Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Apr 11;22(7):1305–1312. doi: 10.1093/nar/22.7.1305

Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain.

W K Hoeffler 1, A D Levinson 1, E A Bauer 1
PMCID: PMC523657  PMID: 8165146

Abstract

C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products.

Full text

PDF
1305

Images in this article

1306Image
on p.1306
1307Image
on p.1307
1307Image
on p.1307
1309Image
on p.1309
1309Image
on p.1309

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. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  3. Angel P., Baumann I., Stein B., Delius H., Rahmsdorf H. J., Herrlich P. 12-O-tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol Cell Biol. 1987 Jun;7(6):2256–2266. doi: 10.1128/mcb.7.6.2256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  5. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  6. Auwerx J., Sassone-Corsi P. IP-1: a dominant inhibitor of Fos/Jun whose activity is modulated by phosphorylation. Cell. 1991 Mar 8;64(5):983–993. doi: 10.1016/0092-8674(91)90322-p. [DOI] [PubMed] [Google Scholar]
  7. Baichwal V. R., Park A., Tjian R. v-Src and EJ Ras alleviate repression of c-Jun by a cell-specific inhibitor. Nature. 1991 Jul 11;352(6331):165–168. doi: 10.1038/352165a0. [DOI] [PubMed] [Google Scholar]
  8. Baichwal V. R., Tjian R. Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun. Cell. 1990 Nov 16;63(4):815–825. doi: 10.1016/0092-8674(90)90147-7. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Bohmann D., Bos T. J., Admon A., Nishimura T., Vogt P. K., Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science. 1987 Dec 4;238(4832):1386–1392. doi: 10.1126/science.2825349. [DOI] [PubMed] [Google Scholar]
  11. Boshart M., Weih F., Nichols M., Schütz G. The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase. Cell. 1991 Sep 6;66(5):849–859. doi: 10.1016/0092-8674(91)90432-x. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Cantley L. C., Auger K. R., Carpenter C., Duckworth B., Graziani A., Kapeller R., Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. doi: 10.1016/0092-8674(91)90639-g. [DOI] [PubMed] [Google Scholar]
  14. Fried M., Crothers D. M. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. doi: 10.1093/nar/9.23.6505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gonzalez G. A., Montminy M. R. Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell. 1989 Nov 17;59(4):675–680. doi: 10.1016/0092-8674(89)90013-5. [DOI] [PubMed] [Google Scholar]
  17. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Greenberg M. E., Ziff E. B. Stimulation of 3T3 cells induces transcription of the c-fos proto-oncogene. Nature. 1984 Oct 4;311(5985):433–438. doi: 10.1038/311433a0. [DOI] [PubMed] [Google Scholar]
  19. Hoeffler W. K., Kovelman R., Roeder R. G. Activation of transcription factor IIIC by the adenovirus E1A protein. Cell. 1988 Jun 17;53(6):907–920. doi: 10.1016/s0092-8674(88)90409-6. [DOI] [PubMed] [Google Scholar]
  20. Imler J. L., Ugarte E., Wasylyk C., Wasylyk B. v-jun is a transcriptional activator, but not in all cell-lines. Nucleic Acids Res. 1988 Apr 11;16(7):3005–3012. doi: 10.1093/nar/16.7.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lamb P., McKnight S. L. Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trends Biochem Sci. 1991 Nov;16(11):417–422. doi: 10.1016/0968-0004(91)90167-t. [DOI] [PubMed] [Google Scholar]
  22. Lee C. Q., Yun Y. D., Hoeffler J. P., Habener J. F. Cyclic-AMP-responsive transcriptional activation of CREB-327 involves interdependent phosphorylated subdomains. EMBO J. 1990 Dec;9(13):4455–4465. doi: 10.1002/j.1460-2075.1990.tb07896.x. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  23. Lee W., Mitchell P., Tjian R. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell. 1987 Jun 19;49(6):741–752. doi: 10.1016/0092-8674(87)90612-x. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. McBride A. A., Klausner R. D., Howley P. M. Conserved cysteine residue in the DNA-binding domain of the bovine papillomavirus type 1 E2 protein confers redox regulation of the DNA-binding activity in vitro. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7531–7535. doi: 10.1073/pnas.89.16.7531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Nordeen S. K., Green P. P., 3rd, Fowlkes D. M. A rapid, sensitive, and inexpensive assay for chloramphenicol acetyltransferase. DNA. 1987 Apr;6(2):173–178. doi: 10.1089/dna.1987.6.173. [DOI] [PubMed] [Google Scholar]
  28. Oehler T., Pintzas A., Stumm S., Darling A., Gillespie D., Angel P. Mutation of a phosphorylation site in the DNA-binding domain is required for redox-independent transactivation of AP1-dependent genes by v-Jun. Oncogene. 1993 May;8(5):1141–1147. [PubMed] [Google Scholar]
  29. Sassone-Corsi P., Lamph W. W., Kamps M., Verma I. M. fos-associated cellular p39 is related to nuclear transcription factor AP-1. Cell. 1988 Aug 12;54(4):553–560. doi: 10.1016/0092-8674(88)90077-3. [DOI] [PubMed] [Google Scholar]
  30. Schönthal A., Herrlich P., Rahmsdorf H. J., Ponta H. Requirement for fos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell. 1988 Jul 29;54(3):325–334. doi: 10.1016/0092-8674(88)90195-x. [DOI] [PubMed] [Google Scholar]
  31. Seeburg P. H., Colby W. W., Capon D. J., Goeddel D. V., Levinson A. D. Biological properties of human c-Ha-ras1 genes mutated at codon 12. Nature. 1984 Nov 1;312(5989):71–75. doi: 10.1038/312071a0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Smeal T., Binetruy B., Mercola D., Grover-Bardwick A., Heidecker G., Rapp U. R., Karin M. Oncoprotein-mediated signalling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol. 1992 Aug;12(8):3507–3513. doi: 10.1128/mcb.12.8.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Suva L. J., Winslow G. A., Wettenhall R. E., Hammonds R. G., Moseley J. M., Diefenbach-Jagger H., Rodda C. P., Kemp B. E., Rodriguez H., Chen E. Y. A parathyroid hormone-related protein implicated in malignant hypercalcemia: cloning and expression. Science. 1987 Aug 21;237(4817):893–896. doi: 10.1126/science.3616618. [DOI] [PubMed] [Google Scholar]
  35. Wasylyk C., Imler J. L., Wasylyk B. Transforming but not immortalizing oncogenes activate the transcription factor PEA1. EMBO J. 1988 Aug;7(8):2475–2483. doi: 10.1002/j.1460-2075.1988.tb03094.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wasylyk C., Wasylyk B., Heidecker G., Huleihel M., Rapp U. R. Expression of raf oncogenes activates the PEA1 transcription factor motif. Mol Cell Biol. 1989 May;9(5):2247–2250. doi: 10.1128/mcb.9.5.2247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Whitham S. E., Murphy G., Angel P., Rahmsdorf H. J., Smith B. J., Lyons A., Harris T. J., Reynolds J. J., Herrlich P., Docherty A. J. Comparison of human stromelysin and collagenase by cloning and sequence analysis. Biochem J. 1986 Dec 15;240(3):913–916. doi: 10.1042/bj2400913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xanthoudakis S., Miao G., Wang F., Pan Y. C., Curran T. Redox activation of Fos-Jun DNA binding activity is mediated by a DNA repair enzyme. EMBO J. 1992 Sep;11(9):3323–3335. doi: 10.1002/j.1460-2075.1992.tb05411.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yamamoto K. K., Gonzalez G. A., Biggs W. H., 3rd, Montminy M. R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988 Aug 11;334(6182):494–498. doi: 10.1038/334494a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES