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. 1991 Feb;11(2):954–962. doi: 10.1128/mcb.11.2.954

Intracellular leucine zipper interactions suggest c-Myc hetero-oligomerization.

C V Dang 1, J Barrett 1, M Villa-Garcia 1, L M Resar 1, G J Kato 1, E R Fearon 1
PMCID: PMC359758  PMID: 1990293

Abstract

The physiological significance of in vitro leucine zipper interactions was studied by the use of two strategies which detect specific protein-protein interactions in mammalian cells. Fusion genes were constructed which produce chimeric proteins containing leucine zipper domains from several proteins fused either to the DNA-binding domain of the Saccharomyces cerevisiae GAL4 protein or to the transcriptional activation domain of the herpes simplex virus VP16 protein. Previous studies in mammalian cells have demonstrated that a single chimeric polypeptide containing these two domains will activate transcription of a reporter gene present downstream of the GAL4 DNA-binding site. Similarly, if the GAL4 DNA-binding domain of a chimeric protein could be complexed through leucine zipper interactions with the VP16 activation domain of another chimeric protein, then transcriptional activation of the reporter gene would be detected. Using this strategy for detecting leucine zipper interactions, we observed homo-oligomerization between leucine zipper domains of the yeast protein GCN4 and hetero-oligomerization between leucine zipper regions from the mammalian transcriptional regulating proteins c-Jun and c-Fos. In contrast, homo-oligomerization of the leucine zipper domain from c-Myc was not detectable in cells. The inability of the c-Myc leucine zipper to homo-oligomerize strongly in cells was confirmed independently. The second strategy to detect leucine zipper interactions takes advantage of the observation that the addition of nuclear localization sequences to a cytoplasmic protein will allow the cytoplasmic protein to be transported to and retained in the nucleus. Chimeric genes encoding proteins with sequences from a cytoplasmic protein fused either to the GCN4 or c-Myc leucine zipper domains were constructed. Experiments with the c-Myc chimeric protein failed to demonstrate transport of the cytoplasmic marker protein to the nucleus in cells expressing the wild-type c-Myc protein. In contrast, the cytoplasmic marker was translocated into the nucleus when the GCN4 leucine zippers were present on both the cytoplasmic marker and a nuclear protein, presumably as a result of leucine zipper interaction. These results suggest that c-Myc function requires hetero-oligomerization to an as yet undefined factor.

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Selected References

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  1. Abate C., Luk D., Gagne E., Roeder R. G., Curran T. Fos and jun cooperate in transcriptional regulation via heterologous activation domains. Mol Cell Biol. 1990 Oct;10(10):5532–5535. doi: 10.1128/mcb.10.10.5532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Agre P., Johnson P. F., McKnight S. L. Cognate DNA binding specificity retained after leucine zipper exchange between GCN4 and C/EBP. Science. 1989 Nov 17;246(4932):922–926. doi: 10.1126/science.2530632. [DOI] [PubMed] [Google Scholar]
  3. Beckmann H., Su L. K., Kadesch T. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif. Genes Dev. 1990 Feb;4(2):167–179. doi: 10.1101/gad.4.2.167. [DOI] [PubMed] [Google Scholar]
  4. Bohmann D., Tjian R. Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell. 1989 Nov 17;59(4):709–717. doi: 10.1016/0092-8674(89)90017-2. [DOI] [PubMed] [Google Scholar]
  5. Brendel V., Karlin S. Too many leucine zippers? Nature. 1989 Oct 19;341(6243):574–575. doi: 10.1038/341574a0. [DOI] [PubMed] [Google Scholar]
  6. Bruzik J. P., Van Doren K., Hirsh D., Steitz J. A. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature. 1988 Oct 6;335(6190):559–562. doi: 10.1038/335559a0. [DOI] [PubMed] [Google Scholar]
  7. Carr C. S., Sharp P. A. A helix-loop-helix protein related to the immunoglobulin E box-binding proteins. Mol Cell Biol. 1990 Aug;10(8):4384–4388. doi: 10.1128/mcb.10.8.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Caspar D. L., Cohen C., Longley W. Tropomyosin: crystal structure, polymorphism and molecular interactions. J Mol Biol. 1969 Apr 14;41(1):87–107. doi: 10.1016/0022-2836(69)90128-4. [DOI] [PubMed] [Google Scholar]
  9. Cohen D. R., Curran T. Analysis of dimerization and DNA binding functions in Fos and Jun by domain-swapping: involvement of residues outside the leucine zipper/basic region. Oncogene. 1990 Jun;5(6):929–939. [PubMed] [Google Scholar]
  10. Cohen D. R., Ferreira P. C., Gentz R., Franza B. R., Jr, Curran T. The product of a fos-related gene, fra-1, binds cooperatively to the AP-1 site with Jun: transcription factor AP-1 is comprised of multiple protein complexes. Genes Dev. 1989 Feb;3(2):173–184. doi: 10.1101/gad.3.2.173. [DOI] [PubMed] [Google Scholar]
  11. Cole M. D. The myc oncogene: its role in transformation and differentiation. Annu Rev Genet. 1986;20:361–384. doi: 10.1146/annurev.ge.20.120186.002045. [DOI] [PubMed] [Google Scholar]
  12. Dang C. V., Lee W. M. Identification of the human c-myc protein nuclear translocation signal. Mol Cell Biol. 1988 Oct;8(10):4048–4054. doi: 10.1128/mcb.8.10.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dang C. V., Lee W. M. Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins. J Biol Chem. 1989 Oct 25;264(30):18019–18023. [PubMed] [Google Scholar]
  14. Dang C. V., McGuire M., Buckmire M., Lee W. M. Involvement of the 'leucine zipper' region in the oligomerization and transforming activity of human c-myc protein. Nature. 1989 Feb 16;337(6208):664–666. doi: 10.1038/337664a0. [DOI] [PubMed] [Google Scholar]
  15. Diamond M. I., Miner J. N., Yoshinaga S. K., Yamamoto K. R. Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science. 1990 Sep 14;249(4974):1266–1272. doi: 10.1126/science.2119054. [DOI] [PubMed] [Google Scholar]
  16. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  17. Fraser R. D., MacRae T. P. Structure of alpha-keratin. Nature. 1971 Sep 10;233(5315):138–140. doi: 10.1038/233138a0. [DOI] [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. Gregor P. D., Sawadogo M., Roeder R. G. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev. 1990 Oct;4(10):1730–1740. doi: 10.1101/gad.4.10.1730. [DOI] [PubMed] [Google Scholar]
  20. Halazonetis T. D., Georgopoulos K., Greenberg M. E., Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988 Dec 2;55(5):917–924. doi: 10.1016/0092-8674(88)90147-x. [DOI] [PubMed] [Google Scholar]
  21. Hirai S. I., Ryseck R. P., Mechta F., Bravo R., Yaniv M. Characterization of junD: a new member of the jun proto-oncogene family. EMBO J. 1989 May;8(5):1433–1439. doi: 10.1002/j.1460-2075.1989.tb03525.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hope I. A., Struhl K. GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. EMBO J. 1987 Sep;6(9):2781–2784. doi: 10.1002/j.1460-2075.1987.tb02573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hu Y. F., Lüscher B., Admon A., Mermod N., Tjian R. Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes Dev. 1990 Oct;4(10):1741–1752. doi: 10.1101/gad.4.10.1741. [DOI] [PubMed] [Google Scholar]
  24. Johnson P. F., McKnight S. L. Eukaryotic transcriptional regulatory proteins. Annu Rev Biochem. 1989;58:799–839. doi: 10.1146/annurev.bi.58.070189.004055. [DOI] [PubMed] [Google Scholar]
  25. Jones N. Transcriptional regulation by dimerization: two sides to an incestuous relationship. Cell. 1990 Apr 6;61(1):9–11. doi: 10.1016/0092-8674(90)90207-u. [DOI] [PubMed] [Google Scholar]
  26. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  27. Kato G. J., Barrett J., Villa-Garcia M., Dang C. V. An amino-terminal c-myc domain required for neoplastic transformation activates transcription. Mol Cell Biol. 1990 Nov;10(11):5914–5920. doi: 10.1128/mcb.10.11.5914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Landschulz W. H., Johnson P. F., McKnight S. L. The DNA binding domain of the rat liver nuclear protein C/EBP is bipartite. Science. 1989 Mar 31;243(4899):1681–1688. doi: 10.1126/science.2494700. [DOI] [PubMed] [Google Scholar]
  30. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  31. Lech K., Anderson K., Brent R. DNA-bound Fos proteins activate transcription in yeast. Cell. 1988 Jan 29;52(2):179–184. doi: 10.1016/0092-8674(88)90506-5. [DOI] [PubMed] [Google Scholar]
  32. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  33. Liu F., Green M. R. A specific member of the ATF transcription factor family can mediate transcription activation by the adenovirus E1a protein. Cell. 1990 Jun 29;61(7):1217–1224. doi: 10.1016/0092-8674(90)90686-9. [DOI] [PubMed] [Google Scholar]
  34. Ma J., Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell. 1987 Mar 13;48(5):847–853. doi: 10.1016/0092-8674(87)90081-x. [DOI] [PubMed] [Google Scholar]
  35. Mitchell P. J., Tjian R. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science. 1989 Jul 28;245(4916):371–378. doi: 10.1126/science.2667136. [DOI] [PubMed] [Google Scholar]
  36. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  37. Nakabeppu Y., Ryder K., Nathans D. DNA binding activities of three murine Jun proteins: stimulation by Fos. Cell. 1988 Dec 2;55(5):907–915. doi: 10.1016/0092-8674(88)90146-8. [DOI] [PubMed] [Google Scholar]
  38. O'Shea E. K., Rutkowski R., Kim P. S. Evidence that the leucine zipper is a coiled coil. Science. 1989 Jan 27;243(4890):538–542. doi: 10.1126/science.2911757. [DOI] [PubMed] [Google Scholar]
  39. Ptashne M., Gann A. A. Activators and targets. Nature. 1990 Jul 26;346(6282):329–331. doi: 10.1038/346329a0. [DOI] [PubMed] [Google Scholar]
  40. Ransone L. J., Visvader J., Sassone-Corsi P., Verma I. M. Fos-Jun interaction: mutational analysis of the leucine zipper domain of both proteins. Genes Dev. 1989 Jun;3(6):770–781. doi: 10.1101/gad.3.6.770. [DOI] [PubMed] [Google Scholar]
  41. Rauscher F. J., 3rd, Voulalas P. J., Franza B. R., Jr, Curran T. Fos and Jun bind cooperatively to the AP-1 site: reconstitution in vitro. Genes Dev. 1988 Dec;2(12B):1687–1699. doi: 10.1101/gad.2.12b.1687. [DOI] [PubMed] [Google Scholar]
  42. Sassone-Corsi P., Ransone L. J., Lamph W. W., Verma I. M. Direct interaction between fos and jun nuclear oncoproteins: role of the 'leucine zipper' domain. Nature. 1988 Dec 15;336(6200):692–695. doi: 10.1038/336692a0. [DOI] [PubMed] [Google Scholar]
  43. Seed B., Sheen J. Y. A simple phase-extraction assay for chloramphenicol acyltransferase activity. Gene. 1988 Jul 30;67(2):271–277. doi: 10.1016/0378-1119(88)90403-9. [DOI] [PubMed] [Google Scholar]
  44. Stone J., de Lange T., Ramsay G., Jakobovits E., Bishop J. M., Varmus H., Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. doi: 10.1128/mcb.7.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Struhl K. The DNA-binding domains of the jun oncoprotein and the yeast GCN4 transcriptional activator protein are functionally homologous. Cell. 1987 Sep 11;50(6):841–846. doi: 10.1016/0092-8674(87)90511-3. [DOI] [PubMed] [Google Scholar]
  46. Sullivan N., Green C., Pasdar M., Watt R. Characterization and nuclear localization of the v- and c-myc proteins. Curr Top Microbiol Immunol. 1986;132:355–361. doi: 10.1007/978-3-642-71562-4_52. [DOI] [PubMed] [Google Scholar]
  47. Triezenberg S. J., LaMarco K. L., McKnight S. L. Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16. Genes Dev. 1988 Jun;2(6):730–742. doi: 10.1101/gad.2.6.730. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Vinson C. R., Sigler P. B., McKnight S. L. Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science. 1989 Nov 17;246(4932):911–916. doi: 10.1126/science.2683088. [DOI] [PubMed] [Google Scholar]

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