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. 1993 Sep 25;21(19):4516–4523. doi: 10.1093/nar/21.19.4516

Interaction of the C-terminal region of p105 with the nuclear localisation signal of p50 is required for inhibition of NF-kappa B DNA binding activity.

J R Matthews 1, E Watson 1, S Buckley 1, R T Hay 1
PMCID: PMC311184  PMID: 7694229

Abstract

DNA binding of the homodimeric p50 subunit of NF-kappa B was inhibited by a bacterially expressed protein containing the ankyrin repeats present in the C-terminus of the p105 precursor but not by the I kappa B protein MAD-3. However p50 was retained on protein affinity matrices containing either the C-terminal ankyrin repeats of p105 or MAD-3. To investigate the interaction between p50 and proteins containing ankyrin repeats we have used a number of approaches to probe the accessibility of the p50 nuclear localisation signal in the protein complex. A monoclonal antibody recognising a linear epitope either very close to, or including, the nuclear localisation signal of the p50 protein could immunoprecipitate p50 homodimers but was unable to precipitate the protein when it was bound to the C-terminal region of p105. A close association between the nuclear localisation signal of p50 and the C-terminal region of p105 was also suggested by protease accessibility experiments. While the nuclear localisation signal of free p50 is extremely susceptible to cleavage with trypsin the same site is masked in the presence of the C-terminal ankyrin repeats of p105 and, to a lesser extent MAD-3. Removal of the nuclear localisation signal by trypsin digestion generates a protein that is fully competent for DNA binding but is refractile to inhibition by the C-terminal ankyrin repeats of p105. Addition of DNA destabilises complexes between p50 and ankyrin repeat containing proteins, increasing the susceptibility of the nuclear localisation signal to trypsin cleavage. The data suggest that there is a rapid exchange of p50 between complexes containing DNA or I kappa B proteins via a metastable complex containing DNA, p50 and I kappa B.

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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. 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]
  2. 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]
  3. Baeuerle P. A., Lenardo M., Pierce J. W., Baltimore D. Phorbol-ester-induced activation of the NF-kappa B transcription factor involves dissociation of an apparently cytoplasmic NF-kappa B/inhibitor complex. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):789–798. doi: 10.1101/sqb.1988.053.01.089. [DOI] [PubMed] [Google Scholar]
  4. Beg A. A., Ruben S. M., Scheinman R. I., Haskill S., Rosen C. A., Baldwin A. S., Jr I kappa B interacts with the nuclear localization sequences of the subunits of NF-kappa B: a mechanism for cytoplasmic retention. Genes Dev. 1992 Oct;6(10):1899–1913. doi: 10.1101/gad.6.10.1899. [DOI] [PubMed] [Google Scholar]
  5. Blank V., Kourilsky P., Israël A. Cytoplasmic retention, DNA binding and processing of the NF-kappa B p50 precursor are controlled by a small region in its C-terminus. EMBO J. 1991 Dec;10(13):4159–4167. doi: 10.1002/j.1460-2075.1991.tb04994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bours V., Burd P. R., Brown K., Villalobos J., Park S., Ryseck R. P., Bravo R., Kelly K., Siebenlist U. A novel mitogen-inducible gene product related to p50/p105-NF-kappa B participates in transactivation through a kappa B site. Mol Cell Biol. 1992 Feb;12(2):685–695. doi: 10.1128/mcb.12.2.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bours V., Franzoso G., Azarenko V., Park S., Kanno T., Brown K., Siebenlist U. The oncoprotein Bcl-3 directly transactivates through kappa B motifs via association with DNA-binding p50B homodimers. Cell. 1993 Mar 12;72(5):729–739. doi: 10.1016/0092-8674(93)90401-b. [DOI] [PubMed] [Google Scholar]
  8. Bours V., Villalobos J., Burd P. R., Kelly K., Siebenlist U. Cloning of a mitogen-inducible gene encoding a kappa B DNA-binding protein with homology to the rel oncogene and to cell-cycle motifs. Nature. 1990 Nov 1;348(6296):76–80. doi: 10.1038/348076a0. [DOI] [PubMed] [Google Scholar]
  9. Breeden L., Nasmyth K. Similarity between cell-cycle genes of budding yeast and fission yeast and the Notch gene of Drosophila. Nature. 1987 Oct 15;329(6140):651–654. doi: 10.1038/329651a0. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Davis N., Ghosh S., Simmons D. L., Tempst P., Liou H. C., Baltimore D., Bose H. R., Jr Rel-associated pp40: an inhibitor of the rel family of transcription factors. Science. 1991 Sep 13;253(5025):1268–1271. doi: 10.1126/science.1891714. [DOI] [PubMed] [Google Scholar]
  13. Fujita T., Nolan G. P., Ghosh S., Baltimore D. Independent modes of transcriptional activation by the p50 and p65 subunits of NF-kappa B. Genes Dev. 1992 May;6(5):775–787. doi: 10.1101/gad.6.5.775. [DOI] [PubMed] [Google Scholar]
  14. Geisler R., Bergmann A., Hiromi Y., Nüsslein-Volhard C. cactus, a gene involved in dorsoventral pattern formation of Drosophila, is related to the I kappa B gene family of vertebrates. Cell. 1992 Nov 13;71(4):613–621. doi: 10.1016/0092-8674(92)90595-4. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Grimm S., Baeuerle P. A. The inducible transcription factor NF-kappa B: structure-function relationship of its protein subunits. Biochem J. 1993 Mar 1;290(Pt 2):297–308. doi: 10.1042/bj2900297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Hatada E. N., Nieters A., Wulczyn F. G., Naumann M., Meyer R., Nucifora G., McKeithan T. W., Scheidereit C. The ankyrin repeat domains of the NF-kappa B precursor p105 and the protooncogene bcl-3 act as specific inhibitors of NF-kappa B DNA binding. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2489–2493. doi: 10.1073/pnas.89.6.2489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Henkel T., Zabel U., van Zee K., Müller J. M., Fanning E., Baeuerle P. A. Intramolecular masking of the nuclear location signal and dimerization domain in the precursor for the p50 NF-kappa B subunit. Cell. 1992 Mar 20;68(6):1121–1133. doi: 10.1016/0092-8674(92)90083-o. [DOI] [PubMed] [Google Scholar]
  20. Inoue J., Kerr L. D., Kakizuka A., Verma I. M. I kappa B gamma, a 70 kd protein identical to the C-terminal half of p110 NF-kappa B: a new member of the I kappa B family. Cell. 1992 Mar 20;68(6):1109–1120. doi: 10.1016/0092-8674(92)90082-n. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Liou H. C., Nolan G. P., Ghosh S., Fujita T., Baltimore D. The NF-kappa B p50 precursor, p105, contains an internal I kappa B-like inhibitor that preferentially inhibits p50. EMBO J. 1992 Aug;11(8):3003–3009. doi: 10.1002/j.1460-2075.1992.tb05370.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lux S. E., John K. M., Bennett V. Analysis of cDNA for human erythrocyte ankyrin indicates a repeated structure with homology to tissue-differentiation and cell-cycle control proteins. Nature. 1990 Mar 1;344(6261):36–42. doi: 10.1038/344036a0. [DOI] [PubMed] [Google Scholar]
  25. Matthews J. R., Kaszubska W., Turcatti G., Wells T. N., Hay R. T. Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B. Nucleic Acids Res. 1993 Apr 25;21(8):1727–1734. doi: 10.1093/nar/21.8.1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matthews J. R., Wakasugi N., Virelizier J. L., Yodoi J., Hay R. T. Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res. 1992 Aug 11;20(15):3821–3830. doi: 10.1093/nar/20.15.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meyer R., Hatada E. N., Hohmann H. P., Haiker M., Bartsch C., Röthlisberger U., Lahm H. W., Schlaeger E. J., van Loon A. P., Scheidereit C. Cloning of the DNA-binding subunit of human nuclear factor kappa B: the level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor alpha. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):966–970. doi: 10.1073/pnas.88.3.966. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morin P. J., Gilmore T. D. The C terminus of the NF-kappa B p50 precursor and an I kappa B isoform contain transcription activation domains. Nucleic Acids Res. 1992 May 25;20(10):2453–2458. doi: 10.1093/nar/20.10.2453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Narayanan R., Klement J. F., Ruben S. M., Higgins K. A., Rosen C. A. Identification of a naturally occurring transforming variant of the p65 subunit of NF-kappa B. Science. 1992 Apr 17;256(5055):367–370. doi: 10.1126/science.256.5055.367. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Ruben S. M., Klement J. F., Coleman T. A., Maher M., Chen C. H., Rosen C. A. I-Rel: a novel rel-related protein that inhibits NF-kappa B transcriptional activity. Genes Dev. 1992 May;6(5):745–760. doi: 10.1101/gad.6.5.745. [DOI] [PubMed] [Google Scholar]
  33. Ruben S. M., Narayanan R., Klement J. F., Chen C. H., Rosen C. A. Functional characterization of the NF-kappa B p65 transcriptional activator and an alternatively spliced derivative. Mol Cell Biol. 1992 Feb;12(2):444–454. doi: 10.1128/mcb.12.2.444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ryseck R. P., Bull P., Takamiya M., Bours V., Siebenlist U., Dobrzanski P., Bravo R. RelB, a new Rel family transcription activator that can interact with p50-NF-kappa B. Mol Cell Biol. 1992 Feb;12(2):674–684. doi: 10.1128/mcb.12.2.674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmid R. M., Perkins N. D., Duckett C. S., Andrews P. C., Nabel G. J. Cloning of an NF-kappa B subunit which stimulates HIV transcription in synergy with p65. Nature. 1991 Aug 22;352(6337):733–736. doi: 10.1038/352733a0. [DOI] [PubMed] [Google Scholar]
  36. Sen R., Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell. 1986 Dec 26;47(6):921–928. doi: 10.1016/0092-8674(86)90807-x. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wilhelmsen K. C., Eggleton K., Temin H. M. Nucleic acid sequences of the oncogene v-rel in reticuloendotheliosis virus strain T and its cellular homolog, the proto-oncogene c-rel. J Virol. 1984 Oct;52(1):172–182. doi: 10.1128/jvi.52.1.172-182.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wulczyn F. G., Naumann M., Scheidereit C. Candidate proto-oncogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-kappa B. Nature. 1992 Aug 13;358(6387):597–599. doi: 10.1038/358597a0. [DOI] [PubMed] [Google Scholar]
  40. Zabel U., Baeuerle P. A. Purified human I kappa B can rapidly dissociate the complex of the NF-kappa B transcription factor with its cognate DNA. Cell. 1990 Apr 20;61(2):255–265. doi: 10.1016/0092-8674(90)90806-p. [DOI] [PubMed] [Google Scholar]
  41. Zabel U., Henkel T., Silva M. S., Baeuerle P. A. Nuclear uptake control of NF-kappa B by MAD-3, an I kappa B protein present in the nucleus. EMBO J. 1993 Jan;12(1):201–211. doi: 10.1002/j.1460-2075.1993.tb05646.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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