Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Feb 2;132(4):511–522. doi: 10.1083/jcb.132.4.511

Activation of transcription factor NF-kappaB by the adenovirus E3/19K protein requires its ER retention

PMCID: PMC2199876  PMID: 8647884

Abstract

We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF- kappaB. However, activation of both NF-kappaB DNA binding and kappaB- dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.

Full Text

The Full Text of this article is available as a PDF (2.4 MB).

Selected References

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

  1. Andersson M., Päbo S., Nilsson T., Peterson P. A. Impaired intracellular transport of class I MHC antigens as a possible means for adenoviruses to evade immune surveillance. Cell. 1985 Nov;43(1):215–222. doi: 10.1016/0092-8674(85)90026-1. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Arnold B., Burgert H. G., Archibald A. L., Kvist S. Complete nucleotide sequence of the murine H-2Kk gene. Comparison of three H-2K locus alleles. Nucleic Acids Res. 1984 Dec 21;12(24):9473–9487. doi: 10.1093/nar/12.24.9473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Baeuerle P. A., Henkel T. Function and activation of NF-kappa B in the immune system. Annu Rev Immunol. 1994;12:141–179. doi: 10.1146/annurev.iy.12.040194.001041. [DOI] [PubMed] [Google Scholar]
  6. Beg A. A., Finco T. S., Nantermet P. V., Baldwin A. S., Jr Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993 Jun;13(6):3301–3310. doi: 10.1128/mcb.13.6.3301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Bost K. L., Mason M. J. Thapsigargin and cyclopiazonic acid initiate rapid and dramatic increases of IL-6 mRNA expression and IL-6 secretion in murine peritoneal macrophages. J Immunol. 1995 Jul 1;155(1):285–296. [PubMed] [Google Scholar]
  9. Burgert H. G., Kvist S. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell. 1985 Jul;41(3):987–997. doi: 10.1016/s0092-8674(85)80079-9. [DOI] [PubMed] [Google Scholar]
  10. Burgert H. G., Maryanski J. L., Kvist S. "E3/19K" protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell-surface expression of histocompatibility class I antigens. Proc Natl Acad Sci U S A. 1987 Mar;84(5):1356–1360. doi: 10.1073/pnas.84.5.1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chiou C. Y., Malagodi M. H. Studies on the mechanism of action of a new Ca-2+ antagonist, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride in smooth and skeletal muscles. Br J Pharmacol. 1975 Feb;53(2):279–285. doi: 10.1111/j.1476-5381.1975.tb07359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cosson P., Letourneur F. Coatomer interaction with di-lysine endoplasmic reticulum retention motifs. Science. 1994 Mar 18;263(5153):1629–1631. doi: 10.1126/science.8128252. [DOI] [PubMed] [Google Scholar]
  13. Cox J. H., Bennink J. R., Yewdell J. W. Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation. J Exp Med. 1991 Dec 1;174(6):1629–1637. doi: 10.1084/jem.174.6.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dahllöf B., Wallin M., Kvist S. The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus-2 is microtubule binding. J Biol Chem. 1991 Jan 25;266(3):1804–1808. [PubMed] [Google Scholar]
  15. Deryckere F., Ebenau-Jehle C., Wold W. S., Burgert H. G. Tumor necrosis factor alpha increases expression of adenovirus E3 proteins. Immunobiology. 1995 Jul;193(2-4):186–192. doi: 10.1016/s0171-2985(11)80542-5. [DOI] [PubMed] [Google Scholar]
  16. Fox J. P., Brandt C. D., Wassermann F. E., Hall C. E., Spigland I., Kogon A., Elveback L. R. The virus watch program: a continuing surveillance of viral infections in metropolitan New York families. VI. Observations of adenovirus infections: virus excretion patterns, antibody response, efficiency of surveillance, patterns of infections, and relation to illness. Am J Epidemiol. 1969 Jan;89(1):25–50. doi: 10.1093/oxfordjournals.aje.a120913. [DOI] [PubMed] [Google Scholar]
  17. Fox J. P., Hall C. E., Cooney M. K. The Seattle Virus Watch. VII. Observations of adenovirus infections. Am J Epidemiol. 1977 Apr;105(4):362–386. doi: 10.1093/oxfordjournals.aje.a112394. [DOI] [PubMed] [Google Scholar]
  18. Gabathuler R., Kvist S. The endoplasmic reticulum retention signal of the E3/19K protein of adenovirus type 2 consists of three separate amino acid segments at the carboxy terminus. J Cell Biol. 1990 Nov;111(5 Pt 1):1803–1810. doi: 10.1083/jcb.111.5.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ginsberg H. S., Moldawer L. L., Sehgal P. B., Redington M., Kilian P. L., Chanock R. M., Prince G. A. A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1651–1655. doi: 10.1073/pnas.88.5.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goeger D. E., Riley R. T., Dorner J. W., Cole R. J. Cyclopiazonic acid inhibition of the Ca2+-transport ATPase in rat skeletal muscle sarcoplasmic reticulum vesicles. Biochem Pharmacol. 1988 Mar 1;37(5):978–981. doi: 10.1016/0006-2952(88)90195-5. [DOI] [PubMed] [Google Scholar]
  21. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  22. Grilli M., Chiu J. J., Lenardo M. J. NF-kappa B and Rel: participants in a multiform transcriptional regulatory system. Int Rev Cytol. 1993;143:1–62. doi: 10.1016/s0074-7696(08)61873-2. [DOI] [PubMed] [Google Scholar]
  23. Henkel T., Machleidt T., Alkalay I., Krönke M., Ben-Neriah Y., Baeuerle P. A. Rapid proteolysis of I kappa B-alpha is necessary for activation of transcription factor NF-kappa B. Nature. 1993 Sep 9;365(6442):182–185. doi: 10.1038/365182a0. [DOI] [PubMed] [Google Scholar]
  24. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hérissé J., Courtois G., Galibert F. Nucleotide sequence of the EcoRI D fragment of adenovirus 2 genome. Nucleic Acids Res. 1980 May 24;8(10):2173–2192. doi: 10.1093/nar/8.10.2173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Israël A., Le Bail O., Hatat D., Piette J., Kieran M., Logeat F., Wallach D., Fellous M., Kourilsky P. TNF stimulates expression of mouse MHC class I genes by inducing an NF kappa B-like enhancer binding activity which displaces constitutive factors. EMBO J. 1989 Dec 1;8(12):3793–3800. doi: 10.1002/j.1460-2075.1989.tb08556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jackson M. R., Nilsson T., Peterson P. A. Identification of a consensus motif for retention of transmembrane proteins in the endoplasmic reticulum. EMBO J. 1990 Oct;9(10):3153–3162. doi: 10.1002/j.1460-2075.1990.tb07513.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jackson M. R., Nilsson T., Peterson P. A. Retrieval of transmembrane proteins to the endoplasmic reticulum. J Cell Biol. 1993 Apr;121(2):317–333. doi: 10.1083/jcb.121.2.317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kvist S., Roberts L., Dobberstein B. Mouse histocompatibility genes: structure and organisation of a Kd gene. EMBO J. 1983;2(2):245–254. doi: 10.1002/j.1460-2075.1983.tb01413.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Körner H., Fritzsche U., Burgert H. G. Tumor necrosis factor alpha stimulates expression of adenovirus early region 3 proteins: implications for viral persistence. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11857–11861. doi: 10.1073/pnas.89.24.11857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Letourneur F., Gaynor E. C., Hennecke S., Démollière C., Duden R., Emr S. D., Riezman H., Cosson P. Coatomer is essential for retrieval of dilysine-tagged proteins to the endoplasmic reticulum. Cell. 1994 Dec 30;79(7):1199–1207. doi: 10.1016/0092-8674(94)90011-6. [DOI] [PubMed] [Google Scholar]
  32. Libermann T. A., Baltimore D. Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol. 1990 May;10(5):2327–2334. doi: 10.1128/mcb.10.5.2327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lucocq J., Warren G., Pryde J. Okadaic acid induces Golgi apparatus fragmentation and arrest of intracellular transport. J Cell Sci. 1991 Dec;100(Pt 4):753–759. doi: 10.1242/jcs.100.4.753. [DOI] [PubMed] [Google Scholar]
  34. Malagodi M. H., Chiou C. Y. Pharmacological evaluation of a new Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8): studies in smooth muscles. Eur J Pharmacol. 1974 Jun;27(1):25–33. doi: 10.1016/0014-2999(74)90198-8. [DOI] [PubMed] [Google Scholar]
  35. Meyer M., Caselmann W. H., Schlüter V., Schreck R., Hofschneider P. H., Baeuerle P. A. Hepatitis B virus transactivator MHBst: activation of NF-kappa B, selective inhibition by antioxidants and integral membrane localization. EMBO J. 1992 Aug;11(8):2991–3001. doi: 10.1002/j.1460-2075.1992.tb05369.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meyer M., Schreck R., Baeuerle P. A. H2O2 and antioxidants have opposite effects on activation of NF-kappa B and AP-1 in intact cells: AP-1 as secondary antioxidant-responsive factor. EMBO J. 1993 May;12(5):2005–2015. doi: 10.1002/j.1460-2075.1993.tb05850.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Owen M. J., Kissonerghis A. M., Lodish H. F. Biosynthesis of HLA-A and HLA-B antigens in vivo. J Biol Chem. 1980 Oct 25;255(20):9678–9684. [PubMed] [Google Scholar]
  38. Pahl H. L., Baeuerle P. A. A novel signal transduction pathway from the endoplasmic reticulum to the nucleus is mediated by transcription factor NF-kappa B. EMBO J. 1995 Jun 1;14(11):2580–2588. doi: 10.1002/j.1460-2075.1995.tb07256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pahl H. L., Baeuerle P. A. Expression of influenza virus hemagglutinin activates transcription factor NF-kappa B. J Virol. 1995 Mar;69(3):1480–1484. doi: 10.1128/jvi.69.3.1480-1484.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Pahl H. L., Burn T. C., Tenen D. G. Optimization of transient transfection into human myeloid cell lines using a luciferase reporter gene. Exp Hematol. 1991 Nov;19(10):1038–1041. [PubMed] [Google Scholar]
  41. Plaksin D., Baeuerle P. A., Eisenbach L. KBF1 (p50 NF-kappa B homodimer) acts as a repressor of H-2Kb gene expression in metastatic tumor cells. J Exp Med. 1993 Jun 1;177(6):1651–1662. doi: 10.1084/jem.177.6.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schmidt K. N., Traenckner E. B., Meier B., Baeuerle P. A. Induction of oxidative stress by okadaic acid is required for activation of transcription factor NF-kappa B. J Biol Chem. 1995 Nov 10;270(45):27136–27142. doi: 10.1074/jbc.270.45.27136. [DOI] [PubMed] [Google Scholar]
  43. Sester M., Burgert H. G. Conserved cysteine residues within the E3/19K protein of adenovirus type 2 are essential for binding to major histocompatibility complex antigens. J Virol. 1994 Sep;68(9):5423–5432. doi: 10.1128/jvi.68.9.5423-5432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Sun S. C., Ganchi P. A., Béraud C., Ballard D. W., Greene W. C. Autoregulation of the NF-kappa B transactivator RelA (p65) by multiple cytoplasmic inhibitors containing ankyrin motifs. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1346–1350. doi: 10.1073/pnas.91.4.1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thastrup O., Cullen P. J., Drøbak B. K., Hanley M. R., Dawson A. P. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2466–2470. doi: 10.1073/pnas.87.7.2466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tong-Starkesen S. E., Luciw P. A., Peterlin B. M. Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat. J Immunol. 1989 Jan 15;142(2):702–707. [PubMed] [Google Scholar]
  47. Traenckner E. B., Pahl H. L., Henkel T., Schmidt K. N., Wilk S., Baeuerle P. A. Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli. EMBO J. 1995 Jun 15;14(12):2876–2883. doi: 10.1002/j.1460-2075.1995.tb07287.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Traenckner E. B., Wilk S., Baeuerle P. A. A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B. EMBO J. 1994 Nov 15;13(22):5433–5441. doi: 10.1002/j.1460-2075.1994.tb06878.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Williams J. L., Garcia J., Harrich D., Pearson L., Wu F., Gaynor R. Lymphoid specific gene expression of the adenovirus early region 3 promoter is mediated by NF-kappa B binding motifs. EMBO J. 1990 Dec;9(13):4435–4442. doi: 10.1002/j.1460-2075.1990.tb07894.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES