p38 Mitogen-activated Protein Kinase Regulation of JB6 Cl41 Cell Transformation Promoted by Epidermal Growth Factor

Abstract

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability. The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability. The mitogen-activated protein kinases (MAPKs), 1The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun NH2-terminal kinase; ATF-2, activating transcription factor 2; STAT1, signal transducer and activator of transcription 1; AP-1, activator protein-1; EGF, epidermal growth factor; TPA, 12-O-tetradecanoyl-phorbol-13-acetate; DN-p38, dominant negative mutant p38; MEM, Eagle's minimal essential medium; FBS, fetal bovine serum. including p38 kinase, c-Jun NH2-terminal kinases (JNKs), extracellular signal-regulated kinases (ERKs), and signal transduction cascades, are vital mediators of many cellular functions such as growth, development, proliferation, differentiation, malignant transformation, inflammation, and apoptosis (1Harper S.J. LoGrasso P. Cell. Signal. 2001; 13: 299-310Crossref PubMed Scopus (281) Google Scholar, 2Ballif B.A. Blenis J. Cell Growth & Differ. 2001; 12: 397-408PubMed Google Scholar, 3Papatsoris A.G. Papavassiliou A.G. Trends Mol. Med. 2001; 7: 288-292Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). p38 MAP kinase is activated by cellular stresses including inflammatory cytokines, ultraviolet light, and growth factors, and the activated p38 MAP kinase has been shown to phosphorylate several transcription factors including ATF-2, STAT1, the Max/Myc complex, and myocyte enhancer factor 2 (MEF2) (1Harper S.J. LoGrasso P. Cell. Signal. 2001; 13: 299-310Crossref PubMed Scopus (281) Google Scholar, 4Herlaar E. Brown Z. Mol. Med. Today. 1999; 5: 439-447Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar, 5Tibbles L.A. Woodgett J.R. Cell. Mol. Life Sci. 1999; 55: 1230-1254Crossref PubMed Scopus (555) Google Scholar, 6Whitmarsh, A. J., and Davis, R. J. (1999) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;1999/1/pe1.Google Scholar). On the other hand, ERKs are predominantly activated by mitogenic stimuli, including mitogens and growth factors, and activated ERKs are involved in cell differentiation and development (2Ballif B.A. Blenis J. Cell Growth & Differ. 2001; 12: 397-408PubMed Google Scholar, 3Papatsoris A.G. Papavassiliou A.G. Trends Mol. Med. 2001; 7: 288-292Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Although one major function of the p38 kinase and JNKs pathways is regulation of inflammation and apoptosis, in many cases the biological consequences of p38 kinase and JNK activation overlap with those of ERKs in mediation of cell growth and differentiation (2Ballif B.A. Blenis J. Cell Growth & Differ. 2001; 12: 397-408PubMed Google Scholar, 3Papatsoris A.G. Papavassiliou A.G. Trends Mol. Med. 2001; 7: 288-292Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 6Whitmarsh, A. J., and Davis, R. J. (1999) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;1999/1/pe1.Google Scholar, 7Fan M. Chambers T.C. Drug Resist. Updat. 2001; 4: 253-267Crossref PubMed Scopus (175) Google Scholar). Previously, we studied ERKs regulation of epidermal growth factor (EGF)-induced cell transformation in promotion sensitive (P+) derivatives of the mouse epidermal JB6 cell line (8Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google Scholar). Results showed that inhibition of ERKs appeared to be an important contributor to the tumor promotion-resistant phenotype in JB6 cells. A recent study showed that the p38 kinase and JNKs pathways cooperate to transactivate the vitamin D receptor through the c-Jun/activator protein-1 (AP-1) (9Qi X. Pramanik R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This result indicates that a connection exists between p38 MAP kinase and AP-1. But AP-1 activation is involved in JB6 cell transformation promoted by EGF and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) (10Liu G. Bibus D.M. Bode A.M. Ma W.Y. Holman R.T. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7510-7515Crossref PubMed Scopus (97) Google Scholar, 11Kaminska B. Pyrzynska B. Ciechomska I. Wisniewska M. Acta Neurobiol. Exp. (Warsz.). 2000; 60: 395-402PubMed Google Scholar). AP-1 is a dimeric complex consisting of proteins encoded by the jun and fos gene families, and the AP-1 binding region in these genes is referred to as the TPA response element (TRE). AP-1 induces the transcription of genes that are related to cell proliferation, metastasis, and metabolism (12Curran T. Franza Jr., B.R. Cell. 1988; 55: 395-397Abstract Full Text PDF PubMed Scopus (1313) Google Scholar, 13Huang C. Ma W.Y. Dawson M.I. Rincon M. Flavell R.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5826-5830Crossref PubMed Scopus (206) Google Scholar, 14Lamb R.F. Hennigan R.F. Turnbull K. Katsanakis K.D. MacKenzie E.D. Birnie G.D. Ozanne B.W. Mol. Cell. Biol. 1997; 17: 963-976Crossref PubMed Scopus (155) Google Scholar). Increased AP-1 activity is associated with malignant transformation, and repression of AP-1 activity has been shown to lead to suppression of cell transformation and tumor promotion (15Dong Z. Huang C. Brown R.E. Ma W.Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Activation of MAP kinase cascades is known to play a considerable role in malignant transformation (3Papatsoris A.G. Papavassiliou A.G. Trends Mol. Med. 2001; 7: 288-292Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 8Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google Scholar). However, whether p38 MAP kinase is involved in the regulation of cell transformation is not known. A recent study showed that only p38 MAP kinase, a MAP kinase usually associated with stress responses, growth arrest, and apoptosis, was consistently increased in human non-small cell lung cancer samples compared with the normal tissues examined (16Greenberg A.K. Basu S. Hu J. Yie T.A. Tchou-Wong K.M. Rom W.N. Lee T.C. Am. J. Respir. Cell Mol. Biol. 2002; 26: 558-564Crossref PubMed Scopus (141) Google Scholar). More interesting are the results showing that ERKs and JNKs, the MAP kinase pathways traditionally associated with cell growth and perhaps malignant transformation, were not activated in the human non-small cell lung tumor samples. These results indicate that p38 MAP kinase is probably involved in malignant cell transformation. The purpose of this research project was to explore the mechanism of p38-mediated JB6 cell transformation using CMV-neo plasmid-transfected JB6 cells and dominant negative mutant p38 (DN-p38) plasmid-transfected JB6 cells following EGF stimulation. The results obtained show that p38 MAP kinase plays a critical role in regulation of JB6 Cl41 cell transformation promoted by EGF through the inhibition of the phosphorylation of ATF-2, STAT1, and c-Myc and the repression of AP-1 binding ability. Materials—Eagle's minimal essential medium (MEM), l-glutamine, and LipofectAMINE™ 2000 reagent were from Invitrogen; EGF and SB202190 were from Calbiochem-Novabiochem. Fetal bovine serum (FBS) was from Gemini Bio-Product (Calabasas, CA). Penicillin, streptomycin, and gentamicin sulfate were from BioWhittaker, Inc. (Walkersville, MD). Folin & Ciocalteu's phenol reagent was from Pierce, and polyvinylidene difluoride (PVDF) membrane was from Millipore (Bedford, MA). Antibodies to detect the phosphorylation of p38 kinase, ERKs, JNKs, ATF-2, STAT1, and c-Myc and the non-phosphorylated levels of p38 kinase, ERKs, JNKs, ATF-2, STAT1 and the c-Myc protein were from Upstate Biotechnology (Lake Placid, NY). [α-32P]ATP was from Amersham Biosciences. Cell Culture and Establishment of Stably Transfected JB6 Cells— Using the CMV-neo plasmid and DN-p38 kinase plasmid, we established stable transfectants according to the protocol from Invitrogen. All of these cells were selected in media containing 400 μg/ml of G-418 for 2 weeks, and then the G418 concentration was decreased to 200 μg/ml and maintained. Empty vector CMV-neo plasmid-transfected JB6 cells (Cl41 CMV-neo) and DN-p38 plasmid-transfected JB6 cells (Cl41 DN-p38) were as in with and in a of CMV-neo and Cl41 DN-p38 cells were as and was as M. A. Z. Ma W.Y. K. Dong Z. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). cells were to and then in for in a The media were to the cells were for and the cells were then with EGF for the were then with and The samples were into and for containing an of protein were into of an for and a polyvinylidene difluoride of ERKs, JNKs, ATF-2, STAT1, and c-Myc were by using a and a phosphorylation of p38 kinase ERKs JNKs STAT1 and c-Myc (Thr58/Ser62). Antibodies non-phosphorylated levels of p38 kinase, ERKs, JNKs, ATF-2, STAT1, and c-Myc were used as to p38 MAP p38 MAP kinase activity was following the from Cell Inc. Cl41 CMV-neo cells and Cl41 DN-p38 cells were in in a for and then with EGF for various as were with 2 and and protein concentration was by the protein cell were for The cell were with p38 the kinase was for in the of and 2 of an of was by using a specific phosphorylation of AP-1 DNA protein were from cells by a of the of J. X. Dong Z. Mol. Cell. Biol. PubMed Scopus Google Scholar). Cl41 CMV-neo cells and Cl41 DN-p38 cells were in and in in a of the cells were to of EGF for The cells were then and in of A μg/ml μg/ml and 1 a the containing the were with of A The were then in of with and and for a the were into and The DNA binding was for in a containing of 1 of and of a AP-1 The samples were a and the were using the role of p38 MAP kinase in EGF-promoted cell transformation was in Cl41 CMV-neo cells and Cl41 DN-p38 cells. The of the inhibitor SB202190 EGF-promoted cell transformation was in JB6 Cl41 cells. cells were to EGF with SB202190 in 1 of medium containing The were in a for and the cell were as by Colburn G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of DN-p38 JB6 Cl41 cell transformation is as Cl41 CMV-neo cells Cl41 DN-p38 cells in and the of SB202190 JB6 cell transformation is also as the of cells. and of the explore the role of factor STAT1, one of the of p38 MAP kinase in JB6 Cl41 cell transformation, we stable expression of the and expression of only plasmid The JB6 Cl41 cell used JB6 and JB6 cells. All of these cells were to detect cell transformation promoted by the of STAT1, including a was with an and by with and and then into the vector the and The STAT1 with from the was into the of the the in the of the a of and for STAT1 and indicate the a were the were using the the expression the of STAT1 and were using and and then into of All of including expression were by DNA between were by the and EGF of p38 and ERKs play important in the transformation of mouse JB6 Cl41 cells by EGF TPA (8Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google Scholar). Our results showed that EGF phosphorylation of ERKs in and dose-dependent A and whether EGF also to phosphorylation of other of such as p38 kinase and Our results showed that EGF also phosphorylation of p38 kinase in and dose-dependent and not JNKs and These data with other that show that EGF cell growth through and p38 kinase signal transduction pathways S. G.D. E.D. T. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The p38 SB202190 the EGF-induced of p38 explore the role of p38 kinase in the EGF-stimulated signal transduction SB202190, a p38 kinase inhibitor M. A. Ma W.Y. S. G. Dong Z. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. C. M. Dong Z. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, Ma W.Y. Dong Z. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Bode A.M. Ma W.Y. Dong Z. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google was used to JB6 cells for 1 Our results showed that SB202190 the EGF-induced phosphorylation of p38 kinase in a dose-dependent EGF-induced phosphorylation These results indicate that SB202190 EGF-induced p38 kinase The p38 SB202190 JB6 Cl41 Cell by the role of p38 MAP kinase in JB6 cell transformation, we EGF-promoted cell transformation in JB6 Cl41 cells using the p38 kinase inhibitor JB6 cells were with EGF with SB202190 in a in a for The was as and were with JB6 Cl41 cells only by EGF, the were decreased EGF and SB202190 and this inhibition in a dose-dependent These data that p38 MAP kinase to JB6 cell transformation promoted by EGF, and this study is the to that SB202190 inhibits EGF-induced JB6 Cl41 cell transformation. DN-p38 MAP of p38 ERKs SB202190 other p38 kinase, we used stable transfectants of Cl41 CMV-neo cells and Cl41 DN-p38 cells to study the biological activity of p38 kinase Ma W.Y. Dong Z. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, G. Bode A.M. Ma W.Y. Dong Z. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). were with EGF various for and was used to the of phosphorylation of p38 kinase, ERKs, and JNKs using specific Our data showed that EGF phosphorylation of p38 kinase and ERKs in a A and and and phosphorylation of JNKs and protein levels of these kinases not and These data also showed that DN-p38 the EGF-induced phosphorylation of p38 kinase in a and dose-dependent phosphorylation of ERKs Results indicate that ERKs and p38 kinase, not JNKs, are involved in EGF-stimulated cell signal DN-p38 by the results we that p38 kinase a role in JB6 cell transformation. Cell transformation was using our A.M. Ma W.Y. Dong Z. 2001; Google Scholar, and J. Science's STKE Scholar). Cl41 CMV-neo cells and Cl41 DN-p38 cells were with EGF in a in a for The of was by and the was Our results showed that DN-p38 the of EGF-induced compared with cells The inhibition was not only in also in However, Cl41 CMV-neo cells and Cl41 DN-p38 cells showed A and These data indicate that p38 MAP kinase is involved in JB6 cell transformation as a an DN-p38 EGF-induced p38 MAP that the Cl41 DN-p38 stable cells as p38 activity its ATF-2, was were with EGF for the and the cell were with an p38 kinase protein was used as a of p38 MAP kinase, and phosphorylation of was by Our results showed that EGF-induced phosphorylation of was decreased in Cl41 DN-p38 cells compared with Cl41 CMV-neo cells These data indicate that p38 kinase has a role in EGF-induced phosphorylation of DN-p38 EGF-induced of STAT1 in a and is of p38 MAP kinase and is a transcription factor that and growth signals that in various biological responses, including and differentiation Mol. Med. 2002; PubMed Scopus Google Scholar). Cl41 DN-p38 cells and Cl41 CMV-neo cells were for various and various with that EGF phosphorylation of STAT1 in Cl41 CMV-neo the phosphorylation of STAT1 in Cl41 DN-p38 cells was However, phosphorylation of STAT1 decreased in cell levels of STAT1 were the and and EGF-induced phosphorylation of STAT1 increased with EGF with in non-phosphorylated levels of STAT1 DN-p38 of c-Myc the mechanism of p38 MAP kinase in the regulation of JB6 cell transformation, we the phosphorylation of c-Myc by using a specific phosphorylation of c-Myc The c-Myc is associated with cell growth, development, and malignant transformation in human and cell B. R.A. PubMed Scopus Google Scholar, B. S. 7: Google Scholar). The c-Myc also to cell in cells J. Mol. Cell. Biol. 12: PubMed Scopus Google Scholar). this our results showed that phosphorylation of c-Myc was increased CMV-neo JB6 cells were from with EGF phosphorylation of c-Myc in cells was the EGF c-Myc protein levels in Cl41 CMV-neo cells Cl41 DN-p38 cells and Moreover, we studied the of various of EGF phosphorylation of c-Myc Results showed that phosphorylation of c-Myc increased in Cl41 CMV-neo cells was only in Cl41 DN-p38 cells cells were with EGF These data indicate that phosphorylation of c-Myc is by EGF in a and dose-dependent in Cl41 CMV-neo cells and that DN-p38 inhibits the phosphorylation of c-Myc A of STAT1 JB6 Cl41 Cell by JB6 cells of STAT1 and JB6 cells plasmid cells were to detect the cell transformation promoted by were with EGF in a in a for The was as (8Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google Scholar). The were decreased in JB6 cells promoted by EGF, were many in JB6 Cl41 cells and JB6 by EGF the These data indicate that STAT1, one of the of p38 MAP kinase, also to JB6 Cl41 cell transformation promoted by DN-p38 EGF-induced AP-1 induces gene transcription by binding to the TPA response element in the region of its target gene and is for cell proliferation, differentiation, and malignant transformation E. M. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, M. S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). AP-1 were with and assays were used to AP-1 DNA binding Our results showed that AP-1 binding was decreased in Cl41 DN-p38 cells A and and compared with that in Cl41 CMV-neo cells A and and cells were with EGF for was between cells and cells EGF A and 2 and that the was specific for AP-1 a of AP-1 was and results showed that the AP-1 binding was A and These data indicate that DN-p38 inhibits EGF-induced AP-1 binding ability. show that ERKs are involved in the JB6 cell transformation promoted by EGF TPA (8Huang C. Ma W.Y. Young M.R. Colburn N. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 156-161Crossref PubMed Scopus (187) Google an of the role of p38 MAP kinase in cell transformation still remains unclear. this we that p38 MAP kinase plays a critical role in JB6 Cl41 cell transformation promoted by DN-p38 and the p38 MAP kinase inhibitor SB202190 JB6 Cl41 cell transformation promoted by DN-p38 EGF-stimulated phosphorylation of ATF-2, STAT1, and and shift results showed that DN-p38 inhibits AP-1 binding ability. These results indicate that p38 MAP kinase is involved in regulation of JB6 Cl41 cell transformation promoted by EGF through the inhibition of phosphorylation of its factors, including ATF-2, STAT1, and also AP-1 DNA indicate that EGF induces the phosphorylation of ERKs and p38 MAP kinase S. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R. Exp. Cell 1999; PubMed Scopus Google Scholar, B. J. K. Han J. J. Cell. 2001; PubMed Scopus Google Scholar, J. A.K. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The pathway is associated with activation of the EGF receptor and has been shown to play a major role in several tumor However, the JNKs pathway has not been shown to be activated through the EGF receptor is by cellular stresses and S. A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Our present data also show that EGF only induces the phosphorylation of ERKs and p38 MAP kinase not JNKs SB202190 and DN-p38 only EGF-induced phosphorylation of p38 MAP kinase not ERKs 2 and These results indicate that ERKs and p38 MAP kinase, not JNKs, in EGF-stimulated cell signal The p38 MAP kinase is usually associated with stress responses, growth arrest, and apoptosis (1Harper S.J. LoGrasso P. Cell. Signal. 2001; 13: 299-310Crossref PubMed Scopus (281) Google Scholar, 2Ballif B.A. Blenis J. Cell Growth & Differ. 2001; 12: 397-408PubMed Google Scholar, 4Herlaar E. Brown Z. Mol. Med. Today. 1999; 5: 439-447Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). But a recent study (16Greenberg A.K. Basu S. Hu J. Yie T.A. Tchou-Wong K.M. Rom W.N. Lee T.C. Am. J. Respir. Cell Mol. Biol. 2002; 26: 558-564Crossref PubMed Scopus (141) Google showed that was activated in human lung cancer an role for this pathway in malignant cell growth transformation. The present study is the to that p38 MAP kinase is involved in JB6 Cl41 cell transformation promoted by SB202190, a p38 MAP kinase in EGF-promoted JB6 Cl41 cell transformation the of SB202190 other we a stable of JB6 Cl41 cells a dominant negative mutant of p38 kinase and DN-p38 according to the A.K. I. M. M. 2002; PubMed Scopus Google Scholar, Chen N. Dong Z. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). Our results showed that the of in Cl41 DN-p38 cells was decreased compared with the of in Cl41 CMV-neo cells These data indicate that p38 MAP kinase plays a critical role in JB6 Cl41 cell transformation promoted by explore the mechanism of p38 kinase in the regulation of JB6 cell transformation, we whether p38 MAP kinase activity was in Cl41 DN-p38 cells. Our results show that DN-p38 inhibits EGF-induced phosphorylation of is a major phosphorylation for and a of protein with malignant in the mouse P. S. A. A. 2000; PubMed Scopus Google Scholar). recent study showed that EGF transcription factor through a The pathway induces phosphorylation of phosphorylation the MAP kinase pathway D.M. J. C. K. J. 2002; PubMed Scopus (187) Google Scholar). Moreover, between ERKs and p38 kinase was to be essential for activation by EGF G. J. Biol. 2001; PubMed Scopus Google Scholar). of the a role in the regulation of cellular transformation Cell Biol. 2001; 13: PubMed Scopus Google Scholar). A recent study showed that activation is for transformation in tumor mouse cells Wang A. A. Colburn 2002; PubMed Scopus Google Scholar). showed that transformation of normal cells with a activation of STAT1 and transcription factors 1998; Google Scholar). this we that EGF-induced phosphorylation of STAT1 also decreased in Cl41 DN-p38 cells increased in Cl41 CMV-neo cells the cells were with EGF to a mutant of STAT1 of JB6 cells mutant of STAT1 its to cell transformation in JB6 Cl41 cells promoted by EGF These results indicate that EGF-induced phosphorylation of STAT1 is p38 MAP kinase and that STAT1 is involved in JB6 cell transformation promoted by show that the c-Myc is associated with cancer cell and transformation Jr., J. T. M. G. 2001; PubMed Scopus Google Scholar, 2001; PubMed Scopus Google Scholar). this we also that DN-p38 EGF-induced phosphorylation of that p38 MAP kinase EGF-induced c-Myc phosphorylation p38 MAP kinase be involved in cell transformation through phosphorylation of On the other hand, AP-1, a of the proteins and plays a important role in the of transformation and the genes involved in cell proliferation, differentiation, and inflammation M. X. Dong Z. Chen J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. 2002; PubMed Scopus Google Scholar). the tumor activation of AP-1 was shown to transformation in JB6 mouse epidermal cells Z. Colburn Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). recent study M. X. Dong Z. Chen J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google showed that p38 and inhibition with and AP-1 These that AP-1 activation be through the p38 and ERKs pathways M. X. Dong Z. Chen J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the present EGF-induced AP-1 binding decreased in Cl41 DN-p38 cells compared with Cl41 CMV-neo cells These data that EGF-induced AP-1 binding is through p38 MAP kinase and that DN-p38 MAP kinase EGF-induced JB6 Cl41 cell transformation through inhibition of AP-1 DNA binding ability. we for the that p38 MAP kinase EGF-promoted JB6 Cl41 cell transformation through regulation of the phosphorylation of its factors, such as STAT1, ATF-2, and and also AP-1 DNA binding This study that p38 MAP kinase is pathway involved in JB6 Cl41 cell transformation promoted by