Induction of the Nuclear Proto-Oncogene c-fos by the Phorbol Ester TPA and c-H-Ras

  • Kazi, Julhash U. (Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University) ;
  • Soh, Jae-Won (Biomedical Research Center for Signal Transduction Networks, Department of Chemistry, Inha University)
  • Received : 2008.04.15
  • Accepted : 2008.08.14
  • Published : 2008.11.30


TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active $PKC{\alpha}$ and $PKC{\varepsilon}$ showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.




Supported by : Inha University


  1. Bishop, A.L., and Hall, A. (2000). Rho GTPases and their effector proteins. Biochem. J. 348, 241-255
  2. Brose, N., Hofmann, K., Hata, Y., and Sudhof, T.C. (1995). Mammalian homologues of Caenorhabditis elegans unc-13 gene define novel family of C2-domain proteins. J. Biol. Chem. 270, 25273-25280
  3. Busam, K.J., Geiser, A.G., Roberts, A.B., and Sporn, M.B. (1993). Synergistic increase of phorbol ester-induced c-fos mRNA expression by retinoic acid through stabilization of the c-fos message. Oncogene 8, 2267-2273
  4. Feig, L.A., and Cooper, G.M. (1988). Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP. Mol. Cell. Biol. 8, 3235-3243
  5. Kawano, Y., Okamoto, I., Murakami, D., Itoh, H., Yoshida, M., Ueda, S., and Saya, H. (2000). Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins. J. Biol. Chem. 275, 29628-29635
  6. Kazi, J.U., Kabir, N.N., and Soh, J.W. (2008). Bioinformatic prediction and analysis of eukaryotic protein kinases in the rat genome. Gene 410, 147-153
  7. Lee, Y.J., Cho, H.N., Soh, J. W., Jhon, G.J., Cho, C.K., Chung, H.Y., Bae, S., Lee, S.J., and Lee, Y.S. (2003a). Oxidative stressinduced apoptosis is mediated by ERK1/2 phosphorylation. Exp. Cell. Res. 291, 251-266
  8. Marais, R., Light, Y., Mason, C., Paterson, H., Olson, M.F., and Marshall, C.J. (1998). Requirement of Ras-GTP-Raf complexes for activation of Raf-1 by protein kinase C. Science 280, 109-112
  9. Newton, A.C. (2003). Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm. Biochem. J. 370, 361-371
  10. Ohara, M., Kawashima, Y., Kitajima, S., Mitsuoka, C., and Watanabe, H. (2003). Blue light inhibits the growth of skin tumors in the v-Ha-ras transgenic mouse. Cancer Sci. 94, 205-209
  11. Satomi, Y., Bu, P., Okuda, M., Tokuda, H., and Nishino, H. (2003). H-ras mutations at codon 61 or 13 in tumors initiated with a NO donor in mouse skin. Cancer Lett. 196, 17-22
  12. Schiller, M., Bohm, M., Dennler, S., Ehrchen, J.M., and Mauviel, A. (2006). Mitogen- and stress-activated protein kinase 1 is critical for interleukin-1-induced, CREB-mediated, c-fos gene expression in keratinocytes. Oncogene 25, 4449-4457
  13. Soh, J.W., Lee, E.H., Prywes, R., and Weinstein, I.B. (1999). Novel roles of specific isoforms of protein kinase C in activation of the c-fos serum response element. Mol. Cell. Biol. 19, 1313-1324
  14. van Blitterswijk, W.J., and Houssa, B. (2000). Properties and functions of diacylglycerol kinases. Cell Signal. 12, 595-605
  15. Hill, C.S., and Treisman, R. (1995). Differential activation of c-fos promoter elements by serum, lysophosphatidic acid, G proteins and polypeptide growth factors. EMBO J. 14, 5037-5047
  16. Soh, J.W., and Weinstein, I.B. (2003). Roles of specific isoforms of protein kinase C in the transcriptional control of cyclin D1 and related genes. J. Biol. Chem. 278, 34709-34716
  17. Li, W., Whaley, C.D., Bonnevier, J.L., Mondino, A., Martin, M.E., Aagaard-Tillery, K.M., and Mueller, D.L. (2001). CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation. J. Immunol. 167, 827-835
  18. Seternes, O.M., Sorensen, R., Johansen, B., Loennechen, T., Aarbakke, J., and Moens, U. (1998). Synergistic increase in c-fos expression by simultaneous activation of the ras/raf/map kinaseand protein kinase A signaling pathways is mediated by the cfos AP-1 and SRE sites. Biochim. Biophys. Acta 1395, 345-360
  19. Hall, C., Monfries, C., Smith, P., Lim, H.H., Kozma, R., Ahmed, S., Vanniasingham, V., Leung, T., and Lim, L. (1990). Novel human brain cDNA encoding a 34,000 Mr protein n-chimaerin, related to both the regulatory domain of protein kinase C and BCR, the product of the breakpoint cluster region gene. J. Mol. Biol. 211, 11-16
  20. Kazi, J.U., and Soh, J.W. (2007). Isoform-specific translocation of PKC isoforms in NIH3T3 cells by TPA. Biochem. Biophys. Res. Commun. 364, 231-237
  21. Sahai, E., Alberts, A.S., and Treisman, R. (1998). RhoA effector mutants reveal distinct effector pathways for cytoskeletal reorganization, SRF activation and transformation. EMBO J. 17, 1350-1361
  22. Campbell, P.M., and Der, C.J. (2004). Oncogenic Ras and its role in tumor cell invasion and metastasis. Semin. Cancer Biol. 14, 105-114
  23. Park, C., Fukamachi, K., Takasuka, N., Han, B.S., Kim, C.K., Hamaguchi, T., Fujita, K., Ueda, S., and Tsuda, H. (2004). Rapid induction of skin and mammary tumors in human c-Haras proto-oncogene transgenic rats by treatment with 7,12- dimethylbenz[a]anthracene followed by 12-O-tetradecanoylphorbol 13-acetate. Cancer Sci. 95, 205-210
  24. Ebinu, J.O., Bottorff, D.A., Chan, E.Y., Stang, S.L., Dunn, R.J., and Stone, J.C. (1998). RasGRP, a Ras guanyl nucleotide- releasing protein with calcium- and diacylglycerol-binding motifs. Science 280, 1082-1086
  25. Hashiramoto, A., Mizukami, H., and Yamashita, T. (2006). Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1. Oncogene 25, 3948-3955
  26. Mellor, H., and Parker, P.J. (1998). The extended protein kinase C superfamily. Biochem. J. 332, 281-292
  27. Stice, L.L., Forman, L.W., Hahn, C.S., and Faller, D.V. (2002). Desensitization of the PDGFbeta receptor by modulation of the cytoskeleton: the role of p21(Ras) and Rho family GTPases. Exp. Cell. Res. 275, 17-30
  28. Singh, A., Sowjanya, A.P., and Ramakrishna, G. (2005). The wildtype Ras: road ahead. FASEB J. 19, 161-169
  29. Valverde, A.M., Sinnett-Smith, J., Van Lint, J., and Rozengurt, E. (1994). Molecular cloning and characterization of protein kinase D: a target for diacylglycerol and phorbol esters with a distinctive catalytic domain. Proc. Natl. Acad. Sci. USA 91, 8572-8576
  30. Campbell, S.L., Khosravi-Far, R., Rossman, K.L., Clark, G.J., and Der, C.J. (1998). Increasing complexity of Ras signaling. Oncogene 17, 1395-1413
  31. Hirai, S., Izumi, Y., Higa, K., Kaibuchi, K., Mizuno, K., Osada, S., Suzuki, K., and Ohno, S. (1994). Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. EMBO J. 13, 2331-2340
  32. Malliri, A., van der Kammen, R.A., Clark, K., van der Valk, M., Michiels, F., and Collard, J.G. (2002). Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours. Nature 417, 867-871
  33. Soh, J.W., Lee, Y.S., and Weinstein, I.B. (2003). Effects of regulatory domains of specific isoforms of protein kinase C on growth control and apoptosis in MCF-7 breast cancer cells. J. Exp. Ther. Oncol. 3, 115-126
  34. Choi, H.J., Park, Y.G., and Kim, C.H. (2007). Lactosylceramide alpha2,3-sialyltransferase is induced via a PKC/ERK/CREBdependent pathway in K562 human leukemia cells. Mol. Cells 23, 138-144
  35. Lee, Y.J., Soh, J.W., Jeoung, D.I., Cho, C.K., Jhon, G.J., Lee, S.J., and Lee, Y.S. (2003b). PKC epsilon -mediated ERK1/2 activation involved in radiation-induced cell death in NIH3T3 cells. Biochim. Biophys. Acta 1593, 219-229
  36. Carnero, A., Dolfi, F., and Lacal, J.C. (1994). ras-p21 activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis oocytes. J. Cell. Biochem. 54, 478-48
  37. Rivera, V.M., Miranti, C., Misra, R.P., Ginty, D.D., Chen, R.H., Blenis, J., and Greenberg, M.E. (1993). A growth factor-induced kinase phosphorylates the serum response factor at a site that regulates its DNA-binding activity. Mol. Cell. Biol. 13, 6260-6273
  38. Stewart, S., and Guan, K.L. (2000). The dominant negative Ras mutant, N17Ras, can inhibit signaling independently of blocking Ras activation. J. Biol. Chem. 275, 8854-8862
  39. Choi, S.Y., Kim, M.J., Kang, C.M., Bae, S., Cho, C.K., Soh, J.W., Kim, J.H., Kang, S., Chung, H.Y., Lee, Y.S., et al. (2006). Activation of Bak and Bax through c-Abl-protein kinase C{delta}-p38 MAPK signaling in response to ionizing radiation in human nonsmall cell lung cancer cells. J. Biol. Chem. 281, 7049-7059
  40. Liu, J.F., Crepin, M., Liu, J.M., Barritault, D., and Ledoux, D. (2002). FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway. Biochem. Biophys. Res. Commun. 293, 1174-1182
  41. Kolch, W., Heidecker, G., Kochs, G., Hummel, R., Vahidi, H., Mischak, H., Finkenzeller, G., Marme, D., and Rapp, U.R. (1993). Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364, 249-252
  42. Herman, W.H., and Simonson, M.S. (1995). Nuclear signaling by endothelin-1. A Ras pathway for activation of the c-fos serum response element. J. Biol. Chem. 270, 11654-11661
  43. Kazi, J.U., and Soh, J.W. (2008). Role of regulatory domain mutants of PKC isoforms in c-fos induction. Bull. Korean Chem. Soc. 29, 252-254