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• W2058556985 abstract "p38/CSBP, a subgroup member of mitogen-activated protein kinase (MAPK) superfamily molecules, is known to be activated by proinflammatory cytokines and environmental stresses. We report here that p38 is specifically activated by signals that lead to interleukin-2 (IL-2) production in T lymphocytes. A p38 activator MKK6 was also markedly activated by the same stimulation. Pretreatment of cells with SB203580, a specific inhibitor of p38, as well as expression of a dominant-negative mutant of MKK6, suppressed the transcriptional activation of the IL-2 promoter. We also demonstrated that MKK7, a recently described MAPK kinase family member, plays a major role in the activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in T lymphocytes. Moreover, a dominant-negative mutant of MKK7 abrogated the transcriptional activation of the distal nuclear factor of activated T cells response element in the IL-2 promoter. Cyclosporin A, a potent immunosuppressant, inhibited activation of both p38 and SAPK/JNK pathways but not the MAPK/extracellular signal-regulated kinase (ERK) pathway. Our results indicate that both MKK6 to p38 and MKK7 to SAPK/JNK signaling pathways are activated in a cyclosporin A-sensitive manner and contribute to IL-2 gene expression in T lymphocytes. p38/CSBP, a subgroup member of mitogen-activated protein kinase (MAPK) superfamily molecules, is known to be activated by proinflammatory cytokines and environmental stresses. We report here that p38 is specifically activated by signals that lead to interleukin-2 (IL-2) production in T lymphocytes. A p38 activator MKK6 was also markedly activated by the same stimulation. Pretreatment of cells with SB203580, a specific inhibitor of p38, as well as expression of a dominant-negative mutant of MKK6, suppressed the transcriptional activation of the IL-2 promoter. We also demonstrated that MKK7, a recently described MAPK kinase family member, plays a major role in the activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in T lymphocytes. Moreover, a dominant-negative mutant of MKK7 abrogated the transcriptional activation of the distal nuclear factor of activated T cells response element in the IL-2 promoter. Cyclosporin A, a potent immunosuppressant, inhibited activation of both p38 and SAPK/JNK pathways but not the MAPK/extracellular signal-regulated kinase (ERK) pathway. Our results indicate that both MKK6 to p38 and MKK7 to SAPK/JNK signaling pathways are activated in a cyclosporin A-sensitive manner and contribute to IL-2 gene expression in T lymphocytes. The mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MAPKK, MAPK kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; CSBP, CSAIDTMbinding protein; CsA, cyclosporin A; IL-2, interleukin-2; mAb, monoclonal antibody; TCR, T cell receptor; NFAT, nuclear factor of activated T cell; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; Luc, luciferase.1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MAPKK, MAPK kinase; SAPK, stress-activated protein kinase; JNK, c-Jun NH2-terminal kinase; CSBP, CSAIDTMbinding protein; CsA, cyclosporin A; IL-2, interleukin-2; mAb, monoclonal antibody; TCR, T cell receptor; NFAT, nuclear factor of activated T cell; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; Luc, luciferase. pathway is a conserved eukaryotic signalling cascade that mediates the effects of extracellular stimuli on a wide array of biological processes. This pathway consists of three protein kinases, MAPK, MAPK kinase (MAPKK), and MAPKK kinase; MAPKK kinase phosphorylates and activates MAPKK, which in turn phosphorylates and activates MAPK (1Cobb M.H. Boulton T.G. Robbins D.J. Cell Regul. 1991; 2: 965-978Crossref PubMed Scopus (428) Google Scholar, 2Sturgill T.W. Wu J. Biochim. Biophys. Acta. 1991; 1092: 350-357Crossref PubMed Scopus (329) Google Scholar, 3Nishida E. Gotoh Y. Trends Biochem. Sci. 1993; 18: 128-131Abstract Full Text PDF PubMed Scopus (960) Google Scholar, 4Blumer K.J. Johnson G.L. Trends Biochem. Sci. 1994; 19: 236-240Abstract Full Text PDF PubMed Scopus (421) Google Scholar, 5Avruch J. Zhang X.F. Kyriakis J.M. Trends Biochem. Sci. 1994; 19: 279-283Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 6Marshall C.J. Cell. 1995; 80: 179-185Abstract Full Text PDF PubMed Scopus (4230) Google Scholar). Three distinct subgroups of the MAPK superfamily have been identified in mammalian cells (7Davis R.J. Trends Biochem. Sci. 1994; 19: 470-473Abstract Full Text PDF PubMed Scopus (917) Google Scholar). These are classical MAPK (also termed extracellular signal-regulated protein kinase, ERK), stress-activated protein kinase (SAPK; also termed c-Jun NH2-terminal kinase (JNK)), and p38 (also termed MPK2, RK, CSBP, or HOG1). Studies on the MAPK/ERK and SAPK/JNK subgroups have led to insights into their physiological functions (8Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2253) Google Scholar). For example, previous studies have implicated a role of MAPK/ERK and SAPK/JNK in activation of T lymphocytes leading to interleukin-2 (IL-2) production (9Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar, 10Li W. Whaley C.D. Mondino A. Mueller D.L. Science. 1996; 271: 1272-1276Crossref PubMed Scopus (407) Google Scholar, 11Fields P.E. Gajewski T.F. Fitch F.W. Science. 1996; 271: 1276-1278Crossref PubMed Scopus (357) Google Scholar, 12DeSilva D.R. Feese W.S. Tancula E.J. Scherle P.A. J. Exp. Med. 1996; 183: 2017-2023Crossref PubMed Scopus (112) Google Scholar). On the other hand, the contribution of p38 to cellular responses to extracellular stimuli has been poorly defined (13Lee J.C. Young P.R. J. Leukocyte Biol. 1996; 59: 152-157Crossref PubMed Scopus (373) Google Scholar). It has been shown that p38 is activated by proinflammatory cytokines (e.g. IL-1 and tumor necrosis factor-α) and cellular stresses (e.g. osmotic shock and ultraviolet light) (14Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2410) Google Scholar, 15Lee 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. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3133) Google Scholar, 16Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1500) Google Scholar, 17Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (775) Google Scholar, 18Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2040) Google Scholar). p38 has been shown to phosphorylatein vitro MAPKAP kinase-2 (16Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1500) Google Scholar, 17Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (775) Google Scholar) and transcription factors such as ATF-2, Elk-1, and CHOP (18Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2040) Google Scholar, 19Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1409) Google Scholar, 20Wang X-Z. Ron D. Science. 1996; 272: 1347-1349Crossref PubMed Scopus (742) Google Scholar). However, its role in T lymphocyte activation has been unclear. IL-2 production, which is a critical step for T lymphocyte activation and proliferation, requires two coordinate signals; the primary stimulus is the interaction of antigen with the T cell receptor (TCR)·CD3 complex (21Weiss A. Littman D.R. Cell. 1994; 76: 263-274Abstract Full Text PDF PubMed Scopus (1952) Google Scholar), and the secondary stimulus, called costimulation, is generated by the interaction of CD28 auxiliary receptors on T cells with its ligand on antigen-presenting cells (22Schwartz R.H. Cell. 1992; 71: 1065-1068Abstract Full Text PDF PubMed Scopus (1233) Google Scholar). Various studies indicate that activation signals for IL-2 production can be bypassed by phorbol esters (such as TPA) and Ca2+ionophore (such as A23187) (23Koyasu S. Suzuki G. Asano Y. Osawa H. Diamantstein T. Yahara I. J. Biol. Chem. 1987; 262: 4689-4695Abstract Full Text PDF PubMed Google Scholar, 24Crabtree G.R. Science. 1989; 243: 355-361Crossref PubMed Scopus (914) Google Scholar, 25Rao A. Crit. Rev. Immunol. 1991; 10: 495-520PubMed Google Scholar). Here we report that either treatment with TPA and Ca2+ionophore or simultaneous activation of the TCR and CD28 results in synergistic activation of MKK6 (26Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1143) Google Scholar, 27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 28Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) to the p38 pathway in T lymphocytes in a cyclosporin A (29Schreiber S.L. Cell. 1992; 70: 365-368Abstract Full Text PDF PubMed Scopus (300) Google Scholar)-sensitive manner. We also demonstrate that newly identified MKK7 (30Tounier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (340) Google Scholar, 31Lu X. Nemoto S. Lin A. J. Biol. Chem. 1997; 272: 24751-24754Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 32Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 33Lawler S. Cuenda A. Goedert M. Cohen P. FEBS Lett. 1997; 414: 153-158Crossref PubMed Scopus (46) Google Scholar, 34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar) plays a major role as an upstream activator of SAPK/JNK in T cells. Several lines of evidence for involvement of p38 and SAPK/JNK in IL-2 gene expression are presented. Jurkat cells were grown in RPMI 1640 with 10% fetal calf serum, 50 units/ml penicillin, 200 μg/ml kanamycin, and 5 × 10−5mβ-mercaptoethanol. Monoclonal antibodies (mAbs) to human CD3 (NU-T3) and human CD28 (KOLT-2) were purchased from Nichirei. PD98059, SB203580, and CsA were purchased from New England Biolabs, Calbiochem,and Wako, respectively. mAb NU-T3 (5 μg) was immobilized on a 35-mm dish by incubation for 12 h at 4 °C. mAb KOLT-2 (5 μg) was preincubated with 5 μg of rabbit anti-mouse antibodies for 12 h at 4 °C to induce cross-linking and then was added to Jurkat cells (2 × 106), or cells were stimulated with various reagents such as TPA (Sigma) and Ca2+ ionophore A23187 (Wako). After a 24-h treatment of Jurkat cells with various stimuli, IL-2 production was measured using ELISA (Endogen) according to the manufacturer's instructions. Following stimulation, cells were washed once with ice-cold Hepes-buffered saline and then lysed in a buffer consisting of 20 mm Tris-HCl (pH 7.5), 2 mmEGTA, 25 mm β-glycerophosphate, 1% Triton X-100, 2 mm dithiothreitol, 1 mm vanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin A, and 1% aprotinin and were centrifuged at 16,000 ×g for 30 min at 2 °C. The supernatant (approximately 0.1 mg/ml cellular protein) was subjected to in-gel kinase assays or immune complex kinase assays. Immunoprecipitation and immune complex kinase assays were performed as described previously (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 35Matsuda S. Kawasaki H. Moriguchi T. Gotoh Y. Nishida E. J. Biol. Chem. 1995; 270: 12781-12786Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Anti-p38 antibody was purchased from Santa Cruz. To assay for p38 activity, p38 was immunoprecipitated from cell lysates in the presence of 0.1% SDS and assayed for kinase activity using ATF-2 as a substrate. Anti-SEK1 (36Moriguchi T. Kawasaki H. Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1995; 270: 12969-12972Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), anti-MKK6 (28Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 29Schreiber S.L. Cell. 1992; 70: 365-368Abstract Full Text PDF PubMed Scopus (300) Google Scholar), and anti-MKK7 (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar) antibodies were previously produced and characterized. pME-MKK6, an expression plasmid for hemagglutinin (HA)-tagged MKK6 under a control of the SRα promoter, has previously been characterized (28Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). A dominant-negative MKK6 (MKK6(AA)) was constructed by replacing Ser-207 and Thr-211 with alanine residues. The mutation was made by site-directed mutagenesis (Quick ChangeTM, Stratagene) and was confirmed by DNA sequencing. An HA-tagged kinase-negative MKK7 (MKK7(KL)), constructed by replacing ATP-binding lysine with leucine residue, has previously been reported (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). A luciferase reporter plasmid carrying the human IL-2 enhancer/promoter element (encompassing nucleotides −326 to +47), IL-2-Luc, was kindly provided by Dr. M. Iwashima (Mitsubishi Chemical Corporation Yokohama Research Center, Japan). Another luciferase reporter plasmid with three tandem repeats of the NFAT site, NFAT-Luc (37Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar), was kindly provided by Dr. G. Crabtree (Stanford University, Stanford, CA). Transient transfection of Jurkat cells (5 × 106) was performed using SuperFectTM (Qiagen) with luciferase reporter plasmids (either 1 μg of IL-2-Luc or 0.5 μg of NFAT-Luc) along with various amounts of expression plasmids. The total amount of DNA transfected was maintained at 5 μg with pcDNA 3.1+ (Invitrogen). After 24 h of incubation at 37 °C, cells were stimulated for 8 h with indicated reagents and then lysed for luciferase assay. Luciferase in cell lysates was measured in duplicate by a luminometer (LB9507, Berthold), using a luciferase assay system (Promega). The protein concentration was determined with a protein assay kit (Bio-Rad) and used for normalization of the luciferase assays. Expression of HA-tagged proteins was analyzed by immunoblotting with the 12CA5 mouse mAb against the HA epitope. In a human T cell line, Jurkat, the activity of p38 markedly increased in response to signals generated by TPA and Ca2+ ionophore, whereas each stimulus alone resulted in little or no activation (Fig.1 A, shaded bar). Similar to its effect on IL-2 production (37Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar), CsA inhibited the activation of p38 (Fig. 1 A, shaded bar). SAPK/JNK was also activated in a similar manner as reported previously (Fig. 1 A, black bar (9Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar)). In contrast, incubation with TPA was sufficient for efficient activation of MAPK/ERK, which was neither enhanced by Ca2+ ionophore nor inhibited by CsA (Fig. 1 A, white bar). Interestingly, treatment with TPA and Ca2+ionophore did not induce the activation of p38 in non-lymphoid cells such as an epidermoid carcinoma cell line, KB (data not shown). We next examined whether MKK6, an upstream activator of p38 (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 28Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 29Schreiber S.L. Cell. 1992; 70: 365-368Abstract Full Text PDF PubMed Scopus (300) Google Scholar, 38Cuenda A. Alonso G. Morrice N. Jones M. Meier R. Cohen P. Nebreda A.R. EMBO J. 1996; 15: 4156-4164Crossref PubMed Scopus (115) Google Scholar), is activated in parallel with p38 activation during T cell activation. Whereas TPA alone had little effect on MKK6 activity, it induced marked activation of MKK6 in combination with Ca2+ ionophore (Fig.1 B). Furthermore, CsA inhibited the activation of MKK6 as observed in the p38 activation (Fig. 1 B). Although treatment with Ca2+ ionophore alone induced slight activation of MKK6 in the Jurkat cells used here (Fig. 1 B), this is not the case with another Jurkat line, TAg Jurkat (data not shown). The reason for this inconsistency remains to be determined. Whereas incubation of Jurkat cells with anti-CD3 or anti-CD28 mAb alone had little effect on either p38 activity or MKK6 activity, simultaneous incubation with both mAbs resulted in synergistic activation of both MKK6 and p38 and IL-2 production (Fig. 1 C). Activation of MKK6 and p38 induced by simultaneous stimulation with anti-CD3 and anti-CD28 mAbs was also inhibited by CsA (data not shown). One of the MAPKKs, SEK1 (also termed as MKK4 or JNKK) has previously been reported to function as a direct activator of SAPK/JNK (19Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1409) Google Scholar, 39Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (916) Google Scholar, 40Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (709) Google Scholar). In addition to SEK1, we have previously identified an activity to phosphorylate and activate SAPK distinct from SEK1 (36Moriguchi T. Kawasaki H. Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1995; 270: 12969-12972Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). This novel kinase has been identified by cDNA cloning and named MKK7 (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar). We thus examined whether SEK1 or MKK7 plays a major role in SAPK/JNK activation during T cell activation. Whereas incubation of Jurkat cells with either anti-CD3 or anti-CD28 mAb alone had a weak effect on MKK7 activity, simultaneous incubation with both mAbs resulted in strong synergistic activation of MKK7, which was inhibited in the presence of CsA (Fig.2 A). SEK1 activity was only slightly activated in response to TPA and Ca2+ ionophore, whereas activation of MKK7 activity was more prominent to the same stimulation (Fig. 2 B), which was also sensitive to CsA (data not shown). Note that both SEK1 and MKK7 activities markedly increased when Jurkat cells were exposed to hyperosmolar media as expected (Fig.2 B) (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 36Moriguchi T. Kawasaki H. Matsuda S. Gotoh Y. Nishida E. J. Biol. Chem. 1995; 270: 12969-12972Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Similar results were obtained in mouse thymocytes (Fig. 2 C) and splenic T cells (data not shown). These results suggest that MKK7 acts as a major upstream activator of SAPK/JNK during T cell activation, as is the case with tumor necrosis factor-α and Fas signaling pathway (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 41Toyoshima F. Moriguchi T. Nishida E. J. Cell Biol. 1997; 139: 1005-1015Crossref PubMed Scopus (143) Google Scholar). It has recently been reported that SAPK/JNK activation is not induced in thymocytes of SEK1-deficient mice in response to TPA and Ca2+ ionophore (42Nishina H. Fischer K.D. Radvanyi L. Shahinian A. Hakem R. Rubie E.A. Bernstein A. Mak T.W. Woodgett J.R. Penninger J.M. Nature. 1997; 385: 350-353Crossref PubMed Scopus (309) Google Scholar, 43Nishina H. Bachmann M. Oliveria-dos-Santos A.J. Kozieradzki I. Fischer K.D. Odermatt B. Wakeham A. Shahinian A. Takimoto H. Bernstein A. Mak T.W. Woodgett J.R. Ohashi P.S. Penninger J.M. J. Exp. Med. 1997; 186: 941-953Crossref PubMed Scopus (116) Google Scholar). The activation pathway of SAPK/JNK might be developmentally regulated such that SEK1 but not MKK7 activates SAPK/JNK in the thymus. Functions of SEK1 and MKK7 in SAPK/JNK pathway in thymocytes remain to be investigated in further studies. Activation of MAPK/ERK alone was not sufficient to induce IL-2 production (Fig. 1 A) (9Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar). To evaluate the role of MAPK/ERK in IL-2 production directly, we employed PD98059, a specific inhibitor of classical MAPKK (also termed as MEK) (44Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar, 45Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2589) Google Scholar). As shown in Fig. 3 A, preincubation with PD98059 prevented IL-2 production induced by simultaneous stimulation with TPA and Ca2+ ionophore in a dose-dependent manner. The concentration of PD98059, which inhibits IL-2 production by 50% in Jurkat cells (<5 μm), is similar to that inhibiting activation of MAPKK/MEK in vitro and in vivo (44Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3254) Google Scholar). We also found that IL-2 production induced by stimulation via CD3 and CD28 was almost completely inhibited by pretreatment of the cells with 20 μm PD98059 (Fig. 3 B). Furthermore, prevention of MAPK/ERK activation resulted in the suppression of IL-2 promoter-driven transcriptional activity (data not shown (46Whitehurst C.E. Geppert T.D. J. Immunol. 1996; 156: 1020-1029PubMed Google Scholar)). It is noteworthy that preteatment of the cells with 20 μmPD98059 did not prevent the activation of SAPK/JNK or p38 (data not shown). These results clearly indicated that the activation of MAPK/ERK pathway is required for IL-2 production. To evaluate the biological significance of p38 activation in T lymphocytes, we examined the effect of SB203580, a highly specific inhibitor for p38 (47Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar, 48Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (601) Google Scholar), on the IL-2 promoter-driven transcriptional activity. As shown in Fig. 4 A, pretreatment of the Jurkat cells with SB203580 resulted in the reduction of IL-2 promoter-driven luciferase (IL-2-Luc) activity in a dose-dependent manner. IL-2 production induced by simultaneous stimulation with anti-CD3 and anti-CD28 mAbs was also inhibited by SB203580 by 50% (data not shown). We further investigated the effect of SB203580 on the transcriptional activation driven by the distal NFAT site in the IL-2 promoter, which is essential for full transcriptional activation of the IL-2 gene (for review see Ref. 49Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). NFAT-driven luciferase (NFAT-Luc) (37Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar) activity was increased after stimulation with TPA and Ca2+ ionophore as previously shown (50Ullman K.S. Northrop J.P. Verweij C.L. Crabtree G.R. Annu. Rev. Immunol. 1990; 8: 421-452Crossref PubMed Scopus (491) Google Scholar), and pretreatment of the cells with SB203580 led to marked reduction of luciferase activity (Fig. 4 B). The same concentration of SB203580 used in this study does not inhibit the activity of SAPK/JNK or MAPK/ERK (Refs. 47Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1977) Google Scholar and 48Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (601) Google Scholar and data not shown). Furthermore, high concentrations of SB203580 (up to 10 μm) had little effect on SV40 promoter-driven luciferase activity (data not shown). The specificity of SB203580 indicates that the prevention of the IL-2 gene expression resulted from the blockade of p38 activity. To further elucidate whether the p38 signaling pathway functions in the IL-2 gene expression, we examined the effect of a dominant-negative form of MKK6 (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), referred to here as MKK6(AA). The expression of MKK6(AA) reduced the NFAT-Luc activity (Fig.4 C) and the IL-2-Luc activity (data not shown) induced by TPA and Ca2+ ionophore (Fig. 4 C) and by anti-CD3 and anti-CD28 stimulation (data not shown), whereas the expression of wild type MKK6 slightly increased the NFAT-Luc activity (Fig.4 C). These data clearly indicate that p38 is significantly involved in the signaling pathway leading to IL-2 gene expression. It was noted that neither SB203580 nor MKK6(AA) showed complete inhibition of the NFAT- or IL-2-Luc activity. It is possible that recently identified molecules, SAPK3 and SAPK4 (51Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (315) Google Scholar, 52Goedert M. Cuenda A. Craxton M. Jakes R. Cohen P. EMBO J. 1997; 16: 3563-3571Crossref PubMed Scopus (356) Google Scholar), which are resistant to SB203580 treatment, might contribute to the residual transcriptional activity in the presence of SB203580. This possibility seems unlikely, however, since a dominant-negative mutant of MKK6, which can also function upstream of SAPK3 and SAPK4, had the same effect as SB203580 as shown in Fig. 4 C. One possibility is that p38 and SAPK/JNK exhibit redundant functions in vivo. To test this possibility, the roles of MKK7 and SAPK/JNK in the signaling pathway leading to IL-2 gene expression were examined. We employed a kinase-negative mutant of MKK7 (referred to here as MKK7(KL)), which can inhibit the activation of SAPK/JNK induced by tumor necrosis factor-α or Fas (34Moriguchi T. Toyoshima F. Masuyama N. Hanafusa H. Gotoh Y. Nishida E. EMBO J. 1997; 16: 7045-7053Crossref PubMed Google Scholar, 41Toyoshima F. Moriguchi T. Nishida E. J. Cell Biol. 1997; 139: 1005-1015Crossref PubMed Scopus (143) Google Scholar). In Jurkat cells, the expression of MKK7(KL) resulted in the inhibition of the NFAT-Luc activity induced by TPA and Ca2+ ionophore (Fig.4 D) and by anti-CD3 and anti-CD28 stimulation (data not shown) in a dose-dependent manner. In this case, too, MKK7(KL) showed only partial inhibition of the NFAT-Luc activity. We thus evaluated the effect of SB203580 on the NFAT-Luc activity in MKK7(KL)-transfected Jurkat cells. Interestingly, a blockade of both signaling pathways further reduced the NFAT-Luc activity to near basal level (Fig. 4 E). It is therefore likely that the MKK6 to p38 and the MKK7 to SAPK/JNK cascades comprise redundant signaling pathways leading to the IL-2 gene expression. This conclusion is further supported by the fact that p38 and SAPK/JNK have a similar specificity of substrate recognition in vitro, although MAPK/ERK displays a distinct substrate selectivity (26Raingeaud J. Whitmarsh A.J. Barrett T. Derijard B. Davis R.J. Mol. Cell. Biol. 1996; 16: 1247-1255Crossref PubMed Scopus (1143) Google Scholar, 53Brunet A. Pouyssegur J. Science. 1996; 272: 1652-1655Crossref PubMed Scopus (119) Google Scholar, 54Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar). Further studies on downstream targets of p38 and SAPK/JNK in T lymphocytes should elucidate mechanisms by which the p38 signaling pathway affects IL-2 production. It has been widely accepted that CsA binds to cytosolic protein, cyclophilin, and that the CsA-cyclophilin complex blocks the IL-2 production by inhibiting the activity of Ca2+calmodulin-dependent protein phosphatase, calcineurin (55McKeon F. Cell. 1991; 66: 823-826Abstract Full Text PDF PubMed Scopus (105) Google Scholar,56Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1473) Google Scholar). We found that activation of MKK7 and MKK6, but not MAPKK/MEK, was inhibited by CsA (Fig. 1 B, Fig. 2, and data not shown). It is possible that calcineurin positively regulates the activity of MKK7 and MKK6 through a mechanism yet to be determined. Alternatively, CsA-cyclophilin complex may directly inactivate an upstream activator of these MAPKKs. Nevertheless, current studies show that the signaling pathways mediated by SAPK/JNK and p38/CSBP could be alternative targets of CsA. Precise mechanisms of the inhibitory effect of CsA on SAPK/JNK and p38/CSBP are now under investigation. We are grateful to Dr. Y. Gotoh for the initial involvement in this study. We are grateful to Dr. G. Crabtree (Stanford University) and Dr. M. Iwashima (Mitsubishi Chemical Corp.) for NFAT-Luc and IL-2-Luc, respectively. We thank Dr. S. Yonehara (Kyoto University) for critical reading of the manuscript, A. Minowa for technical support, and members of the Nishida laboratory for helpful discussion." @default.
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• W2058556985 title "T Lymphocyte Activation Signals for Interleukin-2 Production Involve Activation of MKK6-p38 and MKK7-SAPK/JNK Signaling Pathways Sensitive to Cyclosporin A" @default.
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• W2058556985 doi "https://doi.org/10.1074/jbc.273.20.12378" @default.
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