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• W1480945793 abstract "Nuclear factor κB (NF-κB) is stored in the cytoplasm as an inactive form through interaction with IκB. Stimulation of cells leads to a rapid phosphorylation of IκBα, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-κB in association with the induction of IκBα phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates IκBα. LPS stimulation transiently enhanced the IκBα-bound kinase activity in THP-1 cells. Mutational analyses of IκBα and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of IκBα. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of IκBα, blocked the LPS-induced NF-κB activation as well as inducible phosphorylation of endogenous IκBα in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of IκBα and subsequent dissociation of the NF-κB•IκBα complex. Nuclear factor κB (NF-κB) is stored in the cytoplasm as an inactive form through interaction with IκB. Stimulation of cells leads to a rapid phosphorylation of IκBα, which is presumed to be important for the subsequent degradation. We have recently reported the establishment of a lipopolysaccharide (LPS)-dependent cell-free activation system of NF-κB in association with the induction of IκBα phosphorylation. In this study, we have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically binds and phosphorylates IκBα. LPS stimulation transiently enhanced the IκBα-bound kinase activity in THP-1 cells. Mutational analyses of IκBα and competition experiments with the synthetic peptides identified major phosphorylation sites by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of IκBα. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of IκBα, blocked the LPS-induced NF-κB activation as well as inducible phosphorylation of endogenous IκBα in a cell-free system using THP-1 cells. These results suggested that the bound kinase is involved in the signaling pathway of LPS by inducing the phosphorylation of the C-terminal region of IκBα and subsequent dissociation of the NF-κB•IκBα complex. NF-κB consists of a family of transcriptional factors that play a key role in the regulation of a number of immune and inflammatory response genes(1Leonardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1245) Google Scholar, 2Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar, 3Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1214) Google Scholar), including several inflammatory cytokines such as IL-8 1The abbreviations used are: ILinterleukinCKcasein kinaseEMSAelectrophoretic mobility shift assayGSTglutathione S-transferaseNFnuclear factorLPSlipopolysaccharideMAPKmitogen-activated protein kinasePAGEpolyacrylamide gel electrophoresisTNFtumor necrosis factorHPLChigh performance liquid chromatographyPCRpolymerase chain reaction. and IL-6(4Collart M. Baeuerle P.A. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar, 5Libermann T.A. Baltimore D. Mol. Cell. Biol. 1990; 10: 2327-2334Crossref PubMed Google Scholar, 6Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1991; 265: 21128-21133Google Scholar). Members of the family include p65 (RelA), RelB, c-Rel, p50 (NF-κB-1), and p52 (NF-κB-2). NF-κB is retained in an inactive form being associated with its inhibitors, IκB, in the cytoplasm in most types of cells (7Baeuerle P.A. Baltimore D. Cell. 1988; 53: 211-217Abstract Full Text PDF PubMed Scopus (785) Google Scholar). Following stimulation of cells with a variety of agents, e.g. IL-1, tumor necrosis factor (TNF), phorbol myristate acetate, and lipopolysaccharide (LPS), NF-κB is released from IκB and is translocated to the nucleus and binds to the NF-κB binding sites, thereby activating the transcription of a set of target genes (1Leonardo M.J. Baltimore D. Cell. 1989; 58: 227-229Abstract Full Text PDF PubMed Scopus (1245) Google Scholar, 2Baeuerle P.A. Biochim. Biophys. Acta. 1991; 1072: 63-80PubMed Google Scholar, 3Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1214) Google Scholar). interleukin casein kinase electrophoretic mobility shift assay glutathione S-transferase nuclear factor lipopolysaccharide mitogen-activated protein kinase polyacrylamide gel electrophoresis tumor necrosis factor high performance liquid chromatography polymerase chain reaction. IκB family proteins possess the ankyrin-like repeats, which are thought to interact with the Rel homology region of NF-κB(8Beg A.A. Baldwin Jr., A.S. Genes & Dev. 1993; 7: 2064-2070Crossref PubMed Scopus (728) Google Scholar). This family includes large precursors of p50 and p52, and p105 and p100, respectively, which contain ankyrin repeats in their C-terminal region (9Fan C.M. Maniatis T. Nature. 1991; 354: 395-398Crossref PubMed Scopus (238) Google Scholar, 10Mercurio F.J.A. DiDonato J.A. Rosette C. Karin M. Genes & Dev. 1993; 7: 705-718Crossref PubMed Scopus (251) Google Scholar, 11Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-254Abstract Full Text PDF PubMed Scopus (339) Google Scholar). Cytoplasmic IκBα, encoded by the MAD-3 gene(12Haskill S. Beg A.A. Tompkins S.M. Morris J.S. Yurochko A.D. Sampsom-Johannes A. Mondal K. Ralph P. Baldwin Jr., A.S. Cell. 1991; 65: 1281-1289Abstract Full Text PDF PubMed Scopus (577) Google Scholar), is thought to be a major target in the signal transduction pathway. IκBα is rapidly phosphorylated in vivo in response to cell stimulation (13Beg A.A. Finco T.S. Nantermet P.V. Baldwin Jr., A.S. Mol. Cell. Biol. 1994; 13: 3301-3310Crossref Google Scholar, 14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar, 15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar, 16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar) and is degraded thereafter by proteasomes (13Beg A.A. Finco T.S. Nantermet P.V. Baldwin Jr., A.S. Mol. Cell. Biol. 1994; 13: 3301-3310Crossref Google Scholar, 14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar, 15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar, 16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 17Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1027) Google Scholar, 18Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar). Recently, while several groups have demonstrated that phosphorylation of IκBα is not sufficient to activate NF-κB in vivo(14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar, 15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar, 16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 17Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1027) Google Scholar, 18Alkalay I. Yaron A. Hatzubai A. Jung S. Avraham A. Gerlitz O. Pashut-Lavon I. Ben-Neriah Y. Mol. Cell. Biol. 1995; 15: 1294-1301Crossref PubMed Google Scholar, 19Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1896) Google Scholar), the inducible phosphorylation of IκBα may be required for converting IκBα into an appropriate proteasome substrate. In contrast, we previously demonstrated that LPS induced NF-κB activation without a significant loss of IκBα in a cell-free system using the monocytic cell line, THP-1(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). Lack of degradation of IκBα was also observed upon TNF-induced NF-κB activation in a cell-free experiment using U937 cells(21Reddy S.A.G. Chaturvedi M.M. Darnay B.G. Chan H. Higuchi M. Aggarwal B.B. J. Biol. Chem. 1994; 269: 25369-25372Abstract Full Text PDF PubMed Google Scholar). These observations would imply that appropriate phosphorylation of IκBα can activate NF-κB by dissociating from IκBα in a signal-dependent manner. In vitro phosphorylation of IκBα by several serine/threonine kinases, including protein kinase C and protein kinase A, prevents its binding to NF-κB(22Shirakawa F. Mizel S.B. Mol. Cell. Biol. 1989; 9: 2424-2430Crossref PubMed Scopus (299) Google Scholar, 23Ghosh S. Baltimore D. Nature. 1990; 344: 678-682Crossref PubMed Scopus (897) Google Scholar, 24Kerr J.D. Inoue I-J. Davis N. Link E. Baeuerle P.A. Bose Jr., H.R. Verma I.M. Genes & Dev. 1991; 5: 1464-1476Crossref PubMed Scopus (117) Google Scholar), suggesting that IκBα phosphorylation on specific phosphorylation sites is sufficient for the dissociation of IκBα from NF-κB. However, none of these kinases tested so far in vitro appears to be responsible for the activation of NF-κB in vivo(25Hohmann H.P. Kolbeck R. Remy R. van Loon A.P.G.M. Mol. Cell. Biol. 1991; 11: 2315-2318Crossref PubMed Google Scholar, 26Feuillard J. Gouy H. Bismuth G. Lee L.M. Debre P. Korner M. Cytokine. 1991; 3: 257-265Crossref PubMed Scopus (54) Google Scholar). To our knowledge, the IκBα kinase, which is responsible for the phosphorylation of IκBα in vivo, has not yet been biochemically identified and characterized. In this study, we have identified a kinase in LPS-stimulated human monocytic cell line (THP-1) extracts that specifically binds and phosphorylates IκBα. Moreover, we have identified the acidic domain in the C-terminal region of IκBα as the phosphorylation target sites for this kinase. A peptide substrate for the bound kinase, corresponding to the acidic domain, inhibited the LPS-mediated NF-κB activation in a cell-free system(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar), suggesting that phosphorylation of IκBα at the acidic domain by the kinase is critical for NF-κB activation. LPS (Escherichia coli, O55 B5, DIFCO, Detroit, MI) was dissolved in phosphate-buffered saline(-) and stored at -20°C. Casein kinase II (CKII) substrate (RRREEETEEE) was purchased from American Peptide Co. Inc. (CA). Mitogen-activated protein kinase (MAPK) substrate (APRTPGGRR) and the wild type AR peptide (MLPESEDEESYDTESEFTEFTEDEL) of IκBα or mutated ones were provided from Chugai Pharmaceutical Co. Ltd. (Gotemba, Japan). The sequences of the mutated AR peptides are AR(S283A), MLPEAEDEESYDTESEFTEFTEDEL; AR(S288A), MLPESEDEEAYDTESEFTEFTEDEL; AR(T291A), MLPESEDEESYDAESEFTEFTEDEL; and AR(S293A), MLPESEDEESYDTEAEFTEFTEDEL. The human IκBα (MAD-3) cDNA was obtained by reverse transcriptase-polymerase chain reaction (PCR) using a set of specific primers, GMD5 (5′-GCGAATTCCATGTTCCAGGCGGCCGAGCG-3′) and GMD3 (5′-GCAGGATCCTCATAACGTCAGACGCTGGC-3′) and human peripheral blood mononuclear cell total RNA as a template(11Rice N.R. MacKichan M.L. Israel A. Cell. 1992; 71: 243-254Abstract Full Text PDF PubMed Scopus (339) Google Scholar). The PCR product was cloned into the EcoRI and BamHI sites of the pGENT2 vector(27Murakami S. Cheong J. Kaneko S. J. Biol. Chem. 1994; 269: 15118-15123Abstract Full Text PDF PubMed Google Scholar), a modified version of pGEX2T (Pharmacia Biotech Inc.). Expression vectors for truncation mutants of GST-IκB fusion proteins were constructed by the PCR reaction. Sequences of the primers are available on request. GST-IκBα mutant K expression vector was also constructed by PCR reaction using primers GMD3 and GMD5 and the mammalian expression vector (28Ernst M.K. Dunn L.L. Rice N. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar) as a template. Nucleotide sequences of the wild type or mutants of IκBα were confirmed by direct DNA sequencing(29Sanger F. Nickelen S. Coulson R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (51949) Google Scholar). The GST fusion protein expression vectors were transformed into E. coli, JM109. Protein induction and purification were as described(30Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5011) Google Scholar). The amount of purified protein was estimated by the Bio-Rad protein assay. The binding/kinase reaction was performed according to the method described by Hibi et al.(31Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes & Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1692) Google Scholar) with some modifications. Cell extracts or partially purified fractions were incubated with glutathione (GSH)-Sepharose beads conjugated with GST-IκBα fusion proteins in the binding buffer (20 mM HEPES (pH 7.7), 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 20 mM β-glycerophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin) at 4°C for 3 h. The mixture was pelleted by centrifugation at 5000 rpm for 10 s. The pelleted beads were extensively washed with the washing buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100) and resuspended in 50 μl of the kinase buffer (20 mM HEPES (pH 7.6), 20 mM MgCl2, 20 mM MnCl2, 20 mM β-glycerophosphate, 0.1 mM Na3VO4), containing 5 μCi of [γ-32P]ATP (Amersham Corp.). After 5 min at 30°C, the reaction was terminated by adding EDTA. Reaction products were eluted with Laemmli sample buffer from beads and analyzed on a 10% SDS-PAGE, followed by autoradiography. For quantitation of phosphorylated protein, the gels were analyzed by an image analyzer (BAS 2000, Fuji Film Co. Ltd., Tokyo). For in vitro kinase reaction, the partially purified kinase was incubated with a substrate in 20 μl of the same kinase buffer containing 5 μCi of [γ-32P]ATP (Amersham) in the presence or absence of inhibitors for 5 min at 30°C. The reaction was terminated by adding EDTA. THP-1 cells were grown in RPMI 1640 medium containing 5% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. THP-1 cells were stimulated by 10 μg/ml LPS for 5 min and collected by centrifugation. Pellets were washed twice with phosphate-buffered saline and then resuspended in the buffer (20 mM HEPES (pH7.9), 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). Cells were disrupted by sonication and then centrifuged at 500 × g for 10 min. Supernatant was dialyzed against 20 mM Tris-HCl buffer (pH 7.5) for 4 h. Resultant cytosolic extracts were loaded onto a Red A-Sepharose column (Amicon), which had been equilibrated with the same buffer, and eluted from the column with a stepwise gradient of NaCl (0, 0.5, and 2 M). The IκBα-bound kinase activities of each fraction were determined using GST-IκBα as a substrate as described above, and the kinase activity was detected in 2 M NaCl fraction. The fraction was dialyzed against 20 mM Tris-HCl buffer (pH 7.5) and loaded onto a column of DEAE-sephacel (Pharmacia), which had been equilibrated with the same buffer, and eluted with a stepwise gradient of NaCl (0, 0.1, 0.25, and 0.5 M). The elute at 0.25 M NaCl from DEAE-sephacel was further fractionated by DEAE-HPLC (Bio-Gel DEAE-5PW, Bio-Rad) with a linear gradient (0-0.5 M NaCl). Cytosolic and membrane fractions of THP-1 cells were prepared as described previously(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). For analysis of NF-κB activation, cytosolic and plasma membrane-enriched fractions (each 20 μg of protein) were incubated in a kinase buffer in the presence or the absence of 20 μg/ml LPS with or without peptide inhibitors. The reactions were terminated by adding EDTA (pH 8.0). Half of each mixture was analyzed by electrophoretic mobility shift assay (EMSA) as described previously (20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar) with 32P-labeled NF-κB binding site of the human IL-8 gene as a probe. For the in vitro kinase reaction in a cell-free system, postnuclear fraction (40 μg of protein) was incubated in the kinase buffer with [γ-32P]ATP at 30°C for 5 min in the presence or the absence of LPS (20 μg/ml) as described previously(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). After the in vitro kinase reaction in a cell-free system using the postnuclear fractions in the presence of LPS (20 μg/ml), samples were incubated with anti-IκBα antiserum (28Ernst M.K. Dunn L.L. Rice N. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar) at 4°C for 3 h. Precipitates were collected on a protein G-Sepharose (Pharmacia) and analyzed by 10% SDS-PAGE. Phosphorylated IκBα, identified by autoradiography, was excised from the gel and subjected to phosphoamino analysis as described(32Cooper J.A. Sefton B.M. Hunter T. Methods Enzymol. 1982; 99: 387-402Crossref Scopus (701) Google Scholar). Many transcriptional factors play pivotal roles in cellular events leading to cell proliferation, differentiation, and immune responses. Phosphorylation is presumed to be a crucial step for the regulation of subcellular localization and transactivation capacity of many of these factors. Some of the key protein kinases, such as Jun kinases, have been recently identified as enzymes associated with their targets(31Hibi M. Lin A. Smeal T. Minden A. Karin M. Genes & Dev. 1993; 7: 2135-2148Crossref PubMed Scopus (1692) Google Scholar), probably due to a highly specific interaction with their substrates in vivo. With regard to the activation of NF-κB in response to LPS and inflammatory cytokines, an inducible phosphorylation of IκBα is thought to be prerequisite for subsequent activation of NF-κB after inactivation of IκBα. To characterize a protein kinase(s) that can specifically bind to IκBα, cytosolic extracts were prepared from LPS-stimulated THP-1 cells and incubated with GST-IκBα-conjugated GSH-Sepharose beads. After washing the GSH beads extensively, a solid phase in vitro kinase reaction was performed. As shown in Fig. 1A, GST-IκBα fusion protein but not GST was phosphorylated only in the presence of cytosolic extract in this binding/kinase reaction. These data implied that the cytosolic fraction contained a protein kinase that phosphorylated IκBα after binding to it. Moreover, LPS transiently enhanced the IκBα-bound kinase activity in intact THP-1 cells (Fig. 1B). The activity was enhanced about 2-fold at 2 min after LPS stimulation compared with a basal level and decreased thereafter. Furthermore, phosphoamino acid analysis of phosphorylated IκBα in the binding/kinase reaction revealed that the IκBα-bound kinase is serine and threonine specific (data not shown). To further characterize the IκBα-bound kinase, we tried to partially purify the bound kinase on the basis of the binding/kinase assay using GST-IκBα as a substrate. The bound kinase was partially purified from cytosolic extract of LPS-stimulated THP-1 cells through three steps: Red-Sepharose, DEAE-sephacel column chromatography, and DEAE-HPLC (see “Materials and Methods”), resulting in a 120-fold purification. To determine the region(s) of IκBα that interacted with the kinase(s), several truncated mutants of GST-IκBα fusion proteins were expressed (Fig. 2A), and the ability of the kinase(s) to phosphorylate these mutated IκBα was tested in a binding/kinase assay. As shown in Fig. 3A, mutant del-4, which lacks a 72-amino acid N-terminal region, or del-5, which lacks both N-terminal region and 3 ankyrin repeats, was still phosphorylated by the partially purified bound kinase(s). In contrast, the deletion of the C-terminal 35 amino acids (del-10) but not of 25 amino acids from C-terminal (del-9) abolished the phosphorylation of IκBα by the kinase(s). These data demonstrated the interaction of the acidic domain within the C-terminal region of IκBα with the bound kinase. To further directly delineate the IκBα phosphorylation sites, the effects of truncations of IκBα were also examined in an in vitro kinase reaction using a partially purified kinase. As shown in Fig. 3B, mutant del-9 was phosphorylated by the bound kinase to a similar level as wild type IκBα. However, when del-10 or del-1 mutant was used as a substrate, no phosphorylation was detected. Furthermore, GST-IκBα mutant K, in which six Ser/Thr residues in the C-terminal acidic domain were changed to Ala (Fig. 2B), was not phosphorylated by the kinase (Fig. 3B). These data indicate that the major phosphorylation sites by the bound kinase resided within the C-terminal acidic domain of IκBα. There are six Ser/Thr residues in the C-terminal acidic domain of IκBα, which are surrounded by acidic residues on both the N-terminal and C-terminal sides. These include potential phosphorylation sites by CKII (Ser-283 and Thr-291). To confirm the C-terminal phosphorylation by the bound kinase, we constructed the peptide AR, corresponding to the C-terminal acidic domain of IκBα, residues 279-303 (Fig. 2B), and performed a competition experiment in an in vitro kinase assay. As shown in Fig. 4A, phosphorylation of the GST-IκBα by the bound kinase was significantly inhibited by the peptide AR but not by a MAPK peptide substrate. These results suggest that the C-terminal acidic region is a major phosphorylation site for the kinase. In addition, a specific substrate for the CKII (33Kuenzel E.A. Mulligan J.A. Sommercorn J. Krebs E.G. J. Biol. Chem. 1987; 262: 9136-9140Abstract Full Text PDF PubMed Google Scholar) did not compete at the same concentration, suggesting the possibility that the bound kinase is distinct from CKII. To determine more precisely the phosphoacceptor sites on the acidic domain of IκBα, we prepared a series of mutated peptides in four Ser/Thr residues within the critical region of peptide AR and tested their capacities to be phosphorylated by the bound kinase. As shown in Fig. 4B, when the Ser-293 was replaced by Ala, phosphorylation of the peptide by the bound kinase was significantly reduced. The substitution of Ser-288 or Thr-291 to Ala decreased the phosphorylation by 20-30%, whereas that of Ser-283 retained a similar level of phosphorylation as the wild type protein. These data suggest that Ser-293 was a major phosphoacceptor site and that Ser-288 and Thr-291 were minor phosphorylation sites. Previously, we established the LPS-dependent NF-κB activation in a cell-free system (20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). This system has a great advantage since highly specific peptide inhibitors can be directly added. We have also demonstrated that an inducible phosphorylation of IκBα correlated with LPS-mediated NF-κB activation in the same assay system(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). Phosphoamino acid analysis revealed that IκBα phosphorylation after LPS stimulation in a cell-free system occurred at serine and threonine residues (Fig. 5), while no phosphorylation of these residues was detected without LPS stimulation (data not shown). These data indicate the activation of serine/threonine kinase by LPS stimulation in a cell-free system using THP-1 cells. To assess the physiological role of the IκBα-bound kinase(s) in the signaling pathway of LPS, we examined in a cell-free system the inhibitory activity of the bound kinase substrate, AR peptide of IκBα, as well as other inhibitory peptides. As shown in Fig. 6A, LPS-mediated NF-κB binding was significantly inhibited by addition of AR peptide of IκBα. In contrast, neither a CKII substrate nor a MAPK substrate affected the NF-κB activation. Furthermore, AR peptide of IκBα also blocked an inducible phosphorylation of IκBα (Fig. 6B) in the same assay. These results strongly suggest that the bound kinase is essential for the LPS-dependent activation of NF-κB in THP-1 cells through an inducible phosphorylation of the C-terminal acidic domain of IκBα. In this study, we have identified a protein kinase in the cytosolic fraction of a human monocytic cell line, THP-1, that can specifically bind and phosphorylate IκBα. The bound kinase is serine/threonine kinase, and its activity was transiently increased by in vivo stimulation with LPS in intact THP-1 cells, indicating that the IκBα-bound kinase is located downstream of the LPS signaling cascade. Mutational analysis of the IκBα substrate revealed that the IκBα-bound kinase phosphorylated the Ser/Thr residues within the C-terminal acidic domain of IκBα. This notion is supported by the competition experiment with the synthetic peptide AR corresponding to the C-terminal acidic domain of IκBα (Fig. 4A). Incomplete competition by AR peptide for IκBα phosphorylation in Fig. 4A may be due to lower affinity of AR peptide to the kinase compared with a full-length IκBα. It is possible that the synthetic peptide AR may be somehow sterically different from native phosphorylation sites and/or that some other parts may affect the interaction with the bound kinase in addition to actual phosphoacceptor sites. Moreover, we have demonstrated here that AR peptide was a substrate for the bound kinase and that the peptide significantly suppressed LPS-mediated NF-κB activation in a cell-free system. Previously, we found a correlation between an inducible phosphorylation of IκBα and the activation of NF-κB in response to LPS without apparent degradation of IκBα in the same system(20Ishikawa Y. Mukaida N. Kuno K. Rice N. Okamoto S. Matsushima K. J. Biol. Chem. 1995; 270: 4158-4164Crossref PubMed Scopus (80) Google Scholar). Furthermore, we show here that the substrate for the bound kinase also blocked the inducible phosphorylation of IκBα in a cell-free system. Taken together, these findings demonstrate that in a cell-free system using a monocytic cell line, THP-1, the IκBα-bound kinase phosphorylates the C-terminal acidic domain of IκBα in response to LPS stimulation, leading to dissociation of IκBα from NF-κB. IκBα is rapidly phosphorylated by cell stimulation, followed by degradation in vivo(13Beg A.A. Finco T.S. Nantermet P.V. Baldwin Jr., A.S. Mol. Cell. Biol. 1994; 13: 3301-3310Crossref Google Scholar, 14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar, 15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar, 16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 17Henkel T. Machleidt T. Alkalay I. Kronke M. Ben-Neriah Y. Baeuerle P.A. Nature. 1993; 365: 182-185Crossref PubMed Scopus (1027) Google Scholar). Several groups argued that phosphorylation of IκBα is not sufficient for NF-κB activation, based on their findings that proteasome inhibitors blocked the activation of NF-κB with accumulation of the phosphorylated form of IκBα (15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar, 16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar) and that the phosphorylated form of IκBα could be coimmunoprecipitated with p65 in several cell lines including Hela cells(14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar, 15Traenckner E.B.-M. Wilk S. Baeuerle P.A. EMBO J. 1994; 13: 5433-5441Crossref PubMed Scopus (646) Google Scholar). Recently, Brown et al.(34Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1302) Google Scholar) demonstrated that two Ser residues in the N-terminal region of human IκBα are important for its degradation in a mouse T cell line, EL-4. On the other hand, a recent study showed that phosphorylation states of IκBα were remarkably different in each cell type(14Naumann M. Scheidereit C. EMBO J. 1994; 13: 4595-4607Crossref Scopus (324) Google Scholar). Thus, we speculate that the discrepancy between these studies and our finding in a cell-free system using THP-1 cells may be ascribed to the difference in the cell types and stimuli. In addition, DiDonato et al.(16DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar) reported that IκBα was phosphorylated at multiple sites after TNFα stimulation in Hela cells. Therefore, the function of IκBα might be differentially regulated by multiple phosphorylation sites. It was shown that the C-terminal region of IκBα, containing a PEST-like motif, was also important for a signal-dependent proteolysis in vitro and in vivo in addition to the N-terminal region(34Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1302) Google Scholar, 35Rodriguez M.S. Michalopoupos I. Arenzana F. Hay R. Mol. Cell. Biol. 1995; 15: 2413-2419Crossref PubMed Google Scholar). However, the role of phosphorylation within the C-terminal acidic domain in an inducible proteolysis remains to be elucidated. It is known that IκB associates with Rel family proteins using its ankyrin repeat. The C-terminal acidic region of IκBα was recently shown to be involved in an inhibitory activity for DNA binding of c-Rel or p65(28Ernst M.K. Dunn L.L. Rice N. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar, 36Hatada E.N. Naumann M. Scheidereit C. EMBO J. 1993; 12: 2781-2788Crossref PubMed Scopus (78) Google Scholar). Here, we show that the peptide substrate for the bound kinase blocked NF-κB activation in a cell-free system. Ernst et al.(28Ernst M.K. Dunn L.L. Rice N. Mol. Cell. Biol. 1995; 15: 872-882Crossref PubMed Google Scholar), however, reported that a synthetic peptide, corresponding to the C-terminal acidic domain, by itself could not inhibit DNA binding activity. Similarly, we observed that the peptide substrate for the bound kinase did not affect NF-κB binding activity in EMSA when nuclear extracts from LPS-stimulated THP-1 cells were used as a source of NF-κB. 2K. Kuno, Y. Ishikawa, and K. Matsushima, unpublished results. These findings exclude the possibility that the acidic domain peptide directly inhibits the DNA binding activity of NF-κB in a cell-free assay. In addition, these studies demonstrated that NF-κB made contact with two sites of IκBα, ankyrin repeats and an acidic domain. Thus, it is likely that phosphorylation within this acidic region of IκBα may change its affinity for NF-κB. Mutational analysis of GST-IκBα and the substrate for the bound kinase revealed that Ser-293 within the acidic domain is a major phosphoacceptor site by the bound kinase and that Ser-288 and Thr-291 are minor ones. The sequence motifs of these phosphorylation sites are distinct from those of the proline-directed protein kinases, including MAPK, Jun kinase, and p38(37Kyriakis J.M. Banerjee P. Nikolakaki E. Dai T. Rubie E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Crossref PubMed Scopus (2398) Google Scholar, 38Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2369) Google Scholar, 39Weinstein S.L. Sanghera J.S. Lemke K. DeFranco A.L. Pelech S.L. J. Biol. Chem. 1992; 267: 14955-14962Abstract Full Text PDF PubMed Google Scholar), which were recently shown to be activated by LPS, IL-1, or TNF stimulation in several cell lines. In addition, the recognition sites of the bound kinase are also distinct from protein kinase C acceptor sites, although several groups reported the involvement of protein kinase C in LPS-signaling pathway (40Geng Y. Zhang B. Lotz M. J. Immunol. 1993; 151: 6692-6700PubMed Google Scholar) and the capacity of protein kinase C to phosphorylate IκBα in vitro, resulting in a dissociation from the NF-κB•IκBα complex. The major phosphorylation site, Ser-293, is distinct from a CKII acceptor site since it lacks an acidic residue at a third position of the C-terminal side, which is essentially required for CKII recognition(33Kuenzel E.A. Mulligan J.A. Sommercorn J. Krebs E.G. J. Biol. Chem. 1987; 262: 9136-9140Abstract Full Text PDF PubMed Google Scholar). On the other hand, one of the minor phosphorylation sites, Thr-291, belongs to a CKII acceptor site. We found that CKII substrate could not inhibit IκBα phosphorylation at the same concentration at which the bound kinase substrate blocked it. At this moment, we cannot exclude the possibility that the acidic domain of IκBα is more physiological substrate for CKII or its related molecules than the typical CKII substrate. Photoaffinity labeling by 8-azido-ATP of a partially purified kinase fraction suggested that the molecular mass of the IκBα-bound kinase is 42 kDa.2 However, further efforts will be required for the purification and molecular cloning of the kinase. NF-κB plays a central role in the regulation of gene activation of inflammatory cytokines such as IL-6 and TNFα. Previously, our group demonstrated that the activation of NF-κB is indispensable for the gene activation of human IL-8(6Mukaida N. Mahe Y. Matsushima K. J. Biol. Chem. 1991; 265: 21128-21133Google Scholar), which is a member of the leukocyte chemotactic cytokine(41Oppenheim J.J. Zachariae C.O.C. Mukaida N. Matsushima K. Annu. Rev. Immunol. 1991; 9: 617-648Crossref PubMed Scopus (1804) Google Scholar). In this study, we have demonstrated that a peptide substrate for the IκBα-bound kinase blocked the activation of NF-κB in a cell-free system. This finding suggests that specific peptides regulating the signaling pathway to induce NF-κB activity, such as the IκBα-bound kinase substrate, can target NF-κB and possibly be developed as a novel class of an anti-inflammatory drug. We thank Dr. Howard Young (NCI, National Institutes of Health) for helpful discussion throughout this work." @default.
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• W1480945793 title "Identification of an IκBα-associated Protein Kinase in a Human Monocytic Cell Line and Determination of Its Phosphorylation Sites on IκBα" @default.
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