©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of an IB-associated Protein Kinase in a Human Monocytic Cell Line and Determination of Its Phosphorylation Sites on IB (*)

(Received for publication, June 9, 1995; and in revised form, August 31, 1995)

Kouji Kuno (1)(§) Yuji Ishikawa (1)(§) Mary K. Ernst (2) Masafumi Ogata (1) Nancy R. Rice (2) Naofumi Mukaida (1) Kouji Matsushima (1)(¶)

From the  (1)Department of Pharmacology, Cancer Research Institute, Kanazawa University, Takara-Machi 13-1, Kanazawa 920, Japan and the (2)Laboratory of Molecular Virology and Carcinogenesis, ABL-Basic Research Program, National Cancer Institute Frederick Cancer Research and Development Center, Frederick, Maryland 21702-1201

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nuclear factor kappaB (NF-kappaB) is stored in the cytoplasm as an inactive form through interaction with IkappaB. Stimulation of cells leads to a rapid phosphorylation of IkappaBalpha, 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-kappaB in association with the induction of IkappaBalpha 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 IkappaBalpha. LPS stimulation transiently enhanced the IkappaBalpha-bound kinase activity in THP-1 cells. Mutational analyses of IkappaBalpha 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 IkappaBalpha. Moreover, we show that the peptide, corresponding to the C-terminal acidic domain of IkappaBalpha, blocked the LPS-induced NF-kappaB activation as well as inducible phosphorylation of endogenous IkappaBalpha 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 IkappaBalpha and subsequent dissociation of the NF-kappaBbulletIkappaBalpha complex.


INTRODUCTION

NF-kappaB consists of a family of transcriptional factors that play a key role in the regulation of a number of immune and inflammatory response genes(1, 2, 3) , including several inflammatory cytokines such as IL-8 (^1)and IL-6(4, 5, 6) . Members of the family include p65 (RelA), RelB, c-Rel, p50 (NF-kappaB-1), and p52 (NF-kappaB-2). NF-kappaB is retained in an inactive form being associated with its inhibitors, IkappaB, in the cytoplasm in most types of cells (7) . Following stimulation of cells with a variety of agents, e.g. IL-1, tumor necrosis factor (TNF), phorbol myristate acetate, and lipopolysaccharide (LPS), NF-kappaB is released from IkappaB and is translocated to the nucleus and binds to the NF-kappaB binding sites, thereby activating the transcription of a set of target genes (1, 2, 3) .

IkappaB family proteins possess the ankyrin-like repeats, which are thought to interact with the Rel homology region of NF-kappaB(8) . This family includes large precursors of p50 and p52, and p105 and p100, respectively, which contain ankyrin repeats in their C-terminal region (9, 10, 11) . Cytoplasmic IkappaBalpha, encoded by the MAD-3 gene(12) , is thought to be a major target in the signal transduction pathway. IkappaBalpha is rapidly phosphorylated in vivo in response to cell stimulation (13, 14, 15, 16) and is degraded thereafter by proteasomes (13, 14, 15, 16, 17, 18) . Recently, while several groups have demonstrated that phosphorylation of IkappaBalpha is not sufficient to activate NF-kappaB in vivo(14, 15, 16, 17, 18, 19) , the inducible phosphorylation of IkappaBalpha may be required for converting IkappaBalpha into an appropriate proteasome substrate. In contrast, we previously demonstrated that LPS induced NF-kappaB activation without a significant loss of IkappaBalpha in a cell-free system using the monocytic cell line, THP-1(20) . Lack of degradation of IkappaBalpha was also observed upon TNF-induced NF-kappaB activation in a cell-free experiment using U937 cells(21) . These observations would imply that appropriate phosphorylation of IkappaBalpha can activate NF-kappaB by dissociating from IkappaBalpha in a signal-dependent manner.

In vitro phosphorylation of IkappaBalpha by several serine/threonine kinases, including protein kinase C and protein kinase A, prevents its binding to NF-kappaB(22, 23, 24) , suggesting that IkappaBalpha phosphorylation on specific phosphorylation sites is sufficient for the dissociation of IkappaBalpha from NF-kappaB. However, none of these kinases tested so far in vitro appears to be responsible for the activation of NF-kappaB in vivo(25, 26) . To our knowledge, the IkappaBalpha kinase, which is responsible for the phosphorylation of IkappaBalpha 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 IkappaBalpha. Moreover, we have identified the acidic domain in the C-terminal region of IkappaBalpha 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-kappaB activation in a cell-free system(20) , suggesting that phosphorylation of IkappaBalpha at the acidic domain by the kinase is critical for NF-kappaB activation.


MATERIALS AND METHODS

Reagents

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 IkappaBalpha 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.

Plasmid and Expression of GST Fusion Proteins

The human IkappaBalpha (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(11) . The PCR product was cloned into the EcoRI and BamHI sites of the pGENT2 vector(27) , a modified version of pGEX2T (Pharmacia Biotech Inc.). Expression vectors for truncation mutants of GST-IkappaB fusion proteins were constructed by the PCR reaction. Sequences of the primers are available on request. GST-IkappaBalpha mutant K expression vector was also constructed by PCR reaction using primers GMD3 and GMD5 and the mammalian expression vector (28) as a template. Nucleotide sequences of the wild type or mutants of IkappaBalpha were confirmed by direct DNA sequencing(29) .

The GST fusion protein expression vectors were transformed into E. coli, JM109. Protein induction and purification were as described(30) . The amount of purified protein was estimated by the Bio-Rad protein assay.

Binding/Kinase Reaction

The binding/kinase reaction was performed according to the method described by Hibi et al.(31) with some modifications. Cell extracts or partially purified fractions were incubated with glutathione (GSH)-Sepharose beads conjugated with GST-IkappaBalpha fusion proteins in the binding buffer (20 mM HEPES (pH 7.7), 75 mM NaCl, 2.5 mM MgCl(2), 0.1 mM EDTA, 0.5 mM dithiothreitol, 20 mM beta-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 MgCl(2), 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 MgCl(2), 20 mM MnCl(2), 20 mM beta-glycerophosphate, 0.1 mM Na(3)VO(4)), containing 5 µCi of [-P]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 [-P]ATP (Amersham) in the presence or absence of inhibitors for 5 min at 30 °C. The reaction was terminated by adding EDTA.

Partial Purification of an IkappaBalpha-bound Kinase

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 times 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 IkappaBalpha-bound kinase activities of each fraction were determined using GST-IkappaBalpha 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).

Analysis of NF-kappaB Activation in a Cell-free System

Cytosolic and membrane fractions of THP-1 cells were prepared as described previously(20) . For analysis of NF-kappaB 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 (20) with P-labeled NF-kappaB 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 [-P]ATP at 30 °C for 5 min in the presence or the absence of LPS (20 µg/ml) as described previously(20) .

Immunoprecipitation and Phosphoamino Acid Analysis

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-IkappaBalpha antiserum (28) at 4 °C for 3 h. Precipitates were collected on a protein G-Sepharose (Pharmacia) and analyzed by 10% SDS-PAGE. Phosphorylated IkappaBalpha, identified by autoradiography, was excised from the gel and subjected to phosphoamino analysis as described(32) .


RESULTS

Identification of a Protein Kinase That Binds and Phosphorylates IkappaBalpha

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(31) , probably due to a highly specific interaction with their substrates in vivo. With regard to the activation of NF-kappaB in response to LPS and inflammatory cytokines, an inducible phosphorylation of IkappaBalpha is thought to be prerequisite for subsequent activation of NF-kappaB after inactivation of IkappaBalpha. To characterize a protein kinase(s) that can specifically bind to IkappaBalpha, cytosolic extracts were prepared from LPS-stimulated THP-1 cells and incubated with GST-IkappaBalpha-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-IkappaBalpha 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 IkappaBalpha after binding to it. Moreover, LPS transiently enhanced the IkappaBalpha-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 IkappaBalpha in the binding/kinase reaction revealed that the IkappaBalpha-bound kinase is serine and threonine specific (data not shown).


Figure 1: Protein kinase(s) specifically bound to IkappaBalpha. A, GSH-Sepharose beads conjugated with 2.5 µg of GST-IkappaBalpha (lanes 3 and 4) or equimolar of GST (lanes 1 and 2) were incubated with (lanes 2 and 4) or without (lanes 1 and 3) the cytosolic extracts from LPS-stimulated THP-1 cells. After extensive washing, kinase reaction was performed in the presence of [-P]ATP as described under ``Materials and Methods.'' Reaction products were analyzed by 10% SDS-PAGE and autoradiography. B, transient activation of IkappaBalpha-bound kinase by LPS stimulation in THP-1 cells. Cytosol extracts were prepared from LPS-stimulated THP-1 cells at the indicated times, and the bound kinase activity was determined by a binding/kinase reaction with a GST-IkappaBalpha protein as a substrate. Reaction products were separated by 10% SDS-PAGE, and the relative density of IkappaBalpha phosphorylation was analyzed by an imaging analyzer. Data shown represent the mean ± S.D. of three independent experiments.



The Bound Kinase(s) Phosphorylates an Acidic Domain in C-terminal Region of IkappaBalpha

To further characterize the IkappaBalpha-bound kinase, we tried to partially purify the bound kinase on the basis of the binding/kinase assay using GST-IkappaBalpha 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 IkappaBalpha that interacted with the kinase(s), several truncated mutants of GST-IkappaBalpha fusion proteins were expressed (Fig. 2A), and the ability of the kinase(s) to phosphorylate these mutated IkappaBalpha 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 IkappaBalpha by the kinase(s). These data demonstrated the interaction of the acidic domain within the C-terminal region of IkappaBalpha with the bound kinase. To further directly delineate the IkappaBalpha phosphorylation sites, the effects of truncations of IkappaBalpha 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 IkappaBalpha. However, when del-10 or del-1 mutant was used as a substrate, no phosphorylation was detected. Furthermore, GST-IkappaBalpha 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 IkappaBalpha.


Figure 2: Schematic diagram of the human IkappaBalpha and construction of deletion mutants (A) and amino acid-substituted mutant (B) of GST-IkappaBalpha fusion protein. A, five ankyrin repeats are indicated by dotted boxes. Acidic domain in the C-terminal region was indicated by the cross-hatched box. A solid line represents the portion present in GST-IkappaBalpha mutants with the amino acid number at the end. B, partial amino acid sequences of wild type and mutant K are shown with the C-terminal end of deletion mutants 9 and 10 by arrows. The position of the synthetic peptide AR (residues, 279-303) is indicated by a cross-hatched bar above the sequence.




Figure 3: The bound kinase phosphorylates the C-terminal acidic domain of IkappaBalpha. A, the effects of mutations of IkappaBalpha on its phosphorylation by the bound kinase in a binding/kinase reaction. Partially purified IkappaBalpha-bound kinase from THP cells was incubated with GSH-Sepharose beads conjugated with 2.5 µg of wild type (wt) GST-IkappaBalpha or equimolar of deletion mutants. After an extensive washing, a kinase reaction was performed in the presence of [-P]ATP as described under ``Materials and Methods.'' Reaction products were analyzed by 10% SDS-PAGE and autoradiography. B, the effects of mutations of IkappaBalpha on its phosphorylation in an in vitro kinase reaction. Partially purified IkappaBalpha-bound kinase from THP-1 cells was incubated with 0.5 µg of wild-type GST-IkappaBalpha protein or equimolar mutated GST-IkappaBalpha proteins in the presence of [-P]ATP as described under ``Materials and Methods.'' The reaction products were analyzed by 10% SDS-PAGE and autoradiography.



Ser-293 Was a Major Phosphoacceptor of IkappaBalpha by the Bound Kinase

There are six Ser/Thr residues in the C-terminal acidic domain of IkappaBalpha, 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 IkappaBalpha, 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-IkappaBalpha 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 (33) did not compete at the same concentration, suggesting the possibility that the bound kinase is distinct from CKII.


Figure 4: Determination of the phosphoacceptor sites within the acidic domain of IkappaBalpha by the bound kinase. A, competition experiments with various kinase substrates in an in vitro kinase assay. Partially purified IkappaBalpha-bound kinase was incubated with 0.5 µg (9 pmol) of the wild type GST-IkappaBalpha as a substrate in 20 µl of the kinase buffer containing [-P]ATP in the presence of a 300-fold molar excess (2.7 nmol) of AR peptide of IkappaBalpha, CKII substrate, or MAPK substrate. The results are expressed as a percentage of GST-IkappaBalpha phosphorylation without a peptide competitor. Data shown represent the mean ± S.D. of three independent experiments. B, effects of amino acid substitutions of AR peptide of IkappaBalpha on its phosphorylation by the kinase. Partially purified IkappaBalpha-bound kinase was incubated with 3 nmol of wild type (wt) or mutated AR peptide in the kinase buffer containing [-P]ATP. The reaction products were analyzed by 15% SDS-PAGE in Tris-Tricine buffer. The results are expressed as a percentage of phosphorylation of wild type peptide. Data shown represent the mean ± S.D. of three independent experiments.



To determine more precisely the phosphoacceptor sites on the acidic domain of IkappaBalpha, 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.

Acidic Domain Peptide Blocks NF-kappaB Activation in a Cell-free System

Previously, we established the LPS-dependent NF-kappaB activation in a cell-free system (20) . This system has a great advantage since highly specific peptide inhibitors can be directly added. We have also demonstrated that an inducible phosphorylation of IkappaBalpha correlated with LPS-mediated NF-kappaB activation in the same assay system(20) . Phosphoamino acid analysis revealed that IkappaBalpha 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.


Figure 5: Phosphoamino acid analysis of IkappaBalpha phosphorylated in the cell-free system after LPS stimulation. After kinase reaction with a postnuclear fraction (100 µg of protein) in a cell-free system in the presence of LPS (20 µg/ml) for 5 min, IkappaBalpha was immunoprecipitated with anti-IkappaBalpha antibody and separated by SDS-PAGE, then subjected to phosphoamino acid analysis as described under ``Materials and Methods.'' The direction of migration for the first (1st) and second (2nd) dimensions are indicated. The(-) and (+) signs show the orientation of the electrophoresis. The (O) and (F) indicate the origin and the forward in the chromatography, respectively. The positions of phosphoserine (S), phosphothreonine (T), and phosphotyrosine (Y) are indicated.



To assess the physiological role of the IkappaBalpha-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 IkappaBalpha, as well as other inhibitory peptides. As shown in Fig. 6A, LPS-mediated NF-kappaB binding was significantly inhibited by addition of AR peptide of IkappaBalpha. In contrast, neither a CKII substrate nor a MAPK substrate affected the NF-kappaB activation. Furthermore, AR peptide of IkappaBalpha also blocked an inducible phosphorylation of IkappaBalpha (Fig. 6B) in the same assay. These results strongly suggest that the bound kinase is essential for the LPS-dependent activation of NF-kappaB in THP-1 cells through an inducible phosphorylation of the C-terminal acidic domain of IkappaBalpha.


Figure 6: The substrate of IkappaBalpha-bound kinase blocks NF-kappaB activation as well as inducible phosphorylation of IkappaBalpha in response to LPS stimulation in a cell-free system. A, plasma membrane-enriched and cytosolic fractions were incubated at 30 °C for 10 min in the absence (lane 2) or presence (lanes 1, 3, 4, and 5) of LPS (20 µg/ml) without (lanes 1 and 2) or with 15 nM of CKII substrate (lane 3), MAPK substrate (lane 4), or peptide AR of IkappaBalpha (lane 5), respectively. Then, EMSA was performed using NF-kappaB binding site of the human IL-8 gene promoter as a probe. B, kinase reaction was performed in a cell-free system with a postnuclear fraction (40 µg of protein) in the absence (lane 1) or presence (lanes 2, 3, 4, and 5) of LPS (20 µg/ml) without (lanes 1 and 2) or with 15 nM of AR peptide of IkappaBalpha (lane 3), MAPK substrate (lane 4), and CKII substrate (lane 5). IkappaBalpha was immunoprecipitated with anti-IkappaBalpha antibody and analyzed by 10% SDS-PAGE.




DISCUSSION

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 IkappaBalpha. 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 IkappaBalpha-bound kinase is located downstream of the LPS signaling cascade.

Mutational analysis of the IkappaBalpha substrate revealed that the IkappaBalpha-bound kinase phosphorylated the Ser/Thr residues within the C-terminal acidic domain of IkappaBalpha. This notion is supported by the competition experiment with the synthetic peptide AR corresponding to the C-terminal acidic domain of IkappaBalpha (Fig. 4A). Incomplete competition by AR peptide for IkappaBalpha phosphorylation in Fig. 4A may be due to lower affinity of AR peptide to the kinase compared with a full-length IkappaBalpha. 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-kappaB activation in a cell-free system. Previously, we found a correlation between an inducible phosphorylation of IkappaBalpha and the activation of NF-kappaB in response to LPS without apparent degradation of IkappaBalpha in the same system(20) . Furthermore, we show here that the substrate for the bound kinase also blocked the inducible phosphorylation of IkappaBalpha in a cell-free system. Taken together, these findings demonstrate that in a cell-free system using a monocytic cell line, THP-1, the IkappaBalpha-bound kinase phosphorylates the C-terminal acidic domain of IkappaBalpha in response to LPS stimulation, leading to dissociation of IkappaBalpha from NF-kappaB.

IkappaBalpha is rapidly phosphorylated by cell stimulation, followed by degradation in vivo(13, 14, 15, 16, 17) . Several groups argued that phosphorylation of IkappaBalpha is not sufficient for NF-kappaB activation, based on their findings that proteasome inhibitors blocked the activation of NF-kappaB with accumulation of the phosphorylated form of IkappaBalpha (15, 16) and that the phosphorylated form of IkappaBalpha could be coimmunoprecipitated with p65 in several cell lines including Hela cells(14, 15) . Recently, Brown et al.(34) demonstrated that two Ser residues in the N-terminal region of human IkappaBalpha 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 IkappaBalpha were remarkably different in each cell type(14) . 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.(16) reported that IkappaBalpha was phosphorylated at multiple sites after TNFalpha stimulation in Hela cells. Therefore, the function of IkappaBalpha might be differentially regulated by multiple phosphorylation sites. It was shown that the C-terminal region of IkappaBalpha, 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(34, 35) . However, the role of phosphorylation within the C-terminal acidic domain in an inducible proteolysis remains to be elucidated.

It is known that IkappaB associates with Rel family proteins using its ankyrin repeat. The C-terminal acidic region of IkappaBalpha was recently shown to be involved in an inhibitory activity for DNA binding of c-Rel or p65(28, 36) . Here, we show that the peptide substrate for the bound kinase blocked NF-kappaB activation in a cell-free system. Ernst et al.(28) , 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-kappaB binding activity in EMSA when nuclear extracts from LPS-stimulated THP-1 cells were used as a source of NF-kappaB. (^2)These findings exclude the possibility that the acidic domain peptide directly inhibits the DNA binding activity of NF-kappaB in a cell-free assay. In addition, these studies demonstrated that NF-kappaB made contact with two sites of IkappaBalpha, ankyrin repeats and an acidic domain. Thus, it is likely that phosphorylation within this acidic region of IkappaBalpha may change its affinity for NF-kappaB.

Mutational analysis of GST-IkappaBalpha 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(37, 38, 39) , 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 (40) and the capacity of protein kinase C to phosphorylate IkappaBalpha in vitro, resulting in a dissociation from the NF-kappaBbulletIkappaBalpha 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(33) . 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 IkappaBalpha 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 IkappaBalpha 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 IkappaBalpha-bound kinase is 42 kDa.^2 However, further efforts will be required for the purification and molecular cloning of the kinase.

NF-kappaB plays a central role in the regulation of gene activation of inflammatory cytokines such as IL-6 and TNFalpha. Previously, our group demonstrated that the activation of NF-kappaB is indispensable for the gene activation of human IL-8(6) , which is a member of the leukocyte chemotactic cytokine(41) . In this study, we have demonstrated that a peptide substrate for the IkappaBalpha-bound kinase blocked the activation of NF-kappaB in a cell-free system. This finding suggests that specific peptides regulating the signaling pathway to induce NF-kappaB activity, such as the IkappaBalpha-bound kinase substrate, can target NF-kappaB and possibly be developed as a novel class of an anti-inflammatory drug.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Contributed equally to this work.

To whom all correspondence should be addressed. Tel.: 81-762-62-8151 (ext. 5450); Fax: 81-762-60-7704.

(^1)
The abbreviations used are: IL, interleukin; CK, casein kinase; EMSA, electrophoretic mobility shift assay; GST, glutathione S-transferase; NF, nuclear factor; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; TNF, tumor necrosis factor; HPLC, high performance liquid chromatography; PCR, polymerase chain reaction.

(^2)
K. Kuno, Y. Ishikawa, and K. Matsushima, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. Howard Young (NCI, National Institutes of Health) for helpful discussion throughout this work.


REFERENCES

  1. Leonardo, M. J., and Baltimore, D. (1989) Cell 58, 227-229 [Medline] [Order article via Infotrieve]
  2. Baeuerle, P. A. (1991) Biochim. Biophys. Acta 1072, 63-80 [CrossRef][Medline] [Order article via Infotrieve]
  3. Thanos, D., and Maniatis, T. (1995) Cell 80, 529-532 [Medline] [Order article via Infotrieve]
  4. Collart, M., Baeuerle, P. A., and Vassalli, P. (1990) Mol. Cell. Biol. 10, 1498-1506 [Medline] [Order article via Infotrieve]
  5. Libermann, T. A., and Baltimore, D. (1990) Mol. Cell. Biol. 10, 2327-2334 [Medline] [Order article via Infotrieve]
  6. Mukaida, N., Mahe, Y., and Matsushima, K. (1991) J. Biol. Chem. 265, 21128-21133 [Abstract/Free Full Text]
  7. Baeuerle, P. A., and Baltimore, D. (1988) Cell 53, 211-217 [Medline] [Order article via Infotrieve]
  8. Beg, A. A., and Baldwin, A. S., Jr. (1993) Genes & Dev. 7, 2064-2070
  9. Fan, C. M., and Maniatis, T. (1991) Nature 354, 395-398 [CrossRef][Medline] [Order article via Infotrieve]
  10. Mercurio, F. J. A., DiDonato, J. A., Rosette, C., and Karin, M. (1993) Genes & Dev. 7, 705-718
  11. Rice, N. R., MacKichan, M. L., and Israel, A. (1992) Cell 71, 243-254 [Medline] [Order article via Infotrieve]
  12. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampsom-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289 [Medline] [Order article via Infotrieve]
  13. Beg., A. A., Finco, T. S., Nantermet, P. V., and Baldwin, A. S., Jr. (1994) Mol. Cell. Biol. 13, 3301-3310 [Abstract]
  14. Naumann, M., and Scheidereit, C. (1994) EMBO J. 13, 4595-4607
  15. Traenckner, E. B.-M., Wilk, S., and Baeuerle, P. A. (1994) EMBO J. 13, 5433-5441 [Abstract]
  16. DiDonato, J. A., Mercurio, F., and Karin, M. (1995) Mol. Cell. Biol. 15, 1302-1311 [Abstract]
  17. Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993) Nature 365, 182-185 [CrossRef][Medline] [Order article via Infotrieve]
  18. Alkalay, I., Yaron, A., Hatzubai, A., Jung, S., Avraham, A., Gerlitz, O., Pashut-Lavon, I., and Ben-Neriah, Y. (1995) Mol. Cell. Biol. 15, 1294-1301 [Abstract]
  19. Palombella, V. J., Rando, O. J., Goldberg, A. L., and Maniatis, T. (1994) Cell 78, 773-785 [Medline] [Order article via Infotrieve]
  20. Ishikawa, Y., Mukaida, N., Kuno, K., Rice, N., Okamoto, S., and Matsushima, K. (1995) J. Biol. Chem. 270, 4158-4164 [Abstract/Free Full Text]
  21. Reddy, S. A. G., Chaturvedi, M. M., Darnay, B. G., Chan, H., Higuchi, M., and Aggarwal, B. B. (1994) J. Biol. Chem. 269, 25369-25372 [Abstract/Free Full Text]
  22. Shirakawa, F., and Mizel, S. B. (1989) Mol. Cell. Biol. 9, 2424-2430 [Medline] [Order article via Infotrieve]
  23. Ghosh, S., and Baltimore, D. (1990) Nature 344, 678-682 [CrossRef][Medline] [Order article via Infotrieve]
  24. Kerr, J. D., Inoue, I-J., Davis, N., Link, E., Baeuerle, P. A., Bose, H. R., Jr., and Verma, I. M. (1991) Genes & Dev. 5, 1464-1476
  25. Hohmann, H. P., Kolbeck, R., Remy, R., and van Loon, A. P. G. M. (1991) Mol. Cell. Biol. 11, 2315-2318 [Medline] [Order article via Infotrieve]
  26. Feuillard, J., Gouy, H., Bismuth, G., Lee, L. M., Debre, P., and Korner, M. (1991) Cytokine 3, 257-265 [CrossRef][Medline] [Order article via Infotrieve]
  27. Murakami, S., Cheong, J., and Kaneko, S. (1994) J. Biol. Chem. 269, 15118-15123 [Abstract/Free Full Text]
  28. Ernst, M. K., Dunn, L. L., and Rice, N. (1995) Mol. Cell. Biol. 15, 872-882 [Abstract]
  29. Sanger, F., Nickelen, S., and Coulson, R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract]
  30. Smith, D. B., and Johnson, K. S. (1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  31. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes & Dev. 7, 2135-2148
  32. Cooper, J. A., Sefton, B. M., and Hunter, T. (1982) Methods Enzymol. 99, 387-402
  33. Kuenzel, E. A., Mulligan, J. A., Sommercorn, J., and Krebs, E. G. (1987) J. Biol. Chem. 262, 9136-9140 [Abstract/Free Full Text]
  34. Brown, K., Gerstberger, S., Carlson, L., Franzoso, G., and Siebenlist, U. (1995) Science 267, 1485-1488 [Medline] [Order article via Infotrieve]
  35. Rodriguez, M. S., Michalopoupos, I., Arenzana, F., and Hay, R. (1995) Mol. Cell. Biol. 15, 2413-2419 [Abstract]
  36. Hatada, E. N., Naumann, M., and Scheidereit, C. (1993) EMBO J. 12, 2781-2788 [Abstract]
  37. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160 [CrossRef][Medline] [Order article via Infotrieve]
  38. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811 [Medline] [Order article via Infotrieve]
  39. Weinstein, S. L., Sanghera, J. S., Lemke, K., DeFranco, A. L., and Pelech, S. L. (1992) J. Biol. Chem. 267, 14955-14962 [Abstract/Free Full Text]
  40. Geng, Y., Zhang, B., and Lotz, M. (1993) J. Immunol. 151, 6692-6700 [Abstract/Free Full Text]
  41. Oppenheim, J. J., Zachariae, C. O. C., Mukaida, N., and Matsushima, K. (1991) Annu. Rev. Immunol. 9, 617-648 [CrossRef][Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.