In vivo stimulation of I kappa B phosphorylation is not sufficient to activate NF-kappa B (original) (raw)

Abstract

NF-kappa B is a major inducible transcription factor in many immune and inflammatory reactions. Its activation involves the dissociation of the inhibitory subunit I kappa B from cytoplasmic NF-kappa B/Rel complexes, following which the Rel proteins are translocated to the nucleus, where they bind to DNA and activate transcription. Phosphorylation of I kappa B in cell-free experiments results in its inactivation and release from the Rel complex, but in vivo NF-kappa B activation is associated with I kappa B degradation. In vivo phosphorylation of I kappa B alpha was demonstrated in several recent studies, but its role is unknown. Our study shows that the T-cell activation results in rapid phosphorylation of I kappa B alpha and that this event is a physiological one, dependent on appropriate lymphocyte costimulation. Inducible I kappa B alpha phosphorylation was abolished by several distinct NF-kappa B blocking reagents, suggesting that it plays an essential role in the activation process. However, the in vivo induction of I kappa B alpha phosphorylation did not cause the inhibitory subunit to dissociate from the Rel complex. We identified several protease inhibitors which allow phosphorylation of I kappa B alpha but prevent its degradation upon cell stimulation, presumably through inhibition of the cytoplasmic proteasome. In the presence of these inhibitors, phosphorylated I kappa B alpha remained bound to the Rel complex in the cytoplasm for an extended period of time, whereas NF-kappa B activation was abolished. It appears that activation of NF-kappa B requires degradation of I kappa B alpha while it is a part of the Rel cytoplasmic complex, with inducible phosphorylation of the inhibitory subunit influencing the rate of degradation.

Full Text

The Full Text of this article is available as a PDF (386.1 KB).

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. 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]
  5. Bauskin A. R., Alkalay I., Ben-Neriah Y. Redox regulation of a protein tyrosine kinase in the endoplasmic reticulum. Cell. 1991 Aug 23;66(4):685–696. doi: 10.1016/0092-8674(91)90114-e. [DOI] [PubMed] [Google Scholar]
  6. Beg A. A., Baldwin A. S., Jr The I kappa B proteins: multifunctional regulators of Rel/NF-kappa B transcription factors. Genes Dev. 1993 Nov;7(11):2064–2070. doi: 10.1101/gad.7.11.2064. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Blank V., Kourilsky P., Israël A. NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats. Trends Biochem Sci. 1992 Apr;17(4):135–140. doi: 10.1016/0968-0004(92)90321-y. [DOI] [PubMed] [Google Scholar]
  10. Brown K., Park S., Kanno T., Franzoso G., Siebenlist U. Mutual regulation of the transcriptional activator NF-kappa B and its inhibitor, I kappa B-alpha. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2532–2536. doi: 10.1073/pnas.90.6.2532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chiao P. J., Miyamoto S., Verma I. M. Autoregulation of I kappa B alpha activity. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):28–32. doi: 10.1073/pnas.91.1.28. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Crabtree G. R. Contingent genetic regulatory events in T lymphocyte activation. Science. 1989 Jan 20;243(4889):355–361. doi: 10.1126/science.2783497. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Figueiredo-Pereira M. E., Banik N., Wilk S. Comparison of the effect of calpain inhibitors on two extralysosomal proteinases: the multicatalytic proteinase complex and m-calpain. J Neurochem. 1994 May;62(5):1989–1994. doi: 10.1046/j.1471-4159.1994.62051989.x. [DOI] [PubMed] [Google Scholar]
  16. Frantz B., Nordby E. C., Bren G., Steffan N., Paya C. V., Kincaid R. L., Tocci M. J., O'Keefe S. J., O'Neill E. A. Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B. EMBO J. 1994 Feb 15;13(4):861–870. doi: 10.1002/j.1460-2075.1994.tb06329.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Gilmore T. D., Morin P. J. The I kappa B proteins: members of a multifunctional family. Trends Genet. 1993 Dec;9(12):427–433. doi: 10.1016/0168-9525(93)90106-r. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. 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]
  22. Hiller R., Schaefer A., Zibirre R., Kaback H. R., Koch G. Factors influencing the accumulation of tetraphenylphosphonium cation in HeLa cells. Mol Cell Biol. 1984 Jan;4(1):199–202. doi: 10.1128/mcb.4.1.199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hochstrasser M., Varshavsky A. In vivo degradation of a transcriptional regulator: the yeast alpha 2 repressor. Cell. 1990 May 18;61(4):697–708. doi: 10.1016/0092-8674(90)90481-s. [DOI] [PubMed] [Google Scholar]
  24. Hough R., Pratt G., Rechsteiner M. Purification of two high molecular weight proteases from rabbit reticulocyte lysate. J Biol Chem. 1987 Jun 15;262(17):8303–8313. [PubMed] [Google Scholar]
  25. Hoyos B., Ballard D. W., Böhnlein E., Siekevitz M., Greene W. C. Kappa B-specific DNA binding proteins: role in the regulation of human interleukin-2 gene expression. Science. 1989 Apr 28;244(4903):457–460. doi: 10.1126/science.2497518. [DOI] [PubMed] [Google Scholar]
  26. Kidd S. Characterization of the Drosophila cactus locus and analysis of interactions between cactus and dorsal proteins. Cell. 1992 Nov 13;71(4):623–635. doi: 10.1016/0092-8674(92)90596-5. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lin W. C., Desiderio S. Cell cycle regulation of V(D)J recombination-activating protein RAG-2. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2733–2737. doi: 10.1073/pnas.91.7.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Linsley P. S., Ledbetter J. A. The role of the CD28 receptor during T cell responses to antigen. Annu Rev Immunol. 1993;11:191–212. doi: 10.1146/annurev.iy.11.040193.001203. [DOI] [PubMed] [Google Scholar]
  30. Liou H. C., Baltimore D. Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system. Curr Opin Cell Biol. 1993 Jun;5(3):477–487. doi: 10.1016/0955-0674(93)90014-h. [DOI] [PubMed] [Google Scholar]
  31. Mattila P. S., Ullman K. S., Fiering S., Emmel E. A., McCutcheon M., Crabtree G. R., Herzenberg L. A. The actions of cyclosporin A and FK506 suggest a novel step in the activation of T lymphocytes. EMBO J. 1990 Dec;9(13):4425–4433. doi: 10.1002/j.1460-2075.1990.tb07893.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mehdi S. Cell-penetrating inhibitors of calpain. Trends Biochem Sci. 1991 Apr;16(4):150–153. doi: 10.1016/0968-0004(91)90058-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Melloni E., Pontremoli S. The calpains. Trends Neurosci. 1989 Nov;12(11):438–444. doi: 10.1016/0166-2236(89)90093-3. [DOI] [PubMed] [Google Scholar]
  35. Mercurio F., DiDonato J. A., Rosette C., Karin M. p105 and p98 precursor proteins play an active role in NF-kappa B-mediated signal transduction. Genes Dev. 1993 Apr;7(4):705–718. doi: 10.1101/gad.7.4.705. [DOI] [PubMed] [Google Scholar]
  36. Nabel G., Baltimore D. An inducible transcription factor activates expression of human immunodeficiency virus in T cells. Nature. 1987 Apr 16;326(6114):711–713. doi: 10.1038/326711a0. [DOI] [PubMed] [Google Scholar]
  37. Orlowski M., Cardozo C., Michaud C. Evidence for the presence of five distinct proteolytic components in the pituitary multicatalytic proteinase complex. Properties of two components cleaving bonds on the carboxyl side of branched chain and small neutral amino acids. Biochemistry. 1993 Feb 16;32(6):1563–1572. doi: 10.1021/bi00057a022. [DOI] [PubMed] [Google Scholar]
  38. Orlowski M., Michaud C. Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity. Biochemistry. 1989 Nov 28;28(24):9270–9278. doi: 10.1021/bi00450a006. [DOI] [PubMed] [Google Scholar]
  39. Orlowski M. The multicatalytic proteinase complex (proteasome) and intracellular protein degradation: diverse functions of an intracellular particle. J Lab Clin Med. 1993 Feb;121(2):187–189. [PubMed] [Google Scholar]
  40. Palombella V. J., Rando O. J., Goldberg A. L., Maniatis T. The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell. 1994 Sep 9;78(5):773–785. doi: 10.1016/s0092-8674(94)90482-0. [DOI] [PubMed] [Google Scholar]
  41. Peters J. M. Proteasomes: protein degradation machines of the cell. Trends Biochem Sci. 1994 Sep;19(9):377–382. doi: 10.1016/0968-0004(94)90115-5. [DOI] [PubMed] [Google Scholar]
  42. Rice N. R., Ernst M. K. In vivo control of NF-kappa B activation by I kappa B alpha. EMBO J. 1993 Dec;12(12):4685–4695. doi: 10.1002/j.1460-2075.1993.tb06157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Rock K. L., Gramm C., Rothstein L., Clark K., Stein R., Dick L., Hwang D., Goldberg A. L. Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell. 1994 Sep 9;78(5):761–771. doi: 10.1016/s0092-8674(94)90462-6. [DOI] [PubMed] [Google Scholar]
  45. Sasaki T., Kishi M., Saito M., Tanaka T., Higuchi N., Kominami E., Katunuma N., Murachi T. Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins. J Enzyme Inhib. 1990;3(3):195–201. doi: 10.3109/14756369009035837. [DOI] [PubMed] [Google Scholar]
  46. Scheinman R. I., Beg A. A., Baldwin A. S., Jr NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule. Mol Cell Biol. 1993 Oct;13(10):6089–6101. doi: 10.1128/mcb.13.10.6089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schreck R., Meier B., Männel D. N., Dröge W., Baeuerle P. A. Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J Exp Med. 1992 May 1;175(5):1181–1194. doi: 10.1084/jem.175.5.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schreiber S. L. Immunophilin-sensitive protein phosphatase action in cell signaling pathways. Cell. 1992 Aug 7;70(3):365–368. doi: 10.1016/0092-8674(92)90158-9. [DOI] [PubMed] [Google Scholar]
  49. Scott M. L., Fujita T., Liou H. C., Nolan G. P., Baltimore D. The p65 subunit of NF-kappa B regulates I kappa B by two distinct mechanisms. Genes Dev. 1993 Jul;7(7A):1266–1276. doi: 10.1101/gad.7.7a.1266. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Shirakawa F., Mizel S. B. In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol. 1989 Jun;9(6):2424–2430. doi: 10.1128/mcb.9.6.2424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Su B., Jacinto E., Hibi M., Kallunki T., Karin M., Ben-Neriah Y. JNK is involved in signal integration during costimulation of T lymphocytes. Cell. 1994 Jun 3;77(5):727–736. doi: 10.1016/0092-8674(94)90056-6. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Sun S. C., Ganchi P. A., Ballard D. W., Greene W. C. NF-kappa B controls expression of inhibitor I kappa B alpha: evidence for an inducible autoregulatory pathway. Science. 1993 Mar 26;259(5103):1912–1915. doi: 10.1126/science.8096091. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Tewari M., Dobrzanski P., Mohn K. L., Cressman D. E., Hsu J. C., Bravo R., Taub R. Rapid induction in regenerating liver of RL/IF-1 (an I kappa B that inhibits NF-kappa B, RelB-p50, and c-Rel-p50) and PHF, a novel kappa B site-binding complex. Mol Cell Biol. 1992 Jun;12(6):2898–2908. doi: 10.1128/mcb.12.6.2898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. Umlauf S. W., Beverly B., Kang S. M., Brorson K., Tran A. C., Schwartz R. H. Molecular regulation of the IL-2 gene: rheostatic control of the immune system. Immunol Rev. 1993 Jun;133:177–197. doi: 10.1111/j.1600-065x.1993.tb01516.x. [DOI] [PubMed] [Google Scholar]
  59. Vinitsky A., Michaud C., Powers J. C., Orlowski M. Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex. Biochemistry. 1992 Oct 6;31(39):9421–9428. doi: 10.1021/bi00154a014. [DOI] [PubMed] [Google Scholar]
  60. Wang K. K., Villalobo A., Roufogalis B. D. Calmodulin-binding proteins as calpain substrates. Biochem J. 1989 Sep 15;262(3):693–706. doi: 10.1042/bj2620693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. 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]
  62. 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]