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• W1971925643 abstract "We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rβ chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rβ chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2. We have shown recently that interleukin (IL)-2 activates the mitogen-activated protein (MAP) kinase family members p38 (HOG1/stress-activated protein kinase II) and p54 (c-Jun N-terminal kinase/stress-activated protein kinase I). Furthermore, the p38 MAP kinase inhibitor SB203580 inhibited IL-2-driven T cell proliferation, suggesting that p38 MAP kinase might be involved in mediating proliferative signals. In this study, using transfected BA/F3 cell lines, it is shown that both the acidic domain and the membrane-proximal serine-rich region of the IL-2Rβ chain are required for p38 and p54 MAP kinase activation and that, as for p42/44 MAP kinase, this activation requires the Tyr338 residue of the acidic domain, the binding site for Shc. It is well established that the acidic domain of the IL-2Rβ chain is dispensable for IL-2-driven proliferation, and thus our observations suggest that neither p38 nor p54 MAP kinase activation is required for IL-2-driven proliferation of BA/F3 cells. In addition, the tetravalent guanylhydrazone inhibitor of proinflammatory cytokine production, CNI-1493, can block the activation of p54 and p38 MAP kinases by IL-2 but has no effect on IL-2-driven proliferation of BA/F3 cells, activated primary T cells, or a cytotoxic T cell line. Furthermore, our observations provide evidence for the existence of an additional, unknown target of the p38 MAP kinase inhibitor SB203580, the activation of which is essential for mitogenic signaling by IL-2. T cell clonal proliferation upon antigenic challenge plays an essential role in mounting an effective immune response. Interleukin (IL) 1The abbreviations used are: IL, interleukin; IL-2R, interleukin-2 receptor; MAP, mitogen-activated protein; MAPK, MAP kinase; GST-ATF2, glutathione S-transferase-activating transcription factor 2 1The abbreviations used are: IL, interleukin; IL-2R, interleukin-2 receptor; MAP, mitogen-activated protein; MAPK, MAP kinase; GST-ATF2, glutathione S-transferase-activating transcription factor 2-2 is a potent T cell mitogen that plays a key role in driving this process (1Smith K.A. Science. 1988; 240: 1169-1176Crossref PubMed Scopus (1896) Google Scholar). The intracellular signal transduction pathways activated by IL-2 and the relative roles of these pathways in mediating the mitogenic signal have been extensively studied but have yet to be fully elucidated. IL-2 exerts its cellular effects through binding to specific cell surface receptors. The high affinity IL-2 receptor (IL-2R) is a heterotrimeric complex consisting of α-, β-, and γc-subunits, the γc subunit being shared with the receptors for the other T cell mitogens, IL-4, IL-7, IL-9, and IL-15 (2Theze J. Alzari P.M. Bertoglio J. Immunol. Today. 1996; 17: 481-486Abstract Full Text PDF PubMed Scopus (215) Google Scholar). The α-subunit is responsible for conferring high affinity cytokine binding, while the β- and γc-subunits recruit cytoplasmic molecules, thereby transducing the proliferative signal. The β-subunit has the larger cytoplasmic tail, consisting of subdomains previously identified as the membrane-proximal serine-rich, acidic, and distal proline-rich regions (3Hatakeyama M. Tsudo M. Minamoto S. Kono T. Doi T. Miyata T. Miyasaka M. Taniguchi T. Science. 1989; 244: 551-556Crossref PubMed Scopus (562) Google Scholar, 4Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar). The IL-2Rβ chain contains six cytoplasmic tyrosine residues: Tyr338, Tyr355, Tyr358, and Tyr361, which lie in the acidic region, and Tyr392 and Tyr510, which lie within the proline-rich region. The presence of at least one of the tyrosines Tyr338, Tyr392, and Tyr510 appears to be sufficient to allow IL-2-driven proliferation (5Friedmann M.C. Migone T.S. Russell S.M. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2077-2082Crossref PubMed Scopus (168) Google Scholar, 6Goldsmith M.A. Lai S.Y. Xu W. Amaral M.C. Kuczek E.S. Parent L.J. Mills G.B. Tarr K.L. Longmore G.D. Greene W.C. J. Biol. Chem. 1995; 270: 21729-21737Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Studies in transfected BA/F3 cells have shown that loss of the IL-2Rβ acidic region has no effect on proliferation (4Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar) or the expression of Myc and Bcl-2, factors essential for proliferation and cell survival (7Miyazaki T. Liu Z.J. Kawahara A. Minami Y. Yamada K. Tsujimoto Y. Barsoumian E.L. Perlmutter R.M. Taniguchi T. Cell. 1995; 81: 223-231Abstract Full Text PDF PubMed Scopus (348) Google Scholar). The tyrosine kinases p56Lck, p72Syk, Jak1, and Jak3 are recruited to the IL-2R and activated upon IL-2 binding (8Minami Y. Kono T. Yamada K. Kobayashi N. Kawahara A. Perlmutter R.M. Taniguchi T. EMBO J. 1993; 12: 759-768Crossref PubMed Scopus (123) Google Scholar, 9Minami Y. Nakagawa Y. Kawahara A. Miyazaki T. Sada K. Yamamura H. Taniguchi T. Immunity. 1995; 2: 89-100Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 10Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z.J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (500) Google Scholar, 11Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalstieg F.C. Ihle J.N. O'Shea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (586) Google Scholar). However, of these only the activation of Jak3, which associates with the IL-2Rγc chain, appears to be an absolute requirement for IL-2-driven proliferation (8Minami Y. Kono T. Yamada K. Kobayashi N. Kawahara A. Perlmutter R.M. Taniguchi T. EMBO J. 1993; 12: 759-768Crossref PubMed Scopus (123) Google Scholar, 10Miyazaki T. Kawahara A. Fujii H. Nakagawa Y. Minami Y. Liu Z.J. Oishi I. Silvennoinen O. Witthuhn B.A. Ihle J.N. Taniguchi T. Science. 1994; 266: 1045-1047Crossref PubMed Scopus (500) Google Scholar, 12Hatakeyama M. Kono T. Kobayashi N. Kawahara A. Levin S.D. Perlmutter R.M. Taniguchi T. Science. 1991; 252: 1523-1528Crossref PubMed Scopus (508) Google Scholar, 13Higuchi M. Asao H. Tanaka N. Oda K. Takeshita T. Nakamura M. Van Snick J. Sugamura K. Eur. J. Immunol. 1996; 26: 1322-1327Crossref PubMed Scopus (41) Google Scholar, 14Nosaka T. van Deursen J.M. Tripp R.A. Thierfelder W.E. Witthuhn B.A. McMickle A.P. Doherty P.C. Grosveld G.C. Ihle J.N. Science. 1995; 270: 800-802Crossref PubMed Scopus (571) Google Scholar, 15Thomis D.C. Gurniak C.B. Tivol E. Sharpe A.H. Berg L.J. Science. 1995; 270: 794-797Crossref PubMed Scopus (473) Google Scholar, 16Turner M. Mee P.J. Costello P.S. Williams O. Price A.A. Duddy L.P. Furlong M.T. Geahlen R.L. Tybulewicz V.L. Nature. 1995; 378: 298-302Crossref PubMed Scopus (644) Google Scholar). It has been suggested that Jak3 mediates IL-2 proliferative signaling through activation of another tyrosine kinase, Pyk2 (17Miyazaki T. Takaoka A. Nogueira L. Dikic I. Fujii H. Tsujino S. Mitani Y. Maeda M. Schlessinger J. Taniguchi T. Genes Dev. 1998; 12: 770-775Crossref PubMed Scopus (67) Google Scholar). Signal transducer and activator of transcription (STAT) 5 is also activated as a result of Jak activation, but its role in proliferation is unclear (6Goldsmith M.A. Lai S.Y. Xu W. Amaral M.C. Kuczek E.S. Parent L.J. Mills G.B. Tarr K.L. Longmore G.D. Greene W.C. J. Biol. Chem. 1995; 270: 21729-21737Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 18Fujii H. Nakagawa Y. Schindler U. Kawahara A. Mori H. Gouilleux F. Groner B. Ihle J.N. Minami Y. Miyazaki T. Taniguchi T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5482-5486Crossref PubMed Scopus (193) Google Scholar, 19Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1071) Google Scholar). IL-2 activates two other major signaling pathways: the phosphatidylinositol 3-kinase pathway and the p42/44 MAP kinase pathway. The activation of phosphatidylinositol 3-kinase (20Augustine J.A. Sutor S.L. Abraham R.T. Mol. Cell. Biol. 1991; 11: 4431-4440Crossref PubMed Scopus (98) Google Scholar, 21Merida I. Diez E. Gaulton G.N. J. Immunol. 1991; 147: 2202-2207PubMed Google Scholar) and the subsequent activation of protein kinase B/Akt (22Reif K. Burgering B.M. Cantrell D.A. J. Biol. Chem. 1997; 272: 14426-14433Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and p70S6 kinase (23Calvo V. Crews C.M. Vik T.A. Bierer B.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7571-7575Crossref PubMed Scopus (166) Google Scholar, 24Kuo C.J. Chung J. Fiorentino D.F. Flanagan W.M. Blenis J. Crabtree G.R. Nature. 1992; 358: 70-73Crossref PubMed Scopus (563) Google Scholar), has been associated with the serine-rich region of the IL-2Rβ chain (25Merida I. Williamson P. Kuziel W.A. Greene W.C. Gaulton G.N. J. Biol. Chem. 1993; 268: 6765-6770Abstract Full Text PDF PubMed Google Scholar), and the activation of these factors results in the phosphorylation of Rb, suggesting a key role for these kinases in proliferation (26Brennan P. Babbage J.W. Burgering B.M. Groner B. Reif K. Cantrell D.A. Immunity. 1997; 7: 679-689Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). IL-2-induced activation of the p42/44 MAP kinase (extracellular signal-regulated kinase) pathway proceeds through the activation of p21ras, Raf, and MAP kinase kinase 1/2 (27Satoh T. Nakafuku M. Miyajima A. Kaziro Y. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3314-3318Crossref PubMed Scopus (294) Google Scholar, 28Turner B. Rapp U. App H. Greene M. Dobashi K. Reed J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1227-1231Crossref PubMed Scopus (143) Google Scholar, 29Zmuidzinas A. Mamon H.J. Roberts T.M. Smith K.A. Mol. Cell. Biol. 1991; 11: 2794-2803Crossref PubMed Scopus (76) Google Scholar) and requires both the acidic and the serine-rich regions of the IL-2Rβ chain (30Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar). Tyr338, within the acidic region of the IL-2Rβ chain, is responsible for the recruitment of Shc (31Evans G.A. Goldsmith M.A. Johnston J.A. Xu W. Weiler S.R. Erwin R. Howard O.M.Z. Abraham R.T. O'Shea J.J. Greene W.C. Farrar W.L. J. Biol. Chem. 1995; 270: 28858-28863Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and the subsequent assembly of the p21ras-activating complex along with Grb2 and SOS (31Evans G.A. Goldsmith M.A. Johnston J.A. Xu W. Weiler S.R. Erwin R. Howard O.M.Z. Abraham R.T. O'Shea J.J. Greene W.C. Farrar W.L. J. Biol. Chem. 1995; 270: 28858-28863Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 32Ravichandran K.S. Burakoff S.J. J. Biol. Chem. 1994; 269: 1599-1602Abstract Full Text PDF PubMed Google Scholar, 33Ravichandran K.S. Lorenz U. Shoelson S.E. Burakoff S.J. Mol. Cell. Biol. 1995; 15: 593-600Crossref PubMed Google Scholar). However, loss of p42/44 MAP kinase signaling does not appear to prevent IL-2-driven cell cycle progression (30Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar, 31Evans G.A. Goldsmith M.A. Johnston J.A. Xu W. Weiler S.R. Erwin R. Howard O.M.Z. Abraham R.T. O'Shea J.J. Greene W.C. Farrar W.L. J. Biol. Chem. 1995; 270: 28858-28863Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 34Crawley J.B. Willcocks J. Foxwell B.M. Eur. J. Immunol. 1996; 26: 2717-2723Crossref PubMed Scopus (42) Google Scholar). Two further MAP kinase family members, p54 (stress-activated protein kinase I/c-Jun N-terminal kinase) and p38 (stress-activated protein kinase II/HOG1), were typically thought to be activated by cellular stress and proinflammatory stimuli (35Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (774) Google Scholar, 36Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2407) Google Scholar, 37Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1708) Google Scholar, 38Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2411) Google Scholar, 39Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fidher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3128) Google Scholar). However, we have recently demonstrated that p38 MAP kinase and p54 MAP kinase are activated by IL-2 in T cells (40Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). We observed that an inhibitor of p38 MAP kinase function, SB203580 (39Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fidher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3128) Google Scholar), was able to inhibit T cell proliferation induced by IL-2, suggesting that p38 MAP kinase activation may be required for cell cycle progression. We have now examined further the role of these kinases in IL-2 proliferative signaling by mapping the regions of the IL-2Rβ chain required for p38 and p54 MAP kinase activation. We observe that both the serine-rich and the acidic regions are required for the activation of p54 and p38 MAP kinases by IL-2 in BA/F3 cells and that the activation of these kinases, like that of p42/44 MAP kinase, is dependent on the presence of Tyr338 in the IL-2Rβ chain and proceeds through the recruitment of Shc. Since the acidic region of the IL-2Rβ chain is dispensable for BA/F3 cell proliferation, our observations indicate that the activation of p38 and p54 MAP kinase is not required for IL-2-driven cell cycle progression. This conclusion is supported by studies using the tetravalent guanylhydrazone CNI-1493 (41Bianchi M. Ulrich P. Bloom O. Meistrell III, M. Zimmerman G.A. Schmidtmayerova H. Bukrinsky M. Donnelley T. Bucala R. Sherry B. Manogue K.R. Tortolani A.J. Cerami A. Tracey K.J. Mol. Med. 1995; 1: 254-266Crossref PubMed Google Scholar), which inhibited IL-2-induced p38 and p54 MAP kinase activation but had no effect on IL-2-driven proliferation of either BA/F3 cells or T cells. Our data therefore suggest that the previously observed inhibition of IL-2-induced proliferation by the inhibitor SB203580 is likely to be due to its action on a target(s) other than p38 MAP kinase, which is required for cell cycle progression. The murine IL-2-dependent T cell line CT6 (kindly provided by Genentech, S. San Francisco, CA) was maintained in glutamine-supplemented RPMI 1640 (BioWhittaker, Verviers, Belgium) with 5% fetal bovine serum (Sigma, Poole, Dorset, UK), 1 unit/ml penicillin/streptomycin (BioWhittaker), and 50 μm2-mercaptoethanol (ICN, Thame, Oxon, UK) with the addition of 5 ng/ml recombinant human IL-2 (generously provided by Dr. P Lomedico, Roche Inc., Nutley, NJ). The stable transformant clones F7, S25, and A15 previously described (4Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar) were initially derived from the BAF-B03 clone of the IL-3-dependent BA/F3 cell line and were maintained in RPMI 1640 supplemented with 5% fetal bovine serum, 1% WEHI-3B conditioned medium (as a source of IL-3) and 0.2 μg/ml G418 (Calbiochem-Novabiochem Ltd., Nottingham, UK). The parental BA/F3 cell line was also transfected with versions of human IL-2Rβ chain bearing the mutations Δ355, Y338F, Δ355:Y338F, and Δ325-Shc described previously (42Lord J.D. McIntosh B.C. Greenberg P.D. Nelson B.H. J. Immunol. 1998; 161: 4627-4633PubMed Google Scholar) under control of the human β-actin promoter in an expression vector also encoding neomycin phosphotransferase. Stably transfected subclones were derived through selection at limiting dilution in the presence of G418 and were maintained in RPMI 1640 supplemented with 5% fetal bovine serum, 1% WEHI-3B conditioned medium, and 0.8 μg/ml G418. Human peripheral T cells were isolated as described previously (43Page T.H. Willcocks J.L. Taylor-Fishwick D.A. Foxwell B.M. J. Immunol. 1993; 151: 4753-4763PubMed Google Scholar). All cells were washed in cytokine-free medium and deprived of cytokine supplements for 16 h prior to experimental use. The tetravalent guanylhydrazone CNI-1493 was synthesized as described previously (41Bianchi M. Ulrich P. Bloom O. Meistrell III, M. Zimmerman G.A. Schmidtmayerova H. Bukrinsky M. Donnelley T. Bucala R. Sherry B. Manogue K.R. Tortolani A.J. Cerami A. Tracey K.J. Mol. Med. 1995; 1: 254-266Crossref PubMed Google Scholar). SB203580 was from Calbiochem-Novabiochem Ltd., and PD098059 was from New England Biolabs (Hitchin, Herts, UK). Rabbit antisera to p54 (SAK10) and p38 (SAK7) MAP kinases were provided by Prof. J Saklatvala (Kennedy Institute of Rheumatology, London) (44Finch A. Holland P. Cooper J. Saklatvala J. Kracht M. FEBS Lett. 1997; 418: 144-148Crossref PubMed Scopus (38) Google Scholar). Antibody to p42 MAP kinase/extracellular signal-regulated kinase 2 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to phosphorylated p42/44, p38, and p54 MAP kinases were from New England Biolabs. Antibodies to Shc and Grb2 and the anti-phosphotyrosine antibody PY-20 were from Transduction Labs (Lexington, KY). 4G10 anti-phosphotyrosine antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY). The cDNA for histidine-tagged MAPK-activated protein kinase 2 was the generous gift of Prof. M. Gaestel (Max Delbruck Center for Molecular Medicine, Berlin, Germany). Cellular DNA synthesis was measured by [3H]thymidine incorporation as described previously (45Willcocks J.L. Hales A. Page T.H. Foxwell B.M. Eur. J. Immunol. 1993; 23: 716-720Crossref PubMed Scopus (9) Google Scholar). p38 MAP kinase and c-Jun N-terminal kinase/stress-activated protein kinase were immunoprecipitated from cleared cell lysates as described previously (40Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Shc immunoprecipitations were performed as described previously (33Ravichandran K.S. Lorenz U. Shoelson S.E. Burakoff S.J. Mol. Cell. Biol. 1995; 15: 593-600Crossref PubMed Google Scholar). In vitro kinase assays for p54 and p38 MAP kinase activity were performed on precipitated immune complexes. p54 MAP kinase assays were performed as described previously using glutathione S-transferase-activating transcription factor 2 (GST-ATF2) as a substrate (40Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In vitro kinase assays for p38 MAP kinase were performed using His6-MAPK-activated protein kinase 2 as a substrate. Immunoprecipitates were incubated with 30 μl of kinase assay buffer (25 mm HEPES, pH 7.5, 25 mm MgCl2, 25 mm β-glycerophosphate) containing 50 μg/ml recombinant His6-MAPK-activated protein kinase 2, 30 μm ATP, and 0.5 μCi of [γ-32P]ATP (Amersham International, Little Chalfont, Buckinghamshire, UK) for 25 min at room temperature. Reactions were terminated by the addition of gel sample buffer and boiling for 5 min. All substrates were separated by SDS-polyacrylamide gel electrophoresis. Gels were dried, and phosphorylated substrates were visualized using a Fuji FLA-2000 Imager (Raytek Scientific Ltd, Sheffield, UK) and by autoradiography at −70 °C. p42/44 MAP kinase (extracellular signal-regulated kinase 1/2) and phosphotyrosine Western blotting was performed as described previously (34Crawley J.B. Willcocks J. Foxwell B.M. Eur. J. Immunol. 1996; 26: 2717-2723Crossref PubMed Scopus (42) Google Scholar). Western blotting for Shc, Grb2, and phosphorylated forms of p42/44, p38, and p54 MAP kinases was performed according to the antibody manufacturer's instructions. IL-3-dependent BA/F3 cells normally express both the α- and the γc-chains, but not the β-chain, of the IL-2R. When transfected with the IL-2Rβ subunit, they become responsive to IL-2 (4Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar). We have used BA/F3 cell lines expressing either the wild type IL-2Rβ chain (F7) or mutant forms of the IL-2Rβ chain, lacking either the serine-rich region (S25) or the acidic region (A15) (Fig. 1A), to examine the role of the IL-2Rβ chain subdomains in the activation of p38 and p54 MAP kinase. The serine-rich and the acidic domains of the β-chain are essential for p42/44 MAP kinase activation (30Minami Y. Oishi I. Liu Z.J. Nakagawa S. Miyazaki T. Taniguchi T. J. Immunol. 1994; 152: 5680-5690PubMed Google Scholar); this is confirmed in Fig. 2A by detection of the phosphorylated kinase by Western blotting using a phosphospecific p42/44 MAP kinase antibody. Using immunokinase assays (for p38 and p54; Fig. 2B) or Western blotting for the phosphorylated, activated form (p38 only, Fig. 2A) to measure p38 and p54 MAP kinase activation, we have shown that these kinases are stimulated by IL-2 in F7 cells (Fig. 2, A and B). However, IL-2 was unable to activate either p38 or p54 MAP kinase in the absence of the serine-rich (S25) or acidic (A15) regions, indicating that both regions are required for kinase activation. IL-3 activated p42, p38, and p54 MAP kinases in all three cell lines and is included as a positive control. Kinetic experiments examining p38 and p54 MAP kinase activation over a 2-h period have established that the activation of these kinases by IL-2 is not simply retarded in the S25 and A15 cell lines (data not shown).Figure 2The serine-rich and acidic domains of the IL-2Rβ chain are required for activation of p38 and p54 MAP kinases by IL-2. F7, S25, and A15 cells were stimulated with IL-2 (20 ng/ml) or IL-3 (5% WEHI supernatant) or were left untreated (Un). MAP kinase activity was assayed as described under “Experimental Procedures” as follows. A, Western blotting. The top panels show activation of p42/44 MAP kinase determined by Western blotting using a phosphospecific antibody, and the second set of panels show the same blots reprobed for total p42/44 MAP kinase to demonstrate equal protein loading. The third set of panels show p38 MAP kinase phosphospecific Western blots, and the bottom panels show the same blots reprobed for total p38 MAP kinase. p54 MAP kinase phosphorylation could not be detected using a phosphospecific anti-stress-activated protein kinase/c-Jun N-terminal kinase antibody in these cells. B, kinase assays. Immunoprecipitated p38 MAP kinase activity was determined using His6-MAPK-activated protein kinase 2 as a substrate. Immunoprecipitated p54 MAP kinase activity was determined using GST-ATF2 as a substrate.View Large Image Figure ViewerDownload Hi-res image Download (PPT) This similarity in the β-chain subdomain requirements led us to examine whether, as for the activation of p42 MAP kinase, Shc is involved in the activation of p38 and p54 MAP kinases. A second panel of BA/F3 clones expressing mutant forms of the IL-2Rβ chain was used (Fig. 1B). In the cell line Δ355, a truncation of the β-chain at amino acid 355 removes the proline-rich region and part of the acidic region to eliminate all of the cytoplasmic tyrosine residues except the Tyr338 residue required for Shc binding. IL-2 is nonetheless able to activate both p38 and p54 MAP kinases in this cell line (Fig. 3A). However, in another BA/F3 cell line expressing a full-length IL-2Rβ chain with a point mutation of Tyr338 to phenylalanine (Y338F), IL-2 fails to activate p38 and p54 MAP kinases, as determined by kinase assay and phosphospecific Western blotting (Fig. 3A). Curiously, there is still a very slight residual activation of all three MAP kinases by IL-2 when the Y338F mutation is combined with the Δ355 truncation in the BA/F3 cell line Δ355:Y338F (Fig. 3, A and B). An additional BA/F3 line, Δ325-Shc, bears a version of the IL-2Rβ chain in which the entire acidic and proline-rich regions, including all cytoplasmic tyrosines of IL-2Rβ, are replaced with a covalently tethered Shc molecule to specifically reconstitute Shc-mediated signals, as described previously (42Lord J.D. McIntosh B.C. Greenberg P.D. Nelson B.H. J. Immunol. 1998; 161: 4627-4633PubMed Google Scholar). IL-2 promoted p38 and p54 MAP kinase activation in these Δ325-Shc cells, suggesting that Shc may mediate the activation of p38 and p54 MAP kinases by IL-2 (Fig. 3A). However, the presence of the receptor fusion protein appears to compromise the ability of IL-3 to activate p38 and p54 MAP kinases. This may indicate that the activation of these kinases by IL-3 also occurs through Shc if the IL-2Rβ-Shc fusion protein is somehow inhibiting a functional interaction between endogenous Shc and the IL-3 receptor. Fig. 3B shows p42 MAP kinase Western blots, confirming the role of the IL-2Rβ chain tyrosine Tyr338 and Shc in p42 MAP kinase phosphorylation and activation. The observation that the IL-2Rβ chain acidic region is dispensable for IL-2-driven proliferation but that both the acidic and the serine-rich regions must be present for p38 and p54 MAP kinase activation in BA/F3 cells indicates that neither kinase is required for proliferation. This conflicts with our previous suggestion (40Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), based on studies using the p38 inhibitor SB203580, that p38 MAP kinase activation is required for IL-2-driven T cell proliferation. We therefore examined whether proliferation of the IL-2-responsive BA/F3 cell lines was also sensitive to this drug. Of the cell lines used, only two did not proliferate in response to IL-2: the S25 cell line, lacking the entire serine-rich region (4Hatakeyama M. Mori H. Doi T. Taniguchi T. Cell. 1989; 59: 837-845Abstract Full Text PDF PubMed Scopus (302) Google Scholar), and the Δ355:Y338F cell line, in which the IL-2Rβ chain lacks all three of the tyrosine residues of which at least one is required for proliferation (5Friedmann M.C. Migone T.S. Russell S.M. Leonard W.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2077-2082Crossref PubMed Scopus (168) Google Scholar, 42Lord J.D. McIntosh B.C. Greenberg P.D. Nelson B.H. J. Immunol. 1998; 161: 4627-4633PubMed Google Scholar). Treatment of the cell line F7, A15, or Y338F with SB203580 (0.1–30 μm) resulted in the inhibition of IL-2-driven DNA synthesis with an IC50 of ∼2–6 μm (Fig. 4). Proliferation of the other cell lines was similarly inhibited in each case (data not shown). The IC50 of SB203580 on IL-2-driven proliferation observed in these cell lines was comparable with that observed previously in T cells (40Crawley J.B. Rawlinson L. Lali F.V. Page T.H. Saklatvala J. Foxwell B.M.J. J. Biol. Chem. 1997; 272: 15023-15027Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). However, since no activation of MAP kinases could be detected in either the A15 or the Y338F cells, in these cell lines the effect of SB203580 on proliferation could not be due to the inhibition of p38 MAP kinase and must instead reflect an effect of SB203580 on an unknown target. It is possible that the discrepancy between the data presented here and our previous studies is due to cell type differences in IL-2 signaling between the CT6 T cell line used previously and the pro-B, BA/F3 cell lines used here. In order to address this possibility, we have made use of a second synthetic inhibitor. CNI-1493, a tetravalent guanylhydrazone compound, has bee" @default.
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• W1971925643 title "Role of Interleukin (IL)-2 Receptor β-Chain Subdomains and Shc in p38 Mitogen-activated Protein (MAP) Kinase and p54 MAP Kinase (Stress-activated Protein Kinase/c-Jun N-terminal Kinase) Activation" @default.
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