, including growth factor withdrawal, cell cycle discordance, loss of cell adhesion, and DNA damage (18.Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 19.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4935) Google Scholar, 20.Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar, 21.Zha J. Harada H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2253) Google Scholar). Thus, a signaling pathway has been defined in which cytokine receptor activation leads to the sequential activation of PI3K and AKT, which then promotes cell survival and blocks apoptosis. Our previous study (22.Stomski F.C. Woodcock J.M. Zacharakis B. Bagley C.J. Sun Q. Lopez A.F. J. Biol. Chem. 1998; 273: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) showed that Cys-86 and Cys-91 but not other Cys residues of human βc (hβc) participated in disulfide-linked receptor subunit heterodimerization. We also showed that this linkage is essential for receptor phosphorylation because alanine substitutions of residues 86 (termed Mc4) and 91 (Mc5), but not 100 (Mc7), abolished not only IL-3-induced disulfide-linked IL-3 receptor subunit dimerization but also tyrosine phosphorylation of hβc, without affecting IL-3 binding. To investigate whether or not disulfide-mediated cross-linking of IL-3 receptor subunits and phosphorylation of hβc are essential for receptor signaling, we have now examined the effects of these cysteine mutations on biological and biochemical activities of the IL-3 receptor when expressed in the murine T cell line CTL-EN. We have found that hβc mutants Mc4 and Mc5 barely induce cell proliferation compared with wild-type (WT) or Mc7. Interestingly, a subpopulation of cells expressing each of the former mutants could be selected for growth in IL-3, which, despite the absence of detectable hβc tyrosine phosphorylation, proliferated at similar rates to cells expressing WT hβc. Our data also show that Mc4 and Mc5 (but not Mc7) impaired IL-3-facilitated protection against apoptosis and failed to induce IL-3-stimulated JAK2, STAT, and AKT activation in unselected cells. However, Mc4 and Mc5 had a less severe effect on IL-3-induced Erk1/2 MAP kinase activation in the unselected cells. In contrast, in the IL-3-selected cells, the cysteine mutations had no effects on IL-3-induced activation of any of the signaling molecules that we examined. The ecotropic ψ2 retrovirus packaging cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mml-glutamine, and antibiotics. The IL-2-dependent mouse T cell line, CTL-EN (a derivative of CTLL-2, described in Ref. 23.Jenkins B.J. Le F. Gonda T.J. J. Biol. Chem. 1999; 274: 8669-8677Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), was maintained in CTLL medium (DMEM supplemented with 10% FCS, 2 mml-glutamine, antibiotics, 50 μm β-mercaptoethanol, 5% conditioned medium from MLA cells, and 100 units/ml bacterially synthesized mouse IL-2. Cysteine mutants Mc4, Mc5, Mc7, or WT hβc cDNAs (22.Stomski F.C. Woodcock J.M. Zacharakis B. Bagley C.J. Sun Q. Lopez A.F. J. Biol. Chem. 1998; 273: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) were inserted between theBamHI and HpaI restriction sites of the pRufHygro retroviral vector. The pRufHygro retroviral vector was constructed by replacing the MC1Neo cassette of pRufNeo (24.Rayner J.R. Gonda T.J. Mol. Cell. Biol. 1994; 14: 880-887Crossref PubMed Scopus (70) Google Scholar) with a phosphoglycerate kinase/hygromycin resistance cassette from pPGKHygro (25.Mortensen R.M. Zubiaur M. Neer E.J. Seidman J.G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7036-7040Crossref PubMed Scopus (73) Google Scholar). The ecotropic packaging cell line ψ2 was transfected by a standard calcium phosphate transfection procedure as described (26.Jenkins B.J. D'Andrea R.J. Gonda T.J. EMBO J. 1995; 14: 4276-4287Crossref PubMed Scopus (75) Google Scholar) with 10 μg of retroviral plasmid containing WT or cysteine mutant hβc per 10-cm dish. After overnight incubation the cells were shocked with 2.5 ml of 15% glycerol in DMEM for 4 min followed by a further 24 h incubation and then selected in hygromycin (Roche Molecular Biochemicals) at 200 μg/ml. The selected cells were sorted for expression of hβc, and collected in DMEM. These sorted ψ2 cells expressing WT or the cysteine mutant hβc were then used to infect CTL-EN cells previously infected with a pRUFNeo retrovirus vector (24.Rayner J.R. Gonda T.J. Mol. Cell. Biol. 1994; 14: 880-887Crossref PubMed Scopus (70) Google Scholar) encoding the α chain of human IL-3 receptor (hIL-3Rα), using procedures described previously (26.Jenkins B.J. D'Andrea R.J. Gonda T.J. EMBO J. 1995; 14: 4276-4287Crossref PubMed Scopus (75) Google Scholar). The infected CTL-EN cells were selected in hygromycin at 600 μg/ml, and expression of WT or cysteine mutant hβc on the surface of the hygromycin-resistant cells was examined by flow cytometry. Cells expressing WT or cysteine mutant hβc were collected by cell sorting on a FACStarPLUS flow cytometer (Coulter, Hialeah, FL). Briefly, cells were washed and resuspended in cold DMEM supplemented with 5% FCS. Cells were incubated with the anti-hβc monoclonal antibody 8B8 (27.Sun Q. Jones K. McClure B. Cambareri B. Zacharakis B. Iversen P.O. Stomski F.C. Woodcock J.M. Bagley C.J. D'Andrea R. Lopez A.F. Blood. 1999; 94: 1943-1951Crossref PubMed Google Scholar) for 20 min on ice, washed, and subsequently incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Silenus, Hawthorn, Victoria, Australia) for 20 min on ice. After washing and resuspension in medium, the cells were sorted, and the positive cells collected in CTLL medium. The procedures used for radiolabeling of human IL-3 with 125I and performing saturation-binding assays have been described previously (28.Stomski F. Sun Q. Bagley C. Woodcock J. Goodall G. Andrews R. Berndt M. Lopez A. Mol. Cell. Biol. 1996; 16: 3035-3046Crossref PubMed Google Scholar, 29.Lopez A.F. Vadas M.A. Woodcock J.M. Milton S.E. Lewis A. Elliott M.J. Gillis D. Ireland R. Olwell E. Park L.S. J. Biol. Chem. 1991; 266: 24741-24747Abstract Full Text PDF PubMed Google Scholar). Infected CTL-EN cells expressing hIL-3Rα and WT or cysteine mutant hβc were washed three times with PBS, and triplicate samples of 5 × 103 cells were cultured in a 96-well microtiter plate with or without IL-3 for 72 h. Cell proliferation was measured by the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). The data were normalized with respect to proliferation of the same cells cultured in 100 units/ml of IL-2. Apoptotic cells were detected using the Annexin-V-Fluos Staining kit (Roche Molecular Biochemicals) as per manufacturer's instructions. Briefly, cells (5 × 105) were washed with PBS and centrifuged at 2,000 ×g for 5 min. The cell pellets were resuspended in 100 μl of labeling solution (1:50 diluted Annexin-V-Fluos labeling reagent in 10 mm HEPES buffer (pH 7.4) and 1 μg/ml of propidium iodide) and then added to 0.4 ml of incubation buffer (10 mg of HEPES (pH 7.4), 140 mm NaCl, 5 mm CaCl2). The cells were analyzed on a flow cytometer using a 488-nm excitation and a 515-nm band pass filter. CTL-EN cells coexpressing hIL-3Rα and WT or cysteine mutant hβc (4 × 107) were cultured overnight in the absence of cytokines. Cells were washed with cold PBS followed by stimulation at 37 °C for 5–15 min as indicated with varying concentrations of IL-3 (0–10 ng/ml) and then lysed on ice in lysis buffer (50 mm HEPES (pH 7.5), 150 mmNaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.1% sodium deoxycholate 2 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 1 mm EDTA, 1 mmEGTA, 2 mg/ml iodoacetamide, 50 mm sodium fluoride, 10 mm sodium pyrophosphate, 0.2 mg/ml trypsin inhibitor (Roche Molecular Biochemicals) and CompleteTM protease inhibitor (Roche Molecular Biochemicals)) for 15 min. Insoluble materials were removed by centrifugation, and cell lysates were incubated with rabbit anti-JAK2 antibody 2T. J. Blake and T. J. Gonda, unpublished data. or anti-hβc monoclonal antibody 8E4 (30.Woodcock J.M. McClure B.J. Stomski F.C. Elliott M.J. Bagley C.J. Lopez A.F. Blood. 1997; 90: 3005-3017Crossref PubMed Google Scholar) for 2 h at 4 °C. Immune complexes were precipitated with 75 μl of protein A-Sepharose (Amersham Pharmacia Biotech) for 90 min at 4 °C, washed twice with lysis buffer, and boiled in 1× reducing SDS loading buffer. Immunoprecipitates, or cell lysates prepared as described above, were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to ProtranR nitrocellulose transfer membranes (Schleicher & Schuell). Membranes were incubated with a blocking solution (3% bovine serum albumin in TBS-T (50 mm Tris-HCl (pH 7.4), 135 mm NaCl and 0.1% Tween 20)) at room temperature for 1 h and then incubated overnight with antibody in the same solution at 4 °C. The antibodies used were anti-JAK2 (as above); anti-phosphotyrosine (4G10; Upstate Biotechnology, Inc., Lake Placid, NY); anti-hβc (1C1; Ref. 28.Stomski F. Sun Q. Bagley C. Woodcock J. Goodall G. Andrews R. Berndt M. Lopez A. Mol. Cell. Biol. 1996; 16: 3035-3046Crossref PubMed Google Scholar); anti-Erk1/2 (Zymed Laboratories Inc., San Francisco, CA); anti-phospho-Erk1/2 (Promega, Madison, WI); and anti-phospho-AKT and anti-AKT (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then washed three times in TBS-T solution and incubated with anti-mouse or anti-rabbit secondary antibodies (as appropriate) coupled with horseradish peroxidase (Pierce). Membranes were washed in TBS-T three times and subjected to enhanced chemiluminescence detection as per the manufacturer's instructions (Pierce). Before reprobing, membranes were stripped in 50 mm Tris (pH 7.4), 2% SDS, 100 mm β-mercaptoethanol at 55 °C for 10 mim, washed three times in TBS-T, and blocked in TBS-T containing 3% bovine serum albumin. Nuclear extracts from CTL-EN cells expressing hIL-3Rα and WT or cysteine mutant hβc were prepared as described by Jenkinset al. (31.Jenkins B.J. Blake T.J. Gonda T.J. Blood. 1998; 92: 1989-2002Crossref PubMed Google Scholar). These were mixed with radiolabeled double-stranded oligonucleotides corresponding to the prolactin-responsive element in the bovine β-casein promoter, and EMSA was performed essentially as described by Barry et al.(32.Barry S.C. Moretti P.A.B. Korpeleinen E. D'Andrea R. Lopez A.F. Vadas M.A. Goodall G.J. Blood. 1997; 89: 842-852Crossref PubMed Google Scholar). To introduce WT or cysteine mutant hβc into CTL-EN cells expressing hIL-3Rα, the cells were infected using ψ2 cells producing the corresponding RUFHygro retroviruses. The infected CTL-EN cells were selected in hygromycin in IL-2-containing medium, and cell surface expression of hβc was assessed by FACS analysis. Fig.1 shows that WT and cysteine mutant βc were expressed at comparable levels. Previous studies demonstrated that the cysteine mutations had no effect on the ligand binding in HEK293T cells (22.Stomski F.C. Woodcock J.M. Zacharakis B. Bagley C.J. Sun Q. Lopez A.F. J. Biol. Chem. 1998; 273: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To confirm this finding in CTL-EN cells, we performed binding assays with 125I-IL-3. The results of Fig.2 show that all receptors, either WT or the cysteine mutants, had similar high affinity binding and were present in similar numbers, in agreement with previous results in HEK293T cells (22.Stomski F.C. Woodcock J.M. Zacharakis B. Bagley C.J. Sun Q. Lopez A.F. J. Biol. Chem. 1998; 273: 1192-1199Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and the FACS analyses of Fig. 1.Figure 2Scatchard transformation of saturation binding studies performed on CTL-EN cells coexpressing hIL-3Rα and WT or cysteine mutant hβc as indicated. Binding assays were performed with 125I-labeled IL-3 over a concentration range of 10 pm to 10 nm. The data were analyzed using the LIGAND program, and the lines indicate the high affinity binding component for WT or Cys mutant hβc as indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The growth of CTL-EN cells expressing WT hβc or the cysteine mutants cultured in hIL-3 (10 ng/ml) was monitored over a 3-day period. The results of Fig.3 A show that cells expressing Mc4 and Mc5 mutants did not exhibit detectable growth, whereas cells expressing the Mc7 mutant grew similarly to those expressing WT hβc. Interestingly, it was found that although most cells expressing Mc4 and Mc5 mutants could not grow or survive in IL-3, a small subset (we estimate ≤1%) of these cells grew out over a period of several weeks from IL-3-containing cultures. To distinguish these subsets, which were maintained in medium containing hIL-3, we will term these cells “IL-3-selected cells,” whereas the bulk population of (mutant) hβc-expressing cells, maintained in IL-2, will hereafter be termed “unselected cells.” It was found that the IL-3-selected cells proliferated at similar rates to the cells expressing WT hβc (Fig. 3 B) and did not grow or survive in medium lacking cytokines (data not shown). Moreover, polymerase chain reaction amplification and sequencing of hβc from genomic DNA of the IL-3-selected cells confirmed the presence of the cysteine mutations,i.e. these cells were not genetic revertants. To examine whether the Mc4 and Mc5 mutations have an effect on cell survival, unselected CTL-EN cells expressing WT or cysteine mutant hβc were cultured in different concentrations of IL-3 for 48 h, following which the proportions of apoptotic cells were determined by annexin-V staining and FACS analysis. The results in Fig.4 A show that IL-3 at either low or high concentrations effectively protected the cells expressing WT or Mc7 from apoptosis in a dose-dependent manner. The cysteine mutations Mc4 and Mc5 significantly impaired, but importantly, did not completely abrogate the ability of IL-3 to protect the cells against apoptosis. This result was confirmed in the time course experiment shown in Fig. 4 B. The impairment by Mc4 and Mc5 of hIL-3-facilitated protection against apoptosis was evident at 48 h and was even more marked by 72 h. We previously showed that cysteine mutations Mc4 and Mc5 abrogated disulfide-linked IL-3 receptor dimerization and abolished tyrosine phosphorylation of hβc in response to IL-3 when expressed in HEK293T cells. To test whether this was also the case in the IL-3-selected CTL-EN cells, we performed Western blot analysis to detect tyrosine phosphorylation of hβc. The results of Fig. 5 show that the cysteine mutations Mc4 and Mc5 abolished IL-3-induced receptor tyrosine phosphorylation, which was, however, readily detected in cells expressing WT or Mc7. The activity of JAK2 is believed to be necessary for all the biological functions of IL-3 and GM-CSF (7.Watanabe S. Itoh T. Arai K. J. Allergy Clin. Immunol. 1996; 98: 183-191Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We therefore examined the effects of the Mc4 and Mc5 mutations on JAK2 and STAT activation. In the case of unselected cells, it was found that phosphorylation of JAK2 was observed in cells expressing WT or Mc7 after stimulating the cells with 10 ng/ml hIL-3 but not in cells expressing the Mc4 or Mc5 mutants (Fig.6 A). Although no JAK2 activation was detected when the cells expressing WT or Mc7 were stimulated with a low concentration of hIL-3 (1 ng/ml), we found that IL-3 at a concentration of 1 ng/ml was able to support proliferation of cells expressing WT or Mc7 (data not shown; see also below). One class of effectors of JAK2 are the STAT transcription factors, which are phosphorylated and activated by JAK2. We therefore examined nuclear extracts from unselected cells expressing WT or mutant hβc for the presence of STAT DNA binding activity by performing EMSAs. After stimulation of the cells with 10 ng/ml IL-3, extracts from cells expressing WT or Mc7 contained a protein complex that specifically bound to a β-casein oligonucleotide probe containing a DNA-binding site for STATs 1, 3, and 5 (Fig. 6 B). However, no STAT activation was detected in cells expressing the Mc4 or Mc5 mutants (Fig. 6 B) after stimulation by IL-3. Weak STAT activation was also detected in cells expressing WT or Mc7 stimulated with 1 ng/ml IL-3. The most likely explanation for this, considering that no JAK2 activation was detected with this concentration of IL-3, is simply that EMSA detection of STAT activity is more sensitive than detection of JAK2 tyrosine phosphorylation. Because, as mentioned above, a subpopulation of cells expressing the cysteine mutants Mc4 or Mc5 could be selected for growth in IL-3, we investigated whether or not JAK2 and STATs were activated in IL-3-selected cells expressing these mutants. Fig. 6, C andD, shows that JAK2 and STAT activation was detected following stimulation with IL-3 at 10 ng/ml not only in cells expressing WT or Mc7 but, in contrast to the unselected cells, also in cells expressing Mc4 or Mc5. As for Mc7 and WT, weak activation of STAT but not JAK2 was also detected when a low concentration of IL-3 was used. One of the major signaling pathways activated in response to cytokines is the Ras-Raf-MAP kinase pathway (11.Sato N. Sakamaki K. Terada N. Arai K. Miyajima A. EMBO J. 1993; 12: 4181-4189Crossref PubMed Scopus (330) Google Scholar, 12.Okuda K. Sanghera J.S. Pelech S.L. Kanakura Y. Hallek M. Griffin J.D. Druker B.J. Blood. 1992; 79: 2880-2887Crossref PubMed Google Scholar). To examine whether the cysteine mutations have an effect on hβc-mediated activation of Erk1/2 MAP kinases, Western blot analyses of cell lysates from both unselected and IL-3-selected cells with an antibody specific for activated, i.e. phosphorylated, Erk1/2 MAP kinases were conducted. Fig. 7 A shows that phosphorylation of Erk1/2 MAP kinases was detected in unselected cells expressing WT or Mc7 mutant hβc when the cells were stimulated with 1 or 10 ng/ml hIL-3. Moreover, Erk1/2 phosphorylation was also clearly detected when cells expressing Mc4 or Mc5 were stimulated with 10 ng/ml hIL-3, although little or no phosphorylation above background was detected with 1 ng/ml hIL-3. In the case of IL-3-selected cells, activation of Erk1/2 MAP kinases was readily detectable in cells expressing WT and all of the cysteine mutant forms of hβc, regardless of whether the cells were stimulated with a low or high concentration of hIL-3 (Fig. 7 B). The activation of the PI3K and its downstream effector AKT has been shown to promote cell survival and suppress apoptosis (18.Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 19.Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4935) Google Scholar, 20.Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (965) Google Scholar). We therefore examined the levels of activated AKT kinase in both unselected and IL-3-selected CTL-EN cells expressing WT or mutant hβc. Western blot analysis of cell lysates from unselected cells using an antibody specific for phosphorylated AKT showed that AKT was activated in cells expressing WT or Mc7 but
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