Abstract
Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.
Full Text
The Full Text of this article is available as a PDF (953.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Alkalay I., Yaron A., Hatzubai A., Jung S., Avraham A., Gerlitz O., Pashut-Lavon I., Ben-Neriah Y. In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B. Mol Cell Biol. 1995 Mar;15(3):1294–1301. doi: 10.1128/mcb.15.3.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Anderson M. T., Staal F. J., Gitler C., Herzenberg L. A., Herzenberg L. A. Separation of oxidant-initiated and redox-regulated steps in the NF-kappa B signal transduction pathway. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11527–11531. doi: 10.1073/pnas.91.24.11527. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Baldi L., Brown K., Franzoso G., Siebenlist U. Critical role for lysines 21 and 22 in signal-induced, ubiquitin-mediated proteolysis of I kappa B-alpha. J Biol Chem. 1996 Jan 5;271(1):376–379. doi: 10.1074/jbc.271.1.376. [DOI] [PubMed] [Google Scholar]
- 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]
- Bass D. A., Parce J. W., Dechatelet L. R., Szejda P., Seeds M. C., Thomas M. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983 Apr;130(4):1910–1917. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Brockman J. A., Scherer D. C., McKinsey T. A., Hall S. M., Qi X., Lee W. Y., Ballard D. W. Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation. Mol Cell Biol. 1995 May;15(5):2809–2818. doi: 10.1128/mcb.15.5.2809. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation. Science. 1995 Mar 10;267(5203):1485–1488. doi: 10.1126/science.7878466. [DOI] [PubMed] [Google Scholar]
- Cahill M. A., Janknecht R., Nordheim A. Signalling pathways: jack of all cascades. Curr Biol. 1996 Jan 1;6(1):16–19. doi: 10.1016/s0960-9822(02)00410-4. [DOI] [PubMed] [Google Scholar]
- Chen Z., Hagler J., Palombella V. J., Melandri F., Scherer D., Ballard D., Maniatis T. Signal-induced site-specific phosphorylation targets I kappa B alpha to the ubiquitin-proteasome pathway. Genes Dev. 1995 Jul 1;9(13):1586–1597. doi: 10.1101/gad.9.13.1586. [DOI] [PubMed] [Google Scholar]
- DiDonato J. A., Mercurio F., Karin M. Phosphorylation of I kappa B alpha precedes but is not sufficient for its dissociation from NF-kappa B. Mol Cell Biol. 1995 Mar;15(3):1302–1311. doi: 10.1128/mcb.15.3.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DiDonato J., Mercurio F., Rosette C., Wu-Li J., Suyang H., Ghosh S., Karin M. Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol Cell Biol. 1996 Apr;16(4):1295–1304. doi: 10.1128/mcb.16.4.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Finco T. S., Baldwin A. S. Mechanistic aspects of NF-kappa B regulation: the emerging role of phosphorylation and proteolysis. Immunity. 1995 Sep;3(3):263–272. doi: 10.1016/1074-7613(95)90112-4. [DOI] [PubMed] [Google Scholar]
- Finco T. S., Beg A. A., Baldwin A. S., Jr Inducible phosphorylation of I kappa B alpha is not sufficient for its dissociation from NF-kappa B and is inhibited by protease inhibitors. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11884–11888. doi: 10.1073/pnas.91.25.11884. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Han J., Lee J. D., Bibbs L., Ulevitch R. J. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science. 1994 Aug 5;265(5173):808–811. doi: 10.1126/science.7914033. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Kanno T., Brown K., Siebenlist U. Evidence in support of a role for human T-cell leukemia virus type I Tax in activating NF-kappa B via stimulation of signaling pathways. J Biol Chem. 1995 May 19;270(20):11745–11748. doi: 10.1074/jbc.270.20.11745. [DOI] [PubMed] [Google Scholar]
- 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]
- Kyriakis J. M., Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays. 1996 Jul;18(7):567–577. doi: 10.1002/bies.950180708. [DOI] [PubMed] [Google Scholar]
- Lee J. D., Kravchenko V., Kirkland T. N., Han J., Mackman N., Moriarty A., Leturcq D., Tobias P. S., Ulevitch R. J. Glycosyl-phosphatidylinositol-anchored or integral membrane forms of CD14 mediate identical cellular responses to endotoxin. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9930–9934. doi: 10.1073/pnas.90.21.9930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Maggirwar S. B., Harhaj E., Sun S. C. Activation of NF-kappa B/Rel by Tax involves degradation of I kappa B alpha and is blocked by a proteasome inhibitor. Oncogene. 1995 Sep 7;11(5):993–998. [PubMed] [Google Scholar]
- Mains P. E., Sibley C. H. LPS-nonresponsive variants of mouse B cell lymphoma, 70Z/3: isolation and characterization. Somatic Cell Genet. 1983 Nov;9(6):699–720. doi: 10.1007/BF01539475. [DOI] [PubMed] [Google Scholar]
- Matthews J. R., Watson E., Buckley S., Hay R. T. 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. Nucleic Acids Res. 1993 Sep 25;21(19):4516–4523. doi: 10.1093/nar/21.19.4516. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKinsey T. A., Brockman J. A., Scherer D. C., Al-Murrani S. W., Green P. L., Ballard D. W. Inactivation of IkappaBbeta by the tax protein of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of NF-kappaB. Mol Cell Biol. 1996 May;16(5):2083–2090. doi: 10.1128/mcb.16.5.2083. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mellits K. H., Hay R. T., Goodbourn S. Proteolytic degradation of MAD3 (I kappa B alpha) and enhanced processing of the NF-kappa B precursor p105 are obligatory steps in the activation of NF-kappa B. Nucleic Acids Res. 1993 Nov 11;21(22):5059–5066. doi: 10.1093/nar/21.22.5059. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Mihm S., Ennen J., Pessara U., Kurth R., Dröge W. Inhibition of HIV-1 replication and NF-kappa B activity by cysteine and cysteine derivatives. AIDS. 1991 May;5(5):497–503. doi: 10.1097/00002030-199105000-00004. [DOI] [PubMed] [Google Scholar]
- Miyamoto S., Maki M., Schmitt M. J., Hatanaka M., Verma I. M. Tumor necrosis factor alpha-induced phosphorylation of I kappa B alpha is a signal for its degradation but not dissociation from NF-kappa B. Proc Natl Acad Sci U S A. 1994 Dec 20;91(26):12740–12744. doi: 10.1073/pnas.91.26.12740. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Muñoz E., Courtois G., Veschambre P., Jalinot P., Israël A. Tax induces nuclear translocation of NF-kappa B through dissociation of cytoplasmic complexes containing p105 or p100 but does not induce degradation of I kappa B alpha/MAD3. J Virol. 1994 Dec;68(12):8035–8044. doi: 10.1128/jvi.68.12.8035-8044.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neri A., Chang C. C., Lombardi L., Salina M., Corradini P., Maiolo A. T., Chaganti R. S., Dalla-Favera R. B cell lymphoma-associated chromosomal translocation involves candidate oncogene lyt-10, homologous to NF-kappa B p50. Cell. 1991 Dec 20;67(6):1075–1087. doi: 10.1016/0092-8674(91)90285-7. [DOI] [PubMed] [Google Scholar]
- Rice N. R., MacKichan M. L., Israël A. The precursor of NF-kappa B p50 has I kappa B-like functions. Cell. 1992 Oct 16;71(2):243–253. doi: 10.1016/0092-8674(92)90353-e. [DOI] [PubMed] [Google Scholar]
- Rooney J. W., Emery D. W., Sibley C. H. 1.3E2, a variant of the B lymphoma 70Z/3, defective in activation of NF-kappa B and OTF-2. Immunogenetics. 1990;31(2):73–78. doi: 10.1007/BF00661216. [DOI] [PubMed] [Google Scholar]
- Rousset R., Desbois C., Bantignies F., Jalinot P. Effects on NF-kappa B1/p105 processing of the interaction between the HTLV-1 transactivator Tax and the proteasome. Nature. 1996 May 23;381(6580):328–331. doi: 10.1038/381328a0. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- 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]
- Scherer D. C., Brockman J. A., Chen Z., Maniatis T., Ballard D. W. Signal-induced degradation of I kappa B alpha requires site-specific ubiquitination. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11259–11263. doi: 10.1073/pnas.92.24.11259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Schmidt K. N., Amstad P., Cerutti P., Baeuerle P. A. The roles of hydrogen peroxide and superoxide as messengers in the activation of transcription factor NF-kappa B. Chem Biol. 1995 Jan;2(1):13–22. doi: 10.1016/1074-5521(95)90076-4. [DOI] [PubMed] [Google Scholar]
- Schreck R., Baeuerle P. A. A role for oxygen radicals as second messengers. Trends Cell Biol. 1991 Aug;1(2-3):39–42. doi: 10.1016/0962-8924(91)90072-h. [DOI] [PubMed] [Google Scholar]
- Schreck R., Rieber P., Baeuerle P. A. Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J. 1991 Aug;10(8):2247–2258. doi: 10.1002/j.1460-2075.1991.tb07761.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shapiro L., Dinarello C. A. Osmotic regulation of cytokine synthesis in vitro. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12230–12234. doi: 10.1073/pnas.92.26.12230. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Siebenlist U., Franzoso G., Brown K. Structure, regulation and function of NF-kappa B. Annu Rev Cell Biol. 1994;10:405–455. doi: 10.1146/annurev.cb.10.110194.002201. [DOI] [PubMed] [Google Scholar]
- Staal F. J., Roederer M., Herzenberg L. A., Herzenberg L. A. Intracellular thiols regulate activation of nuclear factor kappa B and transcription of human immunodeficiency virus. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9943–9947. doi: 10.1073/pnas.87.24.9943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sun S. C., Elwood J., Béraud C., Greene W. C. Human T-cell leukemia virus type I Tax activation of NF-kappa B/Rel involves phosphorylation and degradation of I kappa B alpha and RelA (p65)-mediated induction of the c-rel gene. Mol Cell Biol. 1994 Nov;14(11):7377–7384. doi: 10.1128/mcb.14.11.7377. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sun S., Elwood J., Greene W. C. Both amino- and carboxyl-terminal sequences within I kappa B alpha regulate its inducible degradation. Mol Cell Biol. 1996 Mar;16(3):1058–1065. doi: 10.1128/mcb.16.3.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson J. E., Phillips R. J., Erdjument-Bromage H., Tempst P., Ghosh S. I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell. 1995 Feb 24;80(4):573–582. doi: 10.1016/0092-8674(95)90511-1. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Treisman R. Regulation of transcription by MAP kinase cascades. Curr Opin Cell Biol. 1996 Apr;8(2):205–215. doi: 10.1016/s0955-0674(96)80067-6. [DOI] [PubMed] [Google Scholar]
- Verma I. M., Stevenson J. K., Schwarz E. M., Van Antwerp D., Miyamoto S. Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev. 1995 Nov 15;9(22):2723–2735. doi: 10.1101/gad.9.22.2723. [DOI] [PubMed] [Google Scholar]
- Whiteside S. T., Ernst M. K., LeBail O., Laurent-Winter C., Rice N., Israël A. N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity. Mol Cell Biol. 1995 Oct;15(10):5339–5345. doi: 10.1128/mcb.15.10.5339. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]