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. 1994 Nov;5(11):1177–1184. doi: 10.1091/mbc.5.11.1177

Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

M C Frame 1, K Simpson 1, V J Fincham 1, D H Crouch 1
PMCID: PMC301144  PMID: 7865883

Abstract

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abate C., Patel L., Rauscher F. J., 3rd, Curran T. Redox regulation of fos and jun DNA-binding activity in vitro. Science. 1990 Sep 7;249(4973):1157–1161. doi: 10.1126/science.2118682. [DOI] [PubMed] [Google Scholar]
  2. Bader J. P. Temperature-dependent transformation of cells infected with a mutant of Bryan Rous sarcoma virus. J Virol. 1972 Aug;10(2):267–276. doi: 10.1128/jvi.10.2.267-276.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bell J. G., Wyke J. A., Macpherson I. A. Transformation by a temperature sensitive mutant of Rous sarcoma virus in the absence of serum. J Gen Virol. 1975 May;27(2):127–134. doi: 10.1099/0022-1317-27-2-127. [DOI] [PubMed] [Google Scholar]
  4. Beug H., Claviez M., Jockusch B. M., Graf T. Differential expression of Rous Sarcoma virus-specific transformation parameters in enucleated cells. Cell. 1978 Aug;14(4):843–856. doi: 10.1016/0092-8674(78)90340-9. [DOI] [PubMed] [Google Scholar]
  5. Binétruy B., Smeal T., Karin M. Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature. 1991 May 9;351(6322):122–127. doi: 10.1038/351122a0. [DOI] [PubMed] [Google Scholar]
  6. Biquard J. M., Vigier P. Characteristics of a conditional mutant of Rous sarcoma virus defective in ability to transform cells at high temperature. Virology. 1972 Feb;47(2):444–455. doi: 10.1016/0042-6822(72)90280-2. [DOI] [PubMed] [Google Scholar]
  7. Black E. J., Street A. J., Gillespie D. A. Protein phosphatase 2A reverses phosphorylation of c-Jun specified by the delta domain in vitro: correlation with oncogenic activation and deregulated transactivation activity of v-Jun. Oncogene. 1991 Nov;6(11):1949–1958. [PubMed] [Google Scholar]
  8. Boyle W. J., Smeal T., Defize L. H., Angel P., Woodgett J. R., Karin M., Hunter T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell. 1991 Feb 8;64(3):573–584. doi: 10.1016/0092-8674(91)90241-p. [DOI] [PubMed] [Google Scholar]
  9. Brach M. A., Gruss H. J., Sott C., Herrmann F. The mitogenic response to tumor necrosis factor alpha requires c-Jun/AP-1. Mol Cell Biol. 1993 Jul;13(7):4284–4290. doi: 10.1128/mcb.13.7.4284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown P. H., Alani R., Preis L. H., Szabo E., Birrer M. J. Suppression of oncogene-induced transformation by a deletion mutant of c-jun. Oncogene. 1993 Apr;8(4):877–886. [PubMed] [Google Scholar]
  11. Calothy G., Laugier D., Cross F. R., Jove R., Hanafusa T., Hanafusa H. The membrane-binding domain and myristylation of p60v-src are not essential for stimulation of cell proliferation. J Virol. 1987 May;61(5):1678–1681. doi: 10.1128/jvi.61.5.1678-1681.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Catling A. D., Wyke J. A., Frame M. C. Mitogenesis of quiescent chick fibroblasts by v-Src: dependence on events at the membrane leading to early changes in AP-1. Oncogene. 1993 Jul;8(7):1875–1886. [PubMed] [Google Scholar]
  13. Crouch D. H., Lang C., Gillespie D. A. The leucine zipper domain of avian cMyc is required for transformation and autoregulation. Oncogene. 1990 May;5(5):683–689. [PubMed] [Google Scholar]
  14. Durkin J. P., Whitfield J. F. Partial characterization of the mitogenic action of pp60v-src, the oncogenic protein product of the src gene of avian sarcoma virus. J Cell Physiol. 1984 Aug;120(2):135–145. doi: 10.1002/jcp.1041200205. [DOI] [PubMed] [Google Scholar]
  15. Dérijard B., Hibi M., Wu I. H., Barrett T., Su B., Deng T., Karin M., Davis R. J. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994 Mar 25;76(6):1025–1037. doi: 10.1016/0092-8674(94)90380-8. [DOI] [PubMed] [Google Scholar]
  16. Frame M. C., Wilkie N. M., Darling A. J., Chudleigh A., Pintzas A., Lang J. C., Gillespie D. A. Regulation of AP-1/DNA complex formation in vitro. Oncogene. 1991 Feb;6(2):205–209. [PubMed] [Google Scholar]
  17. Fuerstenberg S., Beug H., Introna M., Khazaie K., Muñoz A., Ness S., Nordström K., Sap J., Stanley I., Zenke M. Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector. J Virol. 1990 Dec;64(12):5891–5902. doi: 10.1128/jvi.64.12.5891-5902.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gentz R., Rauscher F. J., 3rd, Abate C., Curran T. Parallel association of Fos and Jun leucine zippers juxtaposes DNA binding domains. Science. 1989 Mar 31;243(4899):1695–1699. doi: 10.1126/science.2494702. [DOI] [PubMed] [Google Scholar]
  19. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kanner S. B., Reynolds A. B., Vines R. R., Parsons J. T. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A. 1990 May;87(9):3328–3332. doi: 10.1073/pnas.87.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kawai S., Hanafusa H. The effects of reciprocal changes in temperature on the transformed state of cells infected with a rous sarcoma virus mutant. Virology. 1971 Nov;46(2):470–479. doi: 10.1016/0042-6822(71)90047-x. [DOI] [PubMed] [Google Scholar]
  22. Kouzarides T., Ziff E. The role of the leucine zipper in the fos-jun interaction. Nature. 1988 Dec 15;336(6200):646–651. doi: 10.1038/336646a0. [DOI] [PubMed] [Google Scholar]
  23. Kumar V., Green S., Staub A., Chambon P. Localisation of the oestradiol-binding and putative DNA-binding domains of the human oestrogen receptor. EMBO J. 1986 Sep;5(9):2231–2236. doi: 10.1002/j.1460-2075.1986.tb04489.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  25. Ledwith B. J., Manam S., Kraynak A. R., Nichols W. W., Bradley M. O. Antisense-fos RNA causes partial reversion of the transformed phenotypes induced by the c-Ha-ras oncogene. Mol Cell Biol. 1990 Apr;10(4):1545–1555. doi: 10.1128/mcb.10.4.1545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lin A., Frost J., Deng T., Smeal T., al-Alawi N., Kikkawa U., Hunter T., Brenner D., Karin M. Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell. 1992 Sep 4;70(5):777–789. doi: 10.1016/0092-8674(92)90311-y. [DOI] [PubMed] [Google Scholar]
  27. Lloyd A., Yancheva N., Wasylyk B. Transformation suppressor activity of a Jun transcription factor lacking its activation domain. Nature. 1991 Aug 15;352(6336):635–638. doi: 10.1038/352635a0. [DOI] [PubMed] [Google Scholar]
  28. Marais R., Wynne J., Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell. 1993 Apr 23;73(2):381–393. doi: 10.1016/0092-8674(93)90237-k. [DOI] [PubMed] [Google Scholar]
  29. Miao G. G., Curran T. Cell transformation by c-fos requires an extended period of expression and is independent of the cell cycle. Mol Cell Biol. 1994 Jun;14(6):4295–4310. doi: 10.1128/mcb.14.6.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nishikura K., Murray J. M. Antisense RNA of proto-oncogene c-fos blocks renewed growth of quiescent 3T3 cells. Mol Cell Biol. 1987 Feb;7(2):639–649. doi: 10.1128/mcb.7.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Okuno H., Suzuki T., Yoshida T., Hashimoto Y., Curran T., Iba H. Inhibition of jun transformation by a mutated fos gene: design of an anti-oncogene. Oncogene. 1991 Sep;6(9):1491–1497. [PubMed] [Google Scholar]
  32. Pawson T., Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. doi: 10.1016/0960-9822(93)90350-w. [DOI] [PubMed] [Google Scholar]
  33. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  34. Ransone L. J., Visvader J., Wamsley P., Verma I. M. Trans-dominant negative mutants of Fos and Jun. Proc Natl Acad Sci U S A. 1990 May;87(10):3806–3810. doi: 10.1073/pnas.87.10.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schaller M. D., Hildebrand J. D., Shannon J. D., Fox J. W., Vines R. R., Parsons J. T. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol. 1994 Mar;14(3):1680–1688. doi: 10.1128/mcb.14.3.1680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schaller M. D., Parsons J. T. Focal adhesion kinase: an integrin-linked protein tyrosine kinase. Trends Cell Biol. 1993 Aug;3(8):258–262. doi: 10.1016/0962-8924(93)90053-4. [DOI] [PubMed] [Google Scholar]
  38. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  39. Seidel-Dugan C., Meyer B. E., Thomas S. M., Brugge J. S. Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src. Mol Cell Biol. 1992 Apr;12(4):1835–1845. doi: 10.1128/mcb.12.4.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Smeal T., Angel P., Meek J., Karin M. Different requirements for formation of Jun: Jun and Jun: Fos complexes. Genes Dev. 1989 Dec;3(12B):2091–2100. doi: 10.1101/gad.3.12b.2091. [DOI] [PubMed] [Google Scholar]
  41. Smeal T., Binetruy B., Mercola D. A., Birrer M., Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature. 1991 Dec 12;354(6353):494–496. doi: 10.1038/354494a0. [DOI] [PubMed] [Google Scholar]
  42. Smith M. R., DeGudicibus S. J., Stacey D. W. Requirement for c-ras proteins during viral oncogene transformation. Nature. 1986 Apr 10;320(6062):540–543. doi: 10.1038/320540a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Turner R., Tjian R. Leucine repeats and an adjacent DNA binding domain mediate the formation of functional cFos-cJun heterodimers. Science. 1989 Mar 31;243(4899):1689–1694. doi: 10.1126/science.2494701. [DOI] [PubMed] [Google Scholar]
  44. Weber M. J., Friis R. R. Dissociation of transformation parameters using temperature-conditional mutants of Rous sarcoma virus. Cell. 1979 Jan;16(1):25–32. doi: 10.1016/0092-8674(79)90184-3. [DOI] [PubMed] [Google Scholar]
  45. Welham M. J., Wyke J. A., Lang A., Wyke A. W. Mitogenesis induced by pp60v-src is not accompanied by increased expression of immediate early response genes. Oncogene. 1990 Feb;5(2):161–169. [PubMed] [Google Scholar]
  46. Wick M., Lucibello F. C., Müller R. Inhibition of Fos- and Ras-induced transformation by mutant Fos proteins with structural alterations in functionally different domains. Oncogene. 1992 May;7(5):859–867. [PubMed] [Google Scholar]
  47. Wyke A. W., Cushley W., Wyke J. A. Mitogenesis by v-Src: a need for active oncoprotein both in leaving G0 and in completing G1 phases of the cell cycle. Cell Growth Differ. 1993 Aug;4(8):671–678. [PubMed] [Google Scholar]
  48. Yoshida T., Shindo Y., Ohta K., Iba H. Identification of a small region of the v-fos gene product that is sufficient for transforming potential and growth-stimulating activity. Oncogene Res. 1989;5(2):79–89. [PubMed] [Google Scholar]
  49. Zachary I., Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992 Dec 11;71(6):891–894. doi: 10.1016/0092-8674(92)90385-p. [DOI] [PubMed] [Google Scholar]

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