Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1992 Feb;12(2):444–454. doi: 10.1128/mcb.12.2.444

Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative.

S M Ruben 1, R Narayanan 1, J F Klement 1, C H Chen 1, C A Rosen 1
PMCID: PMC364189  PMID: 1732726

Abstract

The NF-kappa B transcription factor complex is composed of two proteins, designated p50 and p65, both having considerable homology to the product of the rel oncogene. We present evidence that the p65 subunit is a potent transcriptional activator in the apparent absence of the p50 subunit, consistent with in vitro results demonstrating that p65 can interact with DNA on its own. To identify the minimal activation domain, chimeric fusion proteins between the DNA binding domain of the yeast transcriptional activator protein GAL4 and regions of the carboxy terminus of p65 were constructed, and their transcriptional activity was assessed by using a GAL4 upstream activation sequence-driven promoter-chloramphenicol acetyltransferase fusion. This analysis suggests that the boundaries of the activation domain lie between amino acids 415 and 550. Moreover, single amino acid changes within residues 435 to 459 greatly diminished activation. Similar to other activation domains, this region contains a leucine zipper-like motif as well as an overall net negative charge. To identify those residues essential for DNA binding, we made use of a naturally occurring derivative of p65, lacking residues 222 to 231 (hereafter referred to as p65 delta), and produced via an alternative splice site. Gel mobility shift analysis using bacterially expressed p65, p65 delta, and various mutants indicates that residues 222 to 231 are important for binding to kappa B DNA. Coimmunoprecipitation analysis suggests that these residues likely contribute to the multimerization function required for homomeric complex formation or heteromeric complex formation with p50 in that no association of p65 delta with itself or with p50 was evident. However, p65 delta was able to form weak heteromeric complexes with p65 that were greatly reduced in their ability to bind DNA. On the basis of these findings, we suggest that subtle changes within the proposed multimerization domain can elicit different effects with the individual Rel-related proteins and that a potential role of p65 delta may be to negatively regulate NF-kappa B function through formation of nonfunctional heteromeric complexes.

Full text

PDF
446

Images in this article

446Image
on p.446
447Image
on p.447
448Image
on p.448
449Image
on p.449
450Image
on p.450
450Image
on p.450
450Image
on p.450

Selected References

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

  1. Baeuerle P. A., Baltimore D. A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB. Genes Dev. 1989 Nov;3(11):1689–1698. doi: 10.1101/gad.3.11.1689. [DOI] [PubMed] [Google Scholar]
  2. Baeuerle P. A., Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988 Apr 22;53(2):211–217. doi: 10.1016/0092-8674(88)90382-0. [DOI] [PubMed] [Google Scholar]
  3. Baeuerle P. A., Baltimore D. I kappa B: a specific inhibitor of the NF-kappa B transcription factor. Science. 1988 Oct 28;242(4878):540–546. doi: 10.1126/science.3140380. [DOI] [PubMed] [Google Scholar]
  4. Ballard D. W., Böhnlein E., Lowenthal J. W., Wano Y., Franza B. R., Greene W. C. HTLV-I tax induces cellular proteins that activate the kappa B element in the IL-2 receptor alpha gene. Science. 1988 Sep 23;241(4873):1652–1655. doi: 10.1126/science.241.4873.1652. [DOI] [PubMed] [Google Scholar]
  5. Brownell E., Mittereder N., Rice N. R. A human rel proto-oncogene cDNA containing an Alu fragment as a potential coding exon. Oncogene. 1989 Jul;4(7):935–942. [PubMed] [Google Scholar]
  6. Bull P., Morley K. L., Hoekstra M. F., Hunter T., Verma I. M. The mouse c-rel protein has an N-terminal regulatory domain and a C-terminal transcriptional transactivation domain. Mol Cell Biol. 1990 Oct;10(10):5473–5485. doi: 10.1128/mcb.10.10.5473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Busch S. J., Sassone-Corsi P. Dimers, leucine zippers and DNA-binding domains. Trends Genet. 1990 Feb;6(2):36–40. doi: 10.1016/0168-9525(90)90071-d. [DOI] [PubMed] [Google Scholar]
  8. Böhnlein E., Lowenthal J. W., Siekevitz M., Ballard D. W., Franza B. R., Greene W. C. The same inducible nuclear proteins regulates mitogen activation of both the interleukin-2 receptor-alpha gene and type 1 HIV. Cell. 1988 Jun 3;53(5):827–836. doi: 10.1016/0092-8674(88)90099-2. [DOI] [PubMed] [Google Scholar]
  9. Capobianco A. J., Simmons D. L., Gilmore T. D. Cloning and expression of a chicken c-rel cDNA: unlike p59v-rel, p68c-rel is a cytoplasmic protein in chicken embryo fibroblasts. Oncogene. 1990 Mar;5(3):257–265. [PubMed] [Google Scholar]
  10. Dexter T. M., Allen T. D., Lajtha L. G. Conditions controlling the proliferation of haemopoietic stem cells in vitro. J Cell Physiol. 1977 Jun;91(3):335–344. doi: 10.1002/jcp.1040910303. [DOI] [PubMed] [Google Scholar]
  11. Dillon P. J., Nelbock P., Perkins A., Rosen C. A. Function of the human immunodeficiency virus types 1 and 2 Rev proteins is dependent on their ability to interact with a structured region present in env gene mRNA. J Virol. 1990 Sep;64(9):4428–4437. doi: 10.1128/jvi.64.9.4428-4437.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dorshkind K. Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol. 1990;8:111–137. doi: 10.1146/annurev.iy.08.040190.000551. [DOI] [PubMed] [Google Scholar]
  13. Felber B. K., Paskalis H., Kleinman-Ewing C., Wong-Staal F., Pavlakis G. N. The pX protein of HTLV-I is a transcriptional activator of its long terminal repeats. Science. 1985 Aug 16;229(4714):675–679. doi: 10.1126/science.2992082. [DOI] [PubMed] [Google Scholar]
  14. Fujisawa J., Seiki M., Kiyokawa T., Yoshida M. Functional activation of the long terminal repeat of human T-cell leukemia virus type I by a trans-acting factor. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2277–2281. doi: 10.1073/pnas.82.8.2277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gentz R., Chen C. H., Rosen C. A. Bioassay for trans-activation using purified human immunodeficiency virus tat-encoded protein: trans-activation requires mRNA synthesis. Proc Natl Acad Sci U S A. 1989 Feb;86(3):821–824. doi: 10.1073/pnas.86.3.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ghosh S., Baltimore D. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature. 1990 Apr 12;344(6267):678–682. doi: 10.1038/344678a0. [DOI] [PubMed] [Google Scholar]
  17. Ghosh S., Gifford A. M., Riviere L. R., Tempst P., Nolan G. P., Baltimore D. Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal. Cell. 1990 Sep 7;62(5):1019–1029. doi: 10.1016/0092-8674(90)90276-k. [DOI] [PubMed] [Google Scholar]
  18. Gilmore T. D., Temin H. M. Different localization of the product of the v-rel oncogene in chicken fibroblasts and spleen cells correlates with transformation by REV-T. Cell. 1986 Mar 14;44(5):791–800. doi: 10.1016/0092-8674(86)90845-7. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Grumont R. J., Gerondakis S. Structure of a mammalian c-rel protein deduced from the nucleotide sequence of murine cDNA clones. Oncogene Res. 1989;4(1):1–8. [PubMed] [Google Scholar]
  21. Gélinas C., Temin H. M. The v-rel oncogene encodes a cell-specific transcriptional activator of certain promoters. Oncogene. 1988 Oct;3(4):349–355. [PubMed] [Google Scholar]
  22. Hannink M., Temin H. M. Transactivation of gene expression by nuclear and cytoplasmic rel proteins. Mol Cell Biol. 1989 Oct;9(10):4323–4336. doi: 10.1128/mcb.9.10.4323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haskill S., Beg A. A., Tompkins S. M., Morris J. S., Yurochko A. D., Sampson-Johannes A., Mondal K., Ralph P., Baldwin A. S., Jr Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity. Cell. 1991 Jun 28;65(7):1281–1289. doi: 10.1016/0092-8674(91)90022-q. [DOI] [PubMed] [Google Scholar]
  24. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  25. Ip Y. T., Kraut R., Levine M., Rushlow C. A. The dorsal morphogen is a sequence-specific DNA-binding protein that interacts with a long-range repression element in Drosophila. Cell. 1991 Jan 25;64(2):439–446. doi: 10.1016/0092-8674(91)90651-e. [DOI] [PubMed] [Google Scholar]
  26. Kakidani H., Ptashne M. GAL4 activates gene expression in mammalian cells. Cell. 1988 Jan 29;52(2):161–167. doi: 10.1016/0092-8674(88)90504-1. [DOI] [PubMed] [Google Scholar]
  27. Kamens J., Richardson P., Mosialos G., Brent R., Gilmore T. Oncogenic transformation by vrel requires an amino-terminal activation domain. Mol Cell Biol. 1990 Jun;10(6):2840–2847. doi: 10.1128/mcb.10.6.2840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kieran M., Blank V., Logeat F., Vandekerckhove J., Lottspeich F., Le Bail O., Urban M. B., Kourilsky P., Baeuerle P. A., Israël A. The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product. Cell. 1990 Sep 7;62(5):1007–1018. doi: 10.1016/0092-8674(90)90275-j. [DOI] [PubMed] [Google Scholar]
  29. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [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. Lenardo M. J., Baltimore D. NF-kappa B: a pleiotropic mediator of inducible and tissue-specific gene control. Cell. 1989 Jul 28;58(2):227–229. doi: 10.1016/0092-8674(89)90833-7. [DOI] [PubMed] [Google Scholar]
  32. Leung K., Nabel G. J. HTLV-1 transactivator induces interleukin-2 receptor expression through an NF-kappa B-like factor. Nature. 1988 Jun 23;333(6175):776–778. doi: 10.1038/333776a0. [DOI] [PubMed] [Google Scholar]
  33. McLeod D. L., Shreeve M. M., Axelrad A. A. Improved plasma culture system for production of erythrocytic colonies in vitro: quantitative assay method for CFU-E. Blood. 1974 Oct;44(4):517–534. [PubMed] [Google Scholar]
  34. Mermod N., O'Neill E. A., Kelly T. J., Tjian R. The proline-rich transcriptional activator of CTF/NF-I is distinct from the replication and DNA binding domain. Cell. 1989 Aug 25;58(4):741–753. doi: 10.1016/0092-8674(89)90108-6. [DOI] [PubMed] [Google Scholar]
  35. Narayanan R., Tare N. S., Benjamin W. R., Gubler U. A sensitive technique to monitor gene transfer and expression in bone marrow stem cells. Exp Hematol. 1989 Aug;17(7):832–835. [PubMed] [Google Scholar]
  36. Nolan G. P., Ghosh S., Liou H. C., Tempst P., Baltimore D. DNA binding and I kappa B inhibition of the cloned p65 subunit of NF-kappa B, a rel-related polypeptide. Cell. 1991 Mar 8;64(5):961–969. doi: 10.1016/0092-8674(91)90320-x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Osborn L., Kunkel S., Nabel G. J. Tumor necrosis factor alpha and interleukin 1 stimulate the human immunodeficiency virus enhancer by activation of the nuclear factor kappa B. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2336–2340. doi: 10.1073/pnas.86.7.2336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Queen C., Baltimore D. Immunoglobulin gene transcription is activated by downstream sequence elements. Cell. 1983 Jul;33(3):741–748. doi: 10.1016/0092-8674(83)90016-8. [DOI] [PubMed] [Google Scholar]
  40. Richardson P. M., Gilmore T. D. vRel is an inactive member of the Rel family of transcriptional activating proteins. J Virol. 1991 Jun;65(6):3122–3130. doi: 10.1128/jvi.65.6.3122-3130.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Roth S., Stein D., Nüsslein-Volhard C. A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo. Cell. 1989 Dec 22;59(6):1189–1202. doi: 10.1016/0092-8674(89)90774-5. [DOI] [PubMed] [Google Scholar]
  42. Ruben S. M., Dillon P. J., Schreck R., Henkel T., Chen C. H., Maher M., Baeuerle P. A., Rosen C. A. Isolation of a rel-related human cDNA that potentially encodes the 65-kD subunit of NF-kappa B. Science. 1991 Mar 22;251(5000):1490–1493. doi: 10.1126/science.2006423. [DOI] [PubMed] [Google Scholar]
  43. Ruben S. M., Rosen C. A. Suppression of signals required for activation of transcription factor NF-kappa B in cells constitutively expressing the HTLV-I Tax protein. New Biol. 1990 Oct;2(10):894–902. [PubMed] [Google Scholar]
  44. Ruben S., Poteat H., Tan T. H., Kawakami K., Roeder R., Haseltine W., Rosen C. A. Cellular transcription factors and regulation of IL-2 receptor gene expression by HTLV-I tax gene product. Science. 1988 Jul 1;241(4861):89–92. doi: 10.1126/science.2838905. [DOI] [PubMed] [Google Scholar]
  45. Rushlow C. A., Han K., Manley J. L., Levine M. The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila. Cell. 1989 Dec 22;59(6):1165–1177. doi: 10.1016/0092-8674(89)90772-1. [DOI] [PubMed] [Google Scholar]
  46. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  48. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  49. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Seiki M., Inoue J., Takeda T., Yoshida M. Direct evidence that p40x of human T-cell leukemia virus type I is a trans-acting transcriptional activator. EMBO J. 1986 Mar;5(3):561–565. doi: 10.1002/j.1460-2075.1986.tb04247.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Slamon D. J., Shimotohno K., Cline M. J., Golde D. W., Chen I. S. Identification of the putative transforming protein of the human T-cell leukemia viruses HTLV-I and HTLV-II. Science. 1984 Oct 5;226(4670):61–65. doi: 10.1126/science.6089351. [DOI] [PubMed] [Google Scholar]
  53. Steward R. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel. Science. 1987 Oct 30;238(4827):692–694. doi: 10.1126/science.3118464. [DOI] [PubMed] [Google Scholar]
  54. Steward R. Relocalization of the dorsal protein from the cytoplasm to the nucleus correlates with its function. Cell. 1989 Dec 22;59(6):1179–1188. doi: 10.1016/0092-8674(89)90773-3. [DOI] [PubMed] [Google Scholar]
  55. Struhl K. The JUN oncoprotein, a vertebrate transcription factor, activates transcription in yeast. Nature. 1988 Apr 14;332(6165):649–650. doi: 10.1038/332649a0. [DOI] [PubMed] [Google Scholar]
  56. Urban M. B., Baeuerle P. A. The 65-kD subunit of NF-kappa B is a receptor for I kappa B and a modulator of DNA-binding specificity. Genes Dev. 1990 Nov;4(11):1975–1984. doi: 10.1101/gad.4.11.1975. [DOI] [PubMed] [Google Scholar]
  57. Urban M. B., Schreck R., Baeuerle P. A. NF-kappa B contacts DNA by a heterodimer of the p50 and p65 subunit. EMBO J. 1991 Jul;10(7):1817–1825. doi: 10.1002/j.1460-2075.1991.tb07707.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Urban M. B., Schreck R., Baeuerle P. A. NF-kappa B contacts DNA by a heterodimer of the p50 and p65 subunit. EMBO J. 1991 Jul;10(7):1817–1825. doi: 10.1002/j.1460-2075.1991.tb07707.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Whitlock C. A., Witte O. N. Long-term culture of B lymphocytes and their precursors from murine bone marrow. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3608–3612. doi: 10.1073/pnas.79.11.3608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Williams T., Admon A., Lüscher B., Tjian R. Cloning and expression of AP-2, a cell-type-specific transcription factor that activates inducible enhancer elements. Genes Dev. 1988 Dec;2(12A):1557–1569. doi: 10.1101/gad.2.12a.1557. [DOI] [PubMed] [Google Scholar]
  61. Ziff E. B. Transcription factors: a new family gathers at the cAMP response site. Trends Genet. 1990 Mar;6(3):69–72. doi: 10.1016/0168-9525(90)90081-g. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES