Pervanadate-induced Nuclear Factor-κB Activation Requires Tyrosine Phosphorylation and Degradation of IκBα

Tumor necrosis factor activates nuclear transcription factor κB (NF-κB) by inducing serine phosphorylation of the inhibitory subunit of NF-κB (IκBα), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-κB and induces tyrosine phosphorylation of IκBα (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049–31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787–798). Whether PV also induces IκBα degradation and whether degradation is required for NF-κB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IκBα degradation and NF-κB activation. PV activated NF-κB, as determined by DNA binding, NF-κB-dependent reporter gene expression, and phosphorylation and degradation of IκBα. Maximum degradation of IκBα occurred at 180 min, followed by NF-κB-dependent IκBα resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IκBα degradation and NF-κB activation, suggesting that the IκBα degradation is required for NF-κB activation. PV did not induce serine phosphorylation of IκBα but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IκBα. Like TNF, however, PV induced phosphorylation and degradation of IκBα, and subsequent NF-κB activation, which could be blocked byN-tosyl-l-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-κB activation and IκBα degradation. Overall, our studies demonstrate that PV activates NF-κB, which, unlike TNF, requires tyrosine phosphorylation of IκBα and its degradation. Tumor necrosis factor activates nuclear transcription factor κB (NF-κB) by inducing serine phosphorylation of the inhibitory subunit of NF-κB (IκBα), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-κB and induces tyrosine phosphorylation of IκBα (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049–31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787–798). Whether PV also induces IκBα degradation and whether degradation is required for NF-κB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IκBα degradation and NF-κB activation. PV activated NF-κB, as determined by DNA binding, NF-κB-dependent reporter gene expression, and phosphorylation and degradation of IκBα. Maximum degradation of IκBα occurred at 180 min, followed by NF-κB-dependent IκBα resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IκBα degradation and NF-κB activation, suggesting that the IκBα degradation is required for NF-κB activation. PV did not induce serine phosphorylation of IκBα but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IκBα. Like TNF, however, PV induced phosphorylation and degradation of IκBα, and subsequent NF-κB activation, which could be blocked byN-tosyl-l-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-κB activation and IκBα degradation. Overall, our studies demonstrate that PV activates NF-κB, which, unlike TNF, requires tyrosine phosphorylation of IκBα and its degradation. nuclear transcription factor-κB N-acetylleucylleucylnorlucinal calf intestine alkaline phosphatase inhibitory subunit of NF-κB polyacrylamide gel electrophoresis pervanadate phosphotyrosine kinase pyrrolidine dithiocarbomate secretory alkaline phosphatase tumor necrosis factor N-tosyl-l-phenylalanine chloromethyl ketone Nuclear transcription factor-κB (NF-κB)1 plays a pivotal role in expression of various inducible target genes related to immune and inflammatory responses, including the type I human immunodeficiency virus (1.Grilli M. Chiu J.J. Lenardo M.J. Int. Rev. Cytol. l. 1993; 143: 1-62Crossref PubMed Scopus (880) Google Scholar, 2.Verma I.M. Stevenson J.K. Schwarz E.M. Van Antwerp D. Miyamoto S. Genes Dev. 1995; 9: 2723-2735Crossref PubMed Scopus (1648) Google Scholar, 3.Nabel G. Baltimore D. 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Inhibitors of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (e.g. PTPase) suppress NF-κB activation (18.Eicher D.M. Tan T.-H. Rice N.T. O'Shea J.J. Kennedy I.C.S. J. Immunol. 1994; 152: 2710-2719PubMed Google Scholar, 19.Imbert V. Farahifar D. Auberger P. Mary M. Rossi B. Peyron J.-F. J. Inflammation. 1996; 46: 65-77PubMed Google Scholar, 20.Natarajan K. Manna S.K. Chaturvedi M.M. Aggarwal B.B. Arch. Biochem. Biophys. 1998; 352: 59-70Crossref PubMed Scopus (87) Google Scholar, 21.Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 10631-10639Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 22.Menon S.D. Guy G.R. Tan Y.H. J. Biol. Chem. 1995; 270: 18881-18887Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 23.Koong A.C. Chen E.Y. Giacca A.J. Cancer Res. 1994; 54: 1425-1430PubMed Google Scholar). 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B. in Scholar), on the proteins to a blocked with and with for was to for and by using using the of IκBα, of to for at was by with and the to electrophoresis and for IκBα. with with and in of protein was with of in of at for and the immune complexes with protein and for with the proteins on to a and with was with and by was as S. Darney B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In the IκBα genes the expression by as by the of and with or TNF or PV for of as was on on to a and with reporter gene expression was on our B.G. J. P.A. Aggarwal B.B. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In in of a and for with and the expression of by the for and with or with TNF or PV for was and for was with of and in a of in a and the alkaline phosphatase was by the at for was on for min, of was to the was at for and was on a with at and at of for was We the of PV on NF-κB and IκBα in the on these are in our and of to PV in our effect on not We the of PV-induced NF-κB activation as by in with PV for the nuclear and them by that activity was at min, a at 180 min, and activated of NF-κB was at NF-κB activation could be as as not the of NF-κB activation by PV to be activation by of nuclear with or in the in complex, or NF-κB induced with PV both the and of the PV-induced was by that the was in the of a of but not by of NF-κB by TNF is Ser-32 and Ser-36 phosphorylation of IκBα followed by and degradation, which in the nuclear translocation of Whether PV-induced NF-κB activation is associated with the degradation of IκBα is not with PV for to using of PV IκBα as to which was degraded Maximum degradation of the was at min, degradation was but of IκBα of IκBα, which is on NF-κB activation, at 180 and a at of PV-induced IκBα degradation with the of activation of NF-κB of IκBα that IκBα been by the activated to the steps of of to the DNA not activation. the of PV to activate NF-κB-dependent reporter gene of induced by PV or TNF are shown in D. PV and TNF activity by that NF-κB activated by PV was and with a inhibitor that the activity of the is to the activity of the proteasome A. C. J.J. M. PubMed Scopus Google Scholar) and the degradation of proteins C. L. K. R. L. D. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar). also the degradation of IκBα its and this of NF-κB activation (15.DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar). activation of NF-κB in PV-induced is associated with the degradation of IκBα, the of the proteasome inhibitor on the degradation of IκBα and subsequent NF-κB activation. shown in blocked PV-induced degradation of IκBα and NF-κB activation as by the of a did not PV-induced of IκBα. in of that a proteasome is in PV-induced NF-κB activation and IκBα degradation and that degradation of IκBα protein is a for NF-κB activation. a protein inhibitor, the of IκBα T. J. M. T. M. H. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). In the have shown that PV IκBα was degraded and in the with PV for in the of and the IκBα of IκBα as IκBα was of degradation by proteasome inhibitor and of by that IκBα is the activation of NF-κB that the of IκBα that PV was to its with of the a was shown in with did not the of IκBα, but the was to the IκBα in that IκBα was by whether was to phosphorylation IκBα with a inhibitor, for and to blocked PV-induced IκBα phosphorylation and degradation, and this with of NF-κB activation that a is activated by PV to IκBα and this phosphorylation is needed for IκBα degradation and subsequent NF-κB activation. inhibitors as are not We and have shown that PV induces phosphorylation of IκBα at tyrosine V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. 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Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in that IκBα degradation was induced by both PV and TNF and that blocked the degradation. also phosphorylation of IκBα induced by both TNF and with of or PV-induced NF-κB activation by that PV induces degradation of IκBα by to the one activated by also that of IκBα degradation PV-induced NF-κB activation. In this report, PV induces NF-κB activation in that PV-induced NF-κB activation was associated with IκBα degradation. NF-κB was as it induced IκBα and activated reporter gene of and that PV the residue of IκBα, the Ser-32 residue and IκBα is not degradation. PV induces tyrosine phosphorylation of IκBα and TNF induces serine various as and calpeptin, which blocked IκBα degradation and NF-κB activation induced by that the degradation of IκBα is required for activation. it was that PV induces NF-κB activation IκBα V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). that the subunit of to the PV-induced IκBα and it the p50-p65 C. Henzel W.J. Baeuerle P. Proc. Natl. Acd. Sci. U. S. A. 1999; 96: 429-434Crossref PubMed Scopus (263) Google Scholar). however, that PV induces tyrosine phosphorylation of IκBα, which in leads to its degradation. is a between our and by V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) is not in be to the in of NF-κB V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) activation of NF-κB are with PV for min, activation with PV for 180 V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) IκBα to of with PV and degradation of IκBα. We that IκBα degradation at and at the also be to type between our and of V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) is that the of IκBα PV however, that the of IκBα at 180 and its PV in is on NF-κB activation, that the the resynthesis. effect of on the of induced by PV be as as V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar) a degradation of IκBα at only to of IκBα was not induced by PV in studies by V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). In with V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 26.Singh S. Darney B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 27.Beraud C. Henzel W.J. Baeuerle P. Proc. Natl. Acd. Sci. U. S. A. 1999; 96: 429-434Crossref PubMed Scopus (263) Google Scholar), our studies demonstrate that PV induces phosphorylation of IκBα at tyrosine residue 42. studies also that tyrosine phosphorylation is required for IκBα degradation. We that of with various inhibitors with of blocked IκBα phosphorylation and degradation a inhibitor, however, blocked PV-induced degradation tyrosine phosphorylation of IκBα, that phosphorylation is not sufficient to induce degradation. inhibitor of and serine and both phosphorylation and degradation of IκBα. that phosphorylation of IκBα is needed but not sufficient for degradation induced by is with for IκBα degradation, although the is phosphorylation of serine at positions and of to activate with IκBα degradation but not serine phosphorylation at the N. Karin M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13012-13017Crossref PubMed Scopus (398) Google Scholar). are to in that serine phosphorylation of IκBα was not required for its degradation PV We that although TNF induces ubiquitination of IκBα, PV in the of IκBα degradation by the We that of with or blocked both IκBα degradation and NF-κB activation induced by that IκBα degradation is required for NF-κB activation by We have also not that specific inhibitor of not PV-induced of IκBα but its degradation and NF-κB activation. these are with the for NF-κB activation, they PV-induced NF-κB activation by V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). C. Henzel W.J. Baeuerle P. Proc. Natl. Acd. Sci. U. S. A. 1999; 96: 429-434Crossref PubMed Scopus (263) Google Scholar) that the subunit of the IκBα to NF-κB activation by that IκBα has to be to activate NF-κB by PV is with our phosphorylates IκBα is not and it is that one is including and have been on the A was to be in PV-induced tyrosine phosphorylation of IκBα and NF-κB activation V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). are with of V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar), that tyrosine phosphorylation of IκBα is required for NF-κB activation. We that PV-induced NF-κB activation and IκBα phosphorylation is as did V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). are including and kinases of the protein kinase family P. J.K. J. Immunol. 1995; Google Scholar). role of a of protein kinase in activation of NF-κB and IκBα phosphorylation by TNF and is Hagler J. Chen Maniatis T. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of in IκBα is in with V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 26.Singh S. Darney B.G. Aggarwal B.B. J. Biol. Chem. 1996; 271: 31049-31054Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). on IκBα that PV did not of IκBα. of with tyrosine phosphorylation by J.A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), as it did in our phosphorylation the degradation of IκBα, it is that these the of the of IκBα. IκBα could be of tyrosine phosphorylation of IκBα. of tyrosine phosphorylation of IκBα of V. Rupec R.A. Antonia L. Pahl H.L. Traenckner E.B.-M. Mueller-Dieckmann C. Farahifar D. Rossi B. Auderger P. Baeuerle P.A. Peyron J.-F. Cell. 1996; 86: 787-798Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar). not of a are reoxygenation, which leads to tyrosine phosphorylation of various is as a inhibitor of PTPase that to the activation of and to tyrosine phosphorylation D. W.J. Y. Biochem. J. PubMed Scopus Google Scholar). tyrosine phosphorylation is not with calpeptin, the inhibitor of and proteasome inhibitor that IκBα is degraded by both and to that for degradation of IκBα Y. S. I. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Overall, our that PV-induced tyrosine phosphorylation leads to the degradation of IκBα and the activation of NF-κB in be to as that activate NF-κB tyrosine