c-Jun N-terminal Kinase but Not Mitogen-activated Protein Kinase Is Sensitive to cAMP Inhibition in T Lymphocytes

The molecular mechanism underlying the cAMP inhibition of nuclear activation events in T lymphocytes is unknown. Recently, the activation of fibroblasts and muscle cells are shown to be antagonized by cAMP through the inhibition of mitogen-activated protein (MAP) kinases signaling pathway. Whether a similar antagonism may account for the late inhibitory effect of cAMP in T cell was examined. Surprisingly, extracellular signal regulated kinase 2 (ERK2) activation was resistant to cAMP inhibition in all the T lymphocytes tested. Different isoforms (ERK1, ERK2, and ERK3) of MAP kinase were poorly inhibited by cAMP. High concentration of cAMP also only weakly antagonized Raf-1 in T cells. The resistance of ERK and Raf-1 to cAMP clearly distinguishes T cells from fibroblasts. In contrast, another MAP kinase homologue c-Jun N-terminal kinase (JNK) was inhibited by cAMP in good correlation with that of IL-2 suppression. Moreover, JNK was antagonized by a delayed kinetics which is characteristic of cAMP inhibition. Despite that both ERK and JNK are essential for T cell activation, selective inhibition by cAMP further supports the specific role of JNK in T cell activation. The molecular mechanism underlying the cAMP inhibition of nuclear activation events in T lymphocytes is unknown. Recently, the activation of fibroblasts and muscle cells are shown to be antagonized by cAMP through the inhibition of mitogen-activated protein (MAP) kinases signaling pathway. Whether a similar antagonism may account for the late inhibitory effect of cAMP in T cell was examined. Surprisingly, extracellular signal regulated kinase 2 (ERK2) activation was resistant to cAMP inhibition in all the T lymphocytes tested. Different isoforms (ERK1, ERK2, and ERK3) of MAP kinase were poorly inhibited by cAMP. High concentration of cAMP also only weakly antagonized Raf-1 in T cells. The resistance of ERK and Raf-1 to cAMP clearly distinguishes T cells from fibroblasts. In contrast, another MAP kinase homologue c-Jun N-terminal kinase (JNK) was inhibited by cAMP in good correlation with that of IL-2 suppression. Moreover, JNK was antagonized by a delayed kinetics which is characteristic of cAMP inhibition. Despite that both ERK and JNK are essential for T cell activation, selective inhibition by cAMP further supports the specific role of JNK in T cell activation. MAP 1The abbreviations used are: MAPmitogen-activated proteinBt2cAMPN6,2’-O-dibutyryladenosine 3’,5’-cyclic monophosphateEGFepidermal growth factorERKextracellular signal regulated kinaseJNKc-Jun N-terminal kinaseMKKMAP kinase kinasePKAprotein kinase ATCRT cell receptorTPA12-O-tetradecanoylphorbol 13-acetatePAGEpolyacrylamide gel electrophoresisILinterleukinCon Aconcanavalin A. 1The abbreviations used are: MAPmitogen-activated proteinBt2cAMPN6,2’-O-dibutyryladenosine 3’,5’-cyclic monophosphateEGFepidermal growth factorERKextracellular signal regulated kinaseJNKc-Jun N-terminal kinaseMKKMAP kinase kinasePKAprotein kinase ATCRT cell receptorTPA12-O-tetradecanoylphorbol 13-acetatePAGEpolyacrylamide gel electrophoresisILinterleukinCon Aconcanavalin A.1 kinases and JNK define two distinct activation pathways in T lymphocytes. Raf and MAP kinase are coupled to p21ras activation, and are induced by T cell receptor (TCR) engagement(1Nel A.E. Hanekom C. Rheeder A. Williams K. Pollack S. 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Epidermal growth factor (EGF) and epidermoid carcinoma A431 (ATCC CRL 1555) were obtained from Dr. Jaulang Huang (Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan). Anti-Raf antiserum (SP63) was a generous gift of Dr. Ulf Rapp (National Cancer Institute, Frederick, MD). GST-c-Jun(1-79), produced by Dr. Michael Karin, was obtained through Dr. Hsin-Fang Y. Yen (Institute of Molecular Biology, Academia Sinica). Bacterially expressed (His)6-MKK(K97M) was a generous gift of Dr. Natalie Ahn (University of Colorado, Boulder, CO), and was purified according to Mansor et al.(30Mansor S. Resing K.A. Candi J.M. Hermann A.S. Gloor J.W. Herskind K.R. Wartmann M. Davis R.J. Ahn N.G. J. Biochem. (Tokyo). 1994; 116: 304-314Crossref PubMed Scopus (88) Google Scholar). Anti-ERK1 C-16, anti-ERK2 C-14, anti-ERK3 D-23, anti-JNK2 N-19, and anti-JNK1,2 FL were obtained from Santa Cruz Biotech (Santa Cruz, CA). N-19 reacted with the 54-kDa JNK2 in T cells but not with the 46-kDa JNK1 claimed by Santa Cruz Biotech. 9C12.7 is a T cell hybridoma that recognizes λ repressor cI 12-26 in the context of I-Ad(31Lai M.-Z. Ross D.T. Guillet J.-G. Briner T.J. Gefter M.L. Smith J.A. J. Immunol. 1987; 139: 3973-3980PubMed Google Scholar, 32Lai M.-Z. Huang S.-Y. Briner T.J. Guillet J.-G. Smith J.A. Gefter M.L. J. Exp. Med. 1988; 168: 1081-1097Crossref PubMed Scopus (55) Google Scholar). EL4 (ATCC TIB39) was a gift of Dr. Nan-Shih Liao (Institute of Molecular Biology, Academia Sinica). Splenic T lymphocytes were purified by nylon wool (Polyscience, Warrington, PA) column and treated with J11d (anti-B cell) followed by rabbit complement (Cedarlane, Ontario). IL-2 was quantitated by the proliferation of IL-2-dependent cell line HT-2 (ATCC CRL 1841) as described previously(31Lai M.-Z. Ross D.T. Guillet J.-G. Briner T.J. Gefter M.L. Smith J.A. J. Immunol. 1987; 139: 3973-3980PubMed Google Scholar, 32Lai M.-Z. Huang S.-Y. Briner T.J. Guillet J.-G. Smith J.A. Gefter M.L. J. Exp. Med. 1988; 168: 1081-1097Crossref PubMed Scopus (55) Google Scholar). Splenocytes (2-3 × 107 cells/sampling point), EL4 cell or T cell hybridomas (2-3 × 106 cells/sampling point) were pretreated or activated as indicated, and washed twice with phosphate-buffered saline. The preparation of cell extracts, precipitation of ERK and Raf-1 with the specific antibody, and assay of the immune complex were performed according to Cook and McCormick(24Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (856) Google Scholar). MAP kinase was assayed by the phosphorylation of myelin basic protein, and the substrate for Raf kinase was (His)6-MKK(K97M). The were by for MAP kinase and by for Raf-1 followed by and quantitated by of and Raf-1 in were performed according to and S. C. J. 1992; 11: PubMed Scopus Google Scholar). The JNK assay was performed by cell with as described by Hibi et M. Lin A. Smeal T. 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A. 1994; PubMed Scopus Google Scholar), Raf-1 was also by the to by to an of Raf-1 kinase was a inhibition of Raf-1 kinase by in which a and a 2 were found with The of inhibition that Raf-1 was to cAMP suppression concentration of Despite the cAMP not effectively Raf-1 In contrast, Raf-1 activity was by in cells IL-2 was to cAMP inhibition Raf-1 kinase activity in T cells the antagonism of Raf-1 not to a similar inhibition of and Raf-1 inhibition was not with the suppression of IL-2 by cAMP. may be that resistance of ERK to cAMP has also in and B.M. Th. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (312) Google Scholar, 27Sevetson B.R. Kong X. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10305-10309Crossref PubMed Scopus (342) Google Scholar). Raf activation is by cAMP in and of cAMP to inhibit ERK is to the of pathway that activates Johnson Mol. Cell. Biol. 1994; 14: PubMed Scopus Google Scholar). Whether the inhibition of Raf-1 the of a similar mechanism in T cells remains to be The inhibitory effect of cAMP on MAP kinase and Raf-1 clearly distinguishes T cells from fibroblasts and other cells and The resistance of MAP kinase to in the of T activation, which signaling pathways of T cell receptor A. Cell. 1994; 76: Full Text PDF PubMed Scopus Google Scholar). the JNK an activation pathway in T B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (844) Google Scholar), we is resistant to cAMP. JNK activity was not T lymphocytes from were treated with for and with cAMP in a suppression of in which a was found with with cAMP inhibited of JNK activity not to suppression not shown for a delayed antagonism of JNK by cAMP is also by the of A inhibition of JNK was found with T EL4 after with with for 2 h also to a similar inhibition of JNK in T lymphocytes The in JNK activity was well with the in IL-2 by cAMP in both T lymphocytes and EL4 inhibition of JNK by cAMP was by and D. A, T cells were pretreated with the for or for 2 h activation with of or was with as were and JNK were performed as described in the protein of JNK were not by cAMP. were from T lymphocytes pretreated with the for 2 and were on and with specific for JNK2 Santa Cruz A similar was obtained with reacted with both JNK1 and JNK2 Santa Cruz the JNK activity and the IL-2 in the of cAMP. JNK activity was in IL-2 secretion was as described in The activated JNK activity and IL-2 secretion are used as is the of A, T lymphocytes from The delayed suppression of JNK in T cells kinetics similar to that for the inhibition on the binding of a on the IL-2 D. Rothenberg E.V. J. Exp. Med. 1994; 179: 931-942Crossref PubMed Scopus (159) Google Scholar). with an correlation the of and the of suppression for both IL-2 and JNK may be that the inhibition of IL-2 by cAMP is by the antagonism of JNK in T lymphocytes. The molecular mechanism underlying the inhibition of JNK remains unclear. JNK1 and JNK2 are in T B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (844) Google Scholar), and protein were not by treatment of for not shown for the inhibition was not to a suppression of JNK by cAMP. In contrast, and protein were for the inhibition of JNK by as by the JNK activity and the reduced of JNK to cAMP inhibition in the of or the suppression of JNK was by the by cAMP. The inhibition of JNK is distinct from the that a cAMP is to effectively in A431 cells inhibition of MAP kinase is to an of Raf-1 by phosphorylation in other of cells J. Dent P. Jelinek T. Wolfman A. Weber M.J. Sturgill T.W. Science. 1993; 262: 1065-1069Crossref PubMed Scopus (816) Google Scholar, 24Cook S.J. McCormick F. Science. 1993; 262: 1069-1072Crossref PubMed Scopus (856) Google Scholar, 25Burgering B.M. Th. Pronk G.J. van Weeren P.C. Chardin P. Bos J.L. EMBO J. 1993; 12: 4211-4220Crossref PubMed Scopus (312) Google Scholar, 29Hafner S. Adler H.S. Mischak H. Janosch P. Heidecker G. Wolfman A. Pippig S. Lohse M. Ueffing M. Kolch W. Mol. Cell. Biol. 1994; 14: 6696-6703Crossref PubMed Scopus (286) Google Scholar). 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