A Role for NF-κB in the Induction of β-R1 by Interferon-β

Abstract

Previous experiments have suggested that induction of the β-R1 gene by interferon (IFN)-β required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-κB (NF-κB) can serve as this component. Site-directed mutagenesis of an NF-κB binding site in the β-R1 promoter or over-expression of an IκBα super-repressor abrogated IFN-β-mediated induction of a β-R1 promoter-reporter. IFN-β treatment did not augment abundance of NF-κB but did lead to phosphorylation of the p65 NF-κB subunit. It is proposed that IFN-β-mediated enhancement of the transactivation competence of NF-κB components is required for inducible transcription of the β-R1 promoter. These results provide a novel insight into the role of NF-κB in the transcriptional response to IFN-β. Previous experiments have suggested that induction of the β-R1 gene by interferon (IFN)-β required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-κB (NF-κB) can serve as this component. Site-directed mutagenesis of an NF-κB binding site in the β-R1 promoter or over-expression of an IκBα super-repressor abrogated IFN-β-mediated induction of a β-R1 promoter-reporter. IFN-β treatment did not augment abundance of NF-κB but did lead to phosphorylation of the p65 NF-κB subunit. It is proposed that IFN-β-mediated enhancement of the transactivation competence of NF-κB components is required for inducible transcription of the β-R1 promoter. These results provide a novel insight into the role of NF-κB in the transcriptional response to IFN-β. interferon-β IFN-stimulated gene IFN-stimulated gene factor-3 IFN-stimulated response element mutant ISRE nuclear factor-κB electrophoretic mobility shift assay tumor necrosis factor polyacrylamide gel electrophoresis glutathione S-transferase wild type phosphatidylinositol 3-kinase signal transducers and activators of transcription-3 Interferons (IFNs)1elicit multiple biological responses mediated by the proteins encoded by interferon-stimulated genes (ISGs) (1Gresser I. J. Invest. Dermatol. 1990; 95 (. S-71S): 66Abstract Full Text PDF PubMed Scopus (93) Google Scholar). IFN-α and -β activate transcription of ISGs through the transcription factor interferon-stimulated gene factor-3 (ISGF-3), which interacts with the interferon-stimulated response element (ISRE) present in the promoters of ISGs (2Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5061) Google Scholar). We previously reported the selective induction of β-R1 by IFN-β but not IFN-α in human fibrosarcoma cells (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). ISGF-3 was essential but not sufficient for IFN-β-dependent induction of β-R1 (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In the current report we show that the transcription factor nuclear factor-κB (NF-κB) is also involved in the induction of this gene by IFN-β. NF-κB is a dimer of members of the Rel family of proteins, including p50, p52, c-Rel, RelA/p65, and RelB. NF-κB dimers bind to DNA segments collectively referred to as κB elements (5Chen F.E. Ghosh G. Oncogene. 1999; 18: 6845-6852Crossref PubMed Scopus (266) Google Scholar). In unstimulated cells, NF-κB is sequestered in the cytosol with oligomeric inhibitory components, termed IκB. Stimulation of cells with appropriate inducers results in the rapid degradation of IκB and translocation of NF-κB to the nucleus, where it binds target sequences to initiate transcription. Increasing evidence suggests that a second signaling pathway, independent of IκBα degradation (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar), culminates in phosphorylation of the p65/RelA subunit in its transactivation domain (11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). A role for NF-κB in the induction of ISG in response to IFNs has not been reported. While studying IFN-β activation of β-R1 promoter, we found that modification of pre-existing NF-κB by phosphorylation was essential for IFN-β-induced transcription of β-R1. The human fibrosarcoma cell line UI.wt was maintained as described (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Purified recombinant IFN-β-1b (2 × 108 IU/mg) was from Berlex Biosciences (Richmond, CA). Purified IFN-γ (1.9 × 107 IU/mg) was from Genentech (South San Francisco, CA). TNF-α, from Collaborative Biomedical Products (Bedford, MA), was used at a final concentration of 50 ng/ml. IFN-β and -γ were used at 1000 and 500 units/ml, respectively, unless mentioned otherwise. Human embryonic kidney 293 cells transfected with interleukin-1R were maintained in Dulbecco's modified Eagle's medium with 10% calf serum (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). Z5, a mutant cell line derived from 293-TK/Zeo cells (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar), 2S. S. Sathe, X. Li, and G. R. Stark, unpublished data. exhibits constitutive NF-κB activity as shown by EMSA assays and expression of an NF-κB-dependent promoter. Total RNA was prepared from IFN-treated cells (13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNase protection experiments were performed as described (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The probes used, β-R1 (protects 400 bases), 6-16 (protects 190 bases), and γ-actin (protects 130 bases), are as described (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Site-directed mutagenesis of the ISRE and κB sequence in pGL3-wt-β-R1 (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) was achieved by multiple rounds of polymerase chain reaction using pGL3-wt-β-R1 as template and appropriate primers (14Zhou Z.H. Chaturvedi P. Han Y.L. Aras S. Li Y.S. Kolattukudy P.E. Ping D. Boss J.M. Ransohoff R.M. J. Immunol. 1998; 160: 3908-3916PubMed Google Scholar). Three rounds of polymerase chain reaction were performed using two sets of primers to obtain mISRE-β-R1. The first pair of primers used to make the pGL3-mISRE mutant were 5′-GAAGAGAACAccACAtAAACTCTTGGAAGC-3′ (forward primer with ISRE sequence underlined and mutagenized nucleotides in lowercase) and BglII reverse primer 5′-TTGGAAGATCTAGTAGAAATG-3′. The second pair of primers wereSacI primer 5′-ATACGAGCTCTCCGCTGC-3′ and 5′-GCTTCCAAGAGTTTaTGTggTGTTCTCTTC-3′ (backward primer with ISRE sequence underlined and mutagenized nucleotides in lowercase). The first pair of primers used to make pGL3-mκB mutant were 5′-GCATGACTCAAAGAGtGAAATTaCTGTGCCAT-3′ (forward primer with NF-κB sequence underlined and mutagenized nucleotides in lowercase) and BglII reverse primer 5′-TTGGAAGATCTAGTAGAAATG-3′. The second pair of primers were SacI primer 5′-ATACGAGCTCTCCGCTGC-3′ and 5′- ATGGCACAGtAATTTCaCTCTTTGAGTCATGC-3′ (backward primer with κB binding sequence underlined and mutagenized nucleotides in lowercase). Plasmid DNA from two clones for each set of cloning was sequenced to verify the nucleotide sequence. Transient transfections were done as described (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), with 10 μg of β-R1 reporter plasmid, 10 μg of test plasmid DNA and 1 μg of pCH110 β-galactosidase plasmid DNA. The plasmid construct pCMV-IκBα (with Ser32 and Ser36 mutated to Ala) was as reported earlier (15DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar); pCMV-50 was kindly provided by Dr. M. Kieran (16Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israel A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (602) Google Scholar). The pCMV-65 plasmid was generously given by Dr. D. W. Ballard (17Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). The p561-luciferase construct containing the ISRE from p56 ISG and the 6xIP-10κB-luciferase construct has been reported earlier (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). For electrophoretic mobility shift assays (EMSA) the NF-κB binding site (5′-GAGCAGAGGGAAATTCCGTAACTT-3′) from thehIP-10 gene was used as a probe. Nuclear extracts were prepared and gel-shift assays performed as described earlier (18Majumder S. Zhou L.Z. Chaturvedi P. Babcock G. Aras S. Ransohoff R.M. J. Immunol. 1998; 161: 4736-4744PubMed Google Scholar). U1.wt cells were labeled with [32P]orthophosphate (200 μCi/ml) as described (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). Cells were stimulated for 5 min with IFN-β (5000 units/ml) or TNF-α (50 ng/ml). Lysis and immunoprecipitations were performed using anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) as described (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar,11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Phosphorylated proteins were separated by 10% SDS-PAGE and autoradiographed. The carboxyl terminus of p65 fused to GST obtained from Dr. H. Sakurai (8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar) was expressed in E. coli and cells lysed in 0.5% Nonidet P-40 lysis buffer as described (19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). In vitro phosphorylation was performed using ∼1 μg of GST-fused p65 protein in 23 μl of kinase buffer containing 3 μCi [γ-32P]ATP (19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Nuclear extracts from IFN-β-, IFN-α-, or TNF-α-treated cells (3 μg of protein in a volume of 2 μl) were added to the reaction and incubated at 30 °C for 30 min (8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Following the kinase reaction, phosphorylated protein was separated by 10% SDS-PAGE and autoradiographed. Western analysis with anti-GST antibody (Amersham Pharmacia Biotech) was used to quantitate the total amount of p65 protein. We performed a functional analysis of the β-R1 ISRE-like element using wt-β-R1 and mutant mISRE-β-R1 (Fig.1 a). In transient transfection assays, IFN-β responsiveness of mISRE-β-R1 was reduced by more than 80% compared with the wt-β-R1 promoter (Fig. 1 a). This result confirmed the requirement of this ISRE-like element for the induction of β-R1 by IFN-β. The induction of mISRE-β-R1 activity by IFN-γ was also significantly reduced by about 70% compared with wt-β-R1 (Fig. 1 a). These results are consistent with our earlier observation in U2A cells (which lack the p48/IRF-9 ISRE recognition factor) of loss of induction of β-R1 by IFN-β or IFN-γ (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We proposed that a second cis-element might be involved in the induction of the β-R1 gene (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We had previously observed 3M. R. S. Rani and R. M. Ransohoff, unpublished data. synergistic induction of β-R1 by IFN-β and TNF-α, the latter being a well characterized activator of transcription factor NF-κB. Hence, a salient choice for analysis was the κB element on the promoter of β-R1. Using transient transfection assays, wt-β-R1 or mκB-β-R1 promoter-reporters were analyzed for IFN-β responsiveness. Mutation of the κB element on the promoter of β-R1 markedly reduced IFN-β induction of the promoter, by 70% (Fig. 1 b). Induction of the wild-type and mκB-β-R1 constructs by IFN-γ differed only slightly (Fig. 1 b). This result indicated that the κB element was specifically required for IFN-β but not IFN-γ-mediated induction. We used EMSAs to address whether the requirement for the κB binding site was associated with IFN-β induced nuclear translocation and DNA binding by NF-κB. Analysis of extracts of U1.wt cells revealed, as previously reported (20Plattner R. Gupta S. Khosravi-Far R. Sato K.Y. Perucho M. Der C.J. Stanbridge E.J. Oncogene. 1999; 18: 1807-1817Crossref PubMed Scopus (50) Google Scholar, 21Wiest J.S. Burnett V.L. Anderson M.W. Reynolds S.H. Oncogene. 1994; 9: 2449-2454PubMed Google Scholar), basal NF-κB DNA binding activity (Fig.2 a). Super-shift experiments showed complexes composed of p65 homodimers, and p65/p50 heterodimers (results not shown). No IFN-β-inducible in DNA binding activity was by analysis of the of the NF-κB complexes to a (Fig.2 b). address the that basal nuclear NF-κB activity might be essential for induction of β-R1 by we analyzed two cell that differed in basal NF-κB. 293 cells did not basal nuclear NF-κB as by cells constitutive NF-κB (Fig. 2 The κB binding in cells of p65 and (Fig.2 IFN-β treatment did not a in NF-κB in cell RNase protection analysis the induction of β-R1 by IFN-β in cells but not in 293 cells (Fig. 2 type gene (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar, Z.H. Chaturvedi P. Han Y.L. Aras S. Li Y.S. Kolattukudy P.E. Ping D. Boss J.M. Ransohoff R.M. J. Immunol. 1998; 160: 3908-3916PubMed Google Scholar, J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israel A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (602) Google Scholar) be induced well in cell TNF-α, in with mediated synergistic induction of β-R1 in and 293 cells, the of an NF-κB in cell We also super-repressor mutant of IκBα in U1.wt cells, in induction of the wt-β-R1 by IFN-β (Fig. 2 of IκBα we analyzed IFN-β-mediated induction of a construct derived from the gene type Induction of the (which a κB was by IκBα (Fig. 2 In transient transfection assays, a construct was not by but TNF-α a induction (Fig. 2 We whether of (which lack a transactivation domain and are were sufficient to β-R1 transcription by of at the κB For was in U1.wt cells with the wt-β-R1 and reporter gene activation in response to IFN-β was IFN-β responsiveness of the promoter was reduced by to a to that obtained by of the NF-κB binding site This result indicated that were to transcription of β-R1 and suggested the requirement of NF-κB of p65 but not for the induction of β-R1 by activity was for transfection as described and expressed in were done as reported reporter activity on transfection of wt-β-R1 in the of treatment was set at This was used to the of treatment with IFN-β or over-expression of NF-κB with wt-β-R1 μg of pCMV-65 was used with 10 μg of wt-β-R1 activity was for cells transfected with p65 in the of wt-β-R1 induction in activity by IFN-β was significantly of wt-β-R1 and pCMV-50 compared to wt-β-R1 with activity was for transfection as described and expressed in were done as reported activity on transfection of wt-β-R1 in the of treatment was set at This was used to the of treatment with IFN-β or over-expression of NF-κB μg of pCMV-65 was used with 10 μg of wt-β-R1 activity was for cells transfected with p65 in the of induction in activity by IFN-β was significantly of wt-β-R1 and pCMV-50 compared to wt-β-R1 with in a of p65 with the wt-β-R1 promoter the basal activity of the promoter by IFN-β treatment promoter activity by results indicated that NF-κB complexes were required for transcription of the β-R1 gene in response to IFN-β. NF-κB activity was essential for IFN-β-mediated transcription of IFN-β treatment did not lead to NF-κB (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that phosphorylation of p65 NF-κB-dependent gene transcription. address IFN-β-mediated phosphorylation of NF-κB proteins, cells were labeled with and cell extracts were with anti-p65 which p50, and IκB (Fig. 3 a). The of the phosphorylated proteins was confirmed by Western analysis (results not shown). This analysis in p65 phosphorylation in cells with or TNF-α a). of p65 in U1.wt cells was as cells basal NF-κB nuclear We also IFN-β-mediated in and IκB phosphorylated IκBα in cells but was in TNF-α-treated 5 min and 1 we observed rapid IκBα degradation in response to TNF-α but not to IFN-β (Fig. 3 using GST-fused p65 that an inducible p65 activity was present in nuclear extracts of or TNF-α cells (Fig. 3 but not in cells (Fig. 3 we address the role of NF-κB in the induction of β-R1 by IFN-β. We had previously that ISGF-3 was essential but not sufficient for the induction of the gene and proposed that an additional might be required (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We NF-κB of the TNF-α and IFN-β for induction of the that TNF-α induced β-R1. Site-directed mutagenesis of the κB element in the β-R1 promoter or over-expression of a super-repressor mutant of IκBα the induction of the β-R1 gene by that NF-κB components were IFN-β did not NF-κB DNA binding activity by IFN-β treatment did not activate transcription from a NF-κB address the of whether the of κB binding activity was sufficient to the transcriptional response of the β-R1 gene to we or of β-R1 over-expression of p65 basal β-R1 promoter activity but a in response to IFN-β. These results confirmed a requirement for NF-κB complexes to transcription of the β-R1 In and cell type was reported to signal to NF-κB a and kinase A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of this markedly NF-κB. NF-κB-dependent gene expression was not in or A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. N. S. M. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. A.S. Mol. Biochem. 1998; PubMed Scopus Google Scholar). The signaling described in this report A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), as a site for signaling is not observed in cells, where activation is for kinase activity and where and not M.R. Leaman D.W. Han Leung S. E. Fish E.N. A. Ransohoff R.M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). signaling of type activation in cells as compared with cells A. L. S. A. PubMed Scopus Google Scholar), it was that NF-κB DNA binding activity not be observed of the of p65 phosphorylation for NF-κB-dependent gene transcription (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and in of we showed that p65 was phosphorylated in A of have been in the phosphorylation of p65 (8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). This report to the multiple of NF-κB. is in NF-κB DNA binding cells an in which to modification of