The p38 Mitogen-activated Protein Kinase Is Required for NF-κB-dependent Gene Expression
1999; Elsevier BV; Volume: 274; Issue: 43 Linguagem: Inglês
10.1074/jbc.274.43.30858
ISSN1083-351X
AutoresA. Brent Carter, Kevin L. Knudtson, Martha M. Monick, Gary W. Hunninghake,
Tópico(s)Immune Response and Inflammation
ResumoEndotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-κB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-κB sites, we hypothesized that the p38 MAP kinase was necessary for NF-κB-dependent gene expression. We found that NF-κB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-κB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-κB-dependent gene transcription, in part, by modulating activation of TFIID (TBP). Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-κB. We have previously shown that the p38 mitogen-activated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-κB sites, we hypothesized that the p38 MAP kinase was necessary for NF-κB-dependent gene expression. We found that NF-κB-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-κB activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-κB-dependent gene transcription, in part, by modulating activation of TFIID (TBP). lipopolysaccharide or endotoxin nuclear factor κB IκB kinase transcription factor IIB transcription factor IID TATA-binding protein mitogen-activated protein extracellular signal-regulated kinase activating transcription factor 2 MAP kinase kinase kinase 1 c-Jun MAP kinase polyacrylamide gel electrophoresis Cytokine gene expression in endotoxin (LPS)1-stimulated macrophages is regulated, at least in part, at the level of transcription. A transcription factor that is necessary for the transcription of many cytokine genes is nuclear factor κB (NF-κB) (1Collart M.A. Baeuerle P. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar, 2Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-2334Crossref PubMed Google Scholar, 3Matsusaka T. Fujikawa K. Nishio Y. Mukaida N. Matsushima K. Kishimoto T. Akira S. Proc. Natl. Acad. Sci. U. S. 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Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3181) Google Scholar). In addition, we found that the p38 MAP kinase also regulated LPS-induced cytokine gene expression at the level of transcription in macrophages (25Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar). The p38 MAP kinase is known to regulate various transcription factors, such as ATF-2, by phosphorylation (27Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar, 28Nick J.A. Avdi N.J. Gerwins P. Johnson G.L. Worthen G.S. J. Immunol. 1996; 156: 4867-4875PubMed Google Scholar, 29Derijard B. Raingeaud J. Barrett T. Wu I.H. Han J. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1431) Google Scholar, 30Cuenda A. Cohen P. Buee-Scherrer V. Goedert M. EMBO J. 1997; 16: 295-305Crossref PubMed Scopus (318) Google Scholar). One study, using tumor necrosis factor as a stimulus, found that inhibition of the p38 MAP kinase with SB 203580 did not alter NF-κB DNA binding (31Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (609) Google Scholar). Another study showed that overexpression of MEKK1, an upstream kinase that activates both the p38 and c-Jun (JNK) MAP kinases, increased NF-κB-driven transcription (32Read M.A. Whitley M.Z. Gupta S. Pierce J.W. Best J. Davis R.J. Collins T. J. Biol. Chem. 1997; 272: 2753-2761Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). MEKK1 has also been found to interact directly with both IKK-α and IKK-β (33Nemoto S. DiDonato J.A. Lin A. Mol. Cell. Biol. 1998; 18: 7336-7343Crossref PubMed Google Scholar). Using a promoter construct driven only by NF-κB, we found that LPS-induced NF-κB-dependent gene expression was inhibited by SB 203580 and a dominant-negative p38 MAP kinase expression vector. We performed electrophoretic mobility shift assays and found that inhibition of the p38 MAP kinase with SB 203580 did not affect NF-κB DNA binding. To confirm these findings, we also found that IκB-β degradation was not affected by p38 MAP kinase inhibition. We next evaluated if the p38 MAP kinase regulated phosphorylation of the p65 subunit by performing in vivo phosphorylation studies and found that SB 203580 did not regulate phosphorylation of the p65 subunit of NF-κB. The inhibition of the p38 MAP kinase with SB 203580 did, however, reduce the DNA binding of TFIID (TBP) to the TATA box, and the dominant-negative p38 MAP kinase expression vector altered the direct interaction of TFIID (TBP) with a co-transfected His-p65 fusion protein. It also interfered with the binding of a co-transfected His-TBP fusion protein with native p65. The p38 MAP kinase was also found to phosphorylate TFIID (TBP) in vitro, and SB 203580 inhibited the phosphorylation of TFIID (TBP) in vivo. These findings suggest that the p38 MAP kinase regulates NF-κB-driven gene expression, in part, by regulation of TFIID (TBP) activation. The THP-1 cell line was obtained from American Type Culture Collection (Manassas, VA). The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with gentamicin and 10% fetal calf serum (Life Technologies, Inc.). In in vivo phosphorylation studies, cells were cultured in phosphate-free RPMI 1640 medium (Life Technologies, Inc.) with the same supplements. NF-κB-dependent gene expression was evaluated using a luciferase reporter gene driven by four tandem copies of the κ enhancer (κB4) in a pUC vector (CLONTECH, Palo Alto, CA). The pCMV-Flag-p38 plasmid has been previously described (a generous gift from Dr. Roger Davis, University of Massachusetts, Worcester, MA) (27Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2061) Google Scholar). The cDNA for the p65 subunit of NF-κB was generated by reverse transcription-polymerase chain reaction from a total RNA preparation (RNA Stat-60, Tel-test B, Friendswood, TX) from THP-1 cells. First-strand synthesis was performed using the primer 5′-GCTTTTGGAGGGCTTCAATC-3′. Amplification of the p65 gene was performed using the primer 5′-CCCGCGGCATGGACGAACTG-3′. A set of nested primers, 5′-CTGGGATCCCTATGGACGAACTGTTCCCCCTC-3′ and 5′-AGACTCGAGTTAGGAGCTGATCTGACTCAGC-3′ were then used in a second polymerase chain reaction to generate an amplicon that consisted of the p65 coding sequence flanked by BamHI and XhoI restriction sites. This amplicon was ligated with the pcDNA3.1/HisA vector (Invitrogen, Carlsbad, CA) to give plasmid pcDNA-His-p65. The cDNA for TBP was generated from THP-1 RNA by reverse transcription-polymerase chain reaction. Amplification of the TBP gene was performed by using the primers 5′-CTAGGATCCAGATGGATCAGAACAACAGCCTGCC-3′ and 5′-CTATCTAGATTACGTCGTCTTCCTGAATCCC-3′. The resulting amplicon was then digested with BamHI and XbaI and ligated into the similarly digested expression vector pcDNA3.1/HisA (Invitrogen) to generate the plasmid pcDNA-His-TBP. The correct reading frame and sequence were verified by fluorescent automated DNA sequencing performed by the University of Iowa DNA Facility. Transfections were performed using the Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's recommendations. Twenty-four h after transfection the cells were stimulated with Escherichia coli serotype 026:B6 LPS (Sigma) at a dose of 100 μg/ml. Luciferase activity, which was normalized to total protein, was measured after 6 h (Promega, Madison, WI), which was determined to be the time of maximal activity. The p38 MAP kinase inhibitor, SB 203580 (Calbiochem), at 0.5 μm, was added 1 h before stimulation with LPS. THP-1 cells were cultured in the presence or absence of SB 203580 for 1 h before stimulation with LPS. After 3 h, the cells were harvested, and nuclear protein was extracted as described previously (5Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1998; 18: 384-391Crossref PubMed Scopus (115) Google Scholar). A consensus NF-κB (5′-AGTTGAGGGGATTTTCCCAGGC-3′) oligonucleotide (Promega) and a TFIID (TBP) (5′-GCAGAGCATATAAAATGAGGTAGGA-3′) oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) were labeled with [γ-32P]ATP (NEN Life Science Products). Binding reactions were performed as described previously (5Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1998; 18: 384-391Crossref PubMed Scopus (115) Google Scholar), and the protein-DNA complexes were separated on a 5% polyacrylamide gel. Whole cell lysates were prepared as described previously (25Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar, 34Monick M.M. Carter A.B. Gudmundsson G. Mallampalli R. Powers L.S. Hunninghake G.W. J. Immunol. 1999; 162: 3005-3012PubMed Google Scholar). The p38, JNK1, or Erk2 MAP kinases were immunoprecipitated from the lysates overnight at 4 °C with either a p38, JNK1, or Erk2 polyclonal rabbit antibodies (Santa Cruz Biotechnology), respectively, bound to Gammabind with Sepharose (Amersham Pharmacia Biotech). Kinase activity was assayed as described previously using ATF-2, c-Jun, or TFIID (TBP) (Santa Cruz Biotechnology) as a substrate (25Carter A.B. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1999; 20: 751-758Crossref PubMed Scopus (284) Google Scholar, 34Monick M.M. Carter A.B. Gudmundsson G. Mallampalli R. Powers L.S. Hunninghake G.W. J. Immunol. 1999; 162: 3005-3012PubMed Google Scholar). For Western blot analysis, SDS-polyacrylamide gels were transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) in 25 mmTris, 192 mm glycine, and 20% methanol. IκB-β, p65, TFIID (TBP), Erk2, JNK1, and p38 MAP kinase rabbit polyclonal antibodies (Santa Cruz Biotechnology) were used at 1:1000 dilutions. The anti-Xpress monoclonal antibody (Invitrogen) recognizes the sequence Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys from the leader peptide in the His-p65 and His-TBP fusion proteins and was used at a 1:5000 dilution. Immunoreactive proteins were developed using a chemiluminescent (Amersham Pharmacia Biotech) or chemifluorescent (JBL Scientific, San Luis Obispo, CA) substrate. The cells were labeled with 1.25 mCi of 32Pi/group (NEN Life Science Products) in phosphate-free RPMI medium with 10% fetal calf serum for 3 h at 37 °C. The cells were harvested and placed in RPMI 1640 medium with 10% fetal calf serum and stimulated with LPS for 1 h at 37 °C. The cells were harvested, resuspended in radioimmunoprecipitation assay lysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 0.01m Na3PO4 (pH 7.2), 2 mmEDTA, 50 mm NaF, 0.2 mmNa3VO4, 1 μm okadaic acid, 100 μg/ml phenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 10 μg/ml leupeptin, 50 μg/ml pepstatin), and sonicated. The p65 subunit of NF-κB or TFIID (TBP) were immunoprecipitated with either the p65 or TFIID (TBP) rabbit polyclonal antibodies (Santa Cruz Biotechnology), respectively, bound to Gammabind with Sepharose (Amersham Pharmacia Biotech) for 2 h to overnight at 4 °C. The Sepharose pellet was washed twice with high salt radioimmunoprecipitation assay buffer (1m NaCl) and twice with radioimmunoprecipitation assay buffer. The samples were separated on a SDS-PAGE discontinuous gel. Before assessing the role of the p38 MAP kinase in regulating NF-κB-driven gene expression, we assured ourselves that SB 203580 was working appropriately in THP-1 cells. To demonstrate that SB 203580 was inhibiting p38 MAP kinase and not Erk- or JNK-mediated signaling, we performed kinase assays and Western blot analysis of the Erk2, JNK1, and p38 MAP kinases. We found that LPS significantly increased Erk2 kinase activity by measuring the phosphorylation of c-Jun, and SB 203580 had no detectable effect on this activity (Fig.1 A). Likewise, LPS increased JNK1 kinase activity, and SB 203580 had no appreciable effect on this activity, which was measured by detecting the phosphorylation of c-Jun (Fig. 1 B). In contrast, although LPS also significantly increased p38 kinase activity, as shown by the phosphorylation of ATF-2, SB 203580 reduced this activity to control levels (Fig.1 C). Western blot analysis for each of these kinase assays showed equal loading of proteins. These studies confirm that SB 203580 is effective and relatively specific for p38 MAP kinase-mediated signaling. To evaluate the role of the p38 MAP kinase in regulating LPS-induced NF-κB-dependent gene expression, we measured NF-κB-dependent promoter activity using luciferase reporter plasmids. We found that LPS significantly increased (greater than 10× control) luciferase activity, and inhibition of the p38 MAP kinase with SB 203580 reduced this activity to near control levels (Fig. 2 A). Due to previous studies that have suggested that the p38 MAP kinase inhibitors have a nonspecific effect (35Borsch-Haubold A.G. Pasquet S. Watson S.P. J. Biol. Chem. 1998; 273: 28766-28772Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar), we performed similar studies using a dominant-negative p38 MAP kinase expression vector. In these studies, either a blank vector or the dominant-negative p38 MAP kinase expression vector was co-transfected with the luciferase reporter plasmid. The luciferase activity was reduced to near control levels in the cells that expressed the dominant-negative p38 MAP kinase compared with cells with the blank vector alone (Fig. 2 B). As an aggregate, these studies show that LPS-induced NF-κB-dependent gene expression requires an active p38 MAP kinase. To determine the role of the p38 MAP kinase in regulating NF-κB-dependent gene expression, we first asked if the p38 MAP kinase regulated NF-κB translocation. LPS caused a significant increase in NF-κB translocation and DNA binding in THP-1 cells, and inhibition of the p38 MAP kinase with SB 203580 had no significant effect on this activation (Fig. 3). To evaluate translocation in a different manner and confirm this finding, we measured IκB-β degradation in LPS-stimulated THP-1 cells since NF-κB-dependent gene expression requires activation of IKK-β (17O'Connell M.A. Bennett B.L. Mercurio F. Manning A.M. Mackman N. J. Biol. Chem. 1998; 273: 30410-30414Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We found that IκB-β degradation was maximal at 60 min (data not shown), and inhibition of the p38 MAP kinase did not affect IκB-β degradation (Fig. 4). We next determined if the p38 MAP kinase altered phosphorylation of the p65 subunit of NF-κB. THP-1 cells were labeled with32Pi, and phosphorylation was measured. LPS induced phosphorylation of native p65, and SB 203580 had no significant effect on this phosphorylation (Fig. 5). Taken together, these findings show that the p38 MAP kinase does not directly regulate degradation of IκB, NF-κB translocation, NF-κB DNA binding, or phosphorylation of the p65 subunit.Figure 4IκB-β degradation. THP-1 cells were cultured for 1 h in the presence or absence of SB 203580 (SB). Cells were then stimulated with LPS for 1 h, which was determined to be the time point of maximal degradation. Samples from the whole cell lysates were separated on a SDS-PAGE gel and transferred to polyvinylidene difluoride membrane. IκB-β rabbit polyclonal antibody was used at a 1:1000 dilution, and immunoreactive proteins were detected by a chemiluminescent substrate. This figure is representative of three different experiments. k = 1000.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Phosphorylation of p65 subunit of NF-κB. Cells were labeled with32Pi in phosphate-free media for 3 h. Cells were stimulated with LPS for 1 h. Whole cell lysates were subjected to immunoprecipitation with a p65 rabbit polyclonal antibody, and samples were separated on a SDS-PAGE gel. This figure is representative of six different experiments. SB, SB 203580.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since NF-κB-driven transcription also depends on activation of basal transcription factors, such as TFIIB and TFIID (TBP), we next evaluated if the p38 MAP kinase regulated this activity. We first determined whether the p38 MAP kinase regulated the direct interaction of the p65 subunit with the basal transcription factors using two reciprocal methods. First, we co-transfected the pcDNA-His-p65 plasmid with either the dominant-negative p38 MAP kinase expression vector or a blank vector. By immunoprecipitating whole cell lysates with anti-Xpress antibody and performing Western blot analysis for the TFIID (TBP), we found that LPS increased this interaction in cells co-transfected with the blank vector. Cells that expressed the dominant-negative p38 kinase had a greater than 50% reduction, as measured by densitometry, in the direct interaction of TFIID (TBP) and the His-p65 fusion protein (Fig.6, A and B). In the second method, we co-transfected the pcDNA-His-TBP with either the dominant-negative p38 MAP kinase expression vector or a blank vector. The His-TBP fusion protein was immunoprecipitated with anti-Xpress antibody. Western blot analysis for the p65 subunit showed that LPS increased the interaction of His-TBP with the native p65 in the cells expressing the blank vector compared with those expressing the dominant-negative p38 MAP kinase, which had a greater than 50% reduction in direct interaction (Fig. 6 C). These findings suggest that an active p38 MAP kinase is necessary for direct binding of these transcription factors. We next evaluated if inhibition of the p38 MAP kinase altered TBP binding to the TATA box. THP-1 cells stimulated with LPS had an increase in TBP DNA binding, and SB 203580 significantly reduced this binding to control levels (Fig. 7). If the p38 MAP kinase regulated TBP binding, it seemed logical that it would regulate binding by phosphorylation. Thus, we determined if the p38 MAP kinase phosphorylated TFIID (TBP) in vitro. We found that TFIID (TBP) was phosphorylated in vitro by LPS-stimulated p38 MAP kinase obtained from THP-1 cells (Fig.8 A). The activation kinetics of p38 MAP kinase was maximal at 15 min, but there was increased activity as early as 5 min and as late as 90 min after exposure to LPS (Fig. 8 B). The binding kinetics of TBP to the TATA box are similar to the activation kinetics of p38 MAP kinase. There was increased binding as early as 5 min, and there was a gradual increase in binding up to 90 min (Fig. 8 C). This suggests that the continuous activity of the p38 MAP kinase results in increased TBP phosphorylation and subsequent binding to the TATA box. To confirm that the phosphorylation of the TFIID (TBP) by the p38 MAP kinase is physiologically relevant, we performed in vivophosphorylation studies. THP-1 cells were labeled with32Pi, and phosphorylation was measured. LPS induced phosphorylation of native TFIID (TBP), and SB 203580 reduced this phosphorylation to control levels (Fig.9 A). Western blot analysis for TFIID (TBP) shows equal loading of each immunoprecipitated protein (Fig. 9 B). As an aggregate, these findings suggest that the phosphorylation of TFIID (TBP) by the p38 MAP kinase regulates the interaction of the TFIID (TBP) with the p65 subunit and the DNA binding to the TATA box.Figure 8TFIID ( TBP ) phosphorylation by the p38 MAP kinase. A, THP-1 cells were cultured in the presence or absence of LPS as indicated for 15 min. Whole cell lysates were subjected to immunoprecipitation with Gammabind alone, as negative control (first lane), or p38 MAP kinase rabbit polyclonal antibody (second and third lanes). In vitrokinase assays were performed using TFIID (TBP) as the substrate. Western blot analysis for p38 protein was performed to confirm equal loading of proteins. B, THP-1 cells were stimulated with LPS at the designated time points. Whole cell lysates were subjected to immunoprecipitation with p38 MAP kinase rabbit polyclonal antibody.In vitro kinase assays were performed using TFIID (TBP) as the substrate. C, THP-1 cells were stimulated with LPS at the designated time points, and nuclear protein was isolated. Binding reactions were performed with the consensus TFIID (TBP) oligonucleotide labeled with [γ-32P]ATP. These figures are representative of three different experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Phosphorylation of TFIID ( TBP ). A, cells were labeled with 32Pi in phosphate-free media for 3 h. Cells were stimulated with LPS for 1 h. Whole cell lysates were subjected to immunoprecipitation with a TFIID (TBP) rabbit polyclonal antibody, and samples were separated on a SDS-PAGE gel. B, Western blot analysis for TFIID (TBP) was performed to confirm equal loading of the immunoprecipitated proteins. These figures are representative of three different experiments.SB, SB 203580.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In these studies, we found that LPS-induced NF-κB-dependent gene expression was significantly reduced by SB 203580, a competitive inhibitor of the p38 MAP kinase, and by a dominant-negative p38 MAP kinase expression vector. These observations showed that the p38 MAP kinase was necessary for NF-κB-dependent gene transcription. To determine the level at which this regulation occurred, we first evaluated NF-κB translocation and DNA binding and found that SB 203580 had no appreciable effect. To confirm these findings in a different manner, we measured IκB-β degradation and found that it was not reduced by p38 MAP kinase inhibition. We next evaluated if the p38 MAP kinase regulated phosphorylation of the p65 subunit of NF-κB by performingin vivo phosphorylation studies. We found that SB 203580 did not regulate phosphorylation of native p65. Due to the interaction of the p65 subunit with basal transcription factors, such as TFIID (TBP), we determined what role the p38 MAP kinase had in regulating its activation. The dominant-negative p38 MAP kinase expression vector altered the interaction of TFIID (TBP) with a co-transfected His-p65 fusion protein. Likewise, the dominant-negative p38 kinase also regulated the interaction of native p65 with a co-transfected His-TBP fusion protein. In addition, we found that inhibition of the p38 MAP kinase reduced TFIID (TBP) binding to the TATA box and that the p38 kinase phosphorylated this basal transcription factor in vitro. We confirmed that these observations were physiologically relevant by showing that inhibition of the p38 MAP kinase decreased the LPS-induced phosphorylation of TFIID (TBP) in vivo. The regulation of NF-κB-dependent gene expression can occur at multiple levels after cell stimulation. Early regulation occurs in the cytoplasm with the activation of IKK and subsequent IκB phosphorylation, ubiquitination, and proteolysis (9Finco T.S. Baldwin A.S. Immunity. 1995; 3: 263-272Abstract Full Text PDF PubMed Scopus (385) Google Scholar, 10Matthews J.R. Hay R.T. Int. J. Biochem. Cell Biol. 1995; 27: 865-879Crossref PubMed Scopus (72) Google Scholar, 11Yin M.J. Yamamoto Y. Gaynor R.B. 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The interaction of NF-κB with the basal transcription factors, such as TFIIB and TBP, is a much more likely scenario. The p65 subunit interacts directly with these basal transcription factors in a manner that activates gene expression in COS7 cells (22Schmitz M.L. Stelzer G. Altmann H. Meisterernst M. Baeuerle P.A. J. Biol. Chem. 1995; 270: 7219-7226Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). TBP is an essential component of transcription initiation in class I, II, and III promoters (40Timmers H.T. Meyers R.E. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8140-8144Crossref PubMed Scopus (58) Google Scholar, 41Brou C. Chaudhary S. Davidson I. Lutz Y. Wu J. Egly J.M. Tora L. Chambon P. EMBO J. 1993; 12: 489-499Crossref PubMed Scopus (155) Google Scholar, 42Lescure A. Lutz Y. Eberhard D. Jacq X. Krol A. Grummt I. Davidson I. Chambon P. Tora L. EMBO J. 1994; 13: 1166-1175Crossref PubMed Scopus (97) Google Scholar, 43Martinez E. Chiang C.M. Ge H. Roeder R.G. EMBO J. 1994; 13: 3115-3126Crossref PubMed Scopus (162) Google Scholar), and it is one of the subunits of TFIID (40Timmers H.T. Meyers R.E. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8140-8144Crossref PubMed Scopus (58) Google Scholar). One study has shown that phosphorylation of both TBP and TFIIB in the amino-terminal regions could regulate the transcription of class II promoters (44Chibazakura T. Watanabe F. Kitajima S. Tsukada K. Yasukochi Y. Teraoka H. Eur. J. Biochem. 1997; 247: 1166-1173Crossref PubMed Scopus (41) Google Scholar). Our data corroborates that phosphorylation of TFIID (TBP) regulates transcription of class II promoters. The novel finding, however, is that the p38 MAP kinase regulates the activation of TFIID (TBP). In fact, we found that inhibition of phosphorylation reduced its binding to the TATA box and its interaction with the p65 subunit of NF-κB. This, in turn, is a plausible mechanism for the regulation of NF-κB-dependent gene expression by the p38 MAP kinase. A phosphorylation substrate for the p38 MAP kinase must have the minimal consensus sequence Ser/Thr-Pro (45Alvarez E. Northwood I.C. Gonzalez F.A. Latour D.A. Seth A. Abate C. Curran T. Davis R.J. J. Biol. Chem. 1991; 266: 15277-15285Abstract Full Text PDF PubMed Google Scholar). One study has shown that another serine/threonine kinase, DNA-dependent protein kinase, phosphorylates TBP in the amino-terminal region (44Chibazakura T. Watanabe F. Kitajima S. Tsukada K. Yasukochi Y. Teraoka H. Eur. J. Biochem. 1997; 247: 1166-1173Crossref PubMed Scopus (41) Google Scholar), so there are several Ser/Thr-Pro motifs present in the first 160 amino acids. Although DNA-dependent protein kinase has a slightly different consensus sequence requirement, both proline-directed and minimal consensus sequences, which are necessary for MAP kinase specificity, are present in the amino-terminal region of TBP. Our data clearly shows that the p38 MAP kinase phosphorylates TFIID (TBP), and this phosphorylation is necessary for TBP binding to the TATA box. Therefore, the p38 MAP kinase regulates NF-κB-dependent transcription in part by modulating activation of basal transcription factors. We thank the University of Iowa DNA Facility for sequencing the plasmids used in this study and R. Similien for outstanding technical assistance.
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