From the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295
Received for publication, February 19, 2003
, and in revised form, March 19, 2003.
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ABSTRACT |
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INTRODUCTION |
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More recent studies of the IB kinase complex have identified a noncatalytic component called IKK
(also known as NEMO, IKKAP1, or FIP-3) (10, 11, 12, 13). IKK
is required for signal-dependent activation of IKK
, leading to its assignment as an essential regulatory subunit of the enzyme (10, 12, 14). Small deletions or point mutations in the gene encoding IKK
can cause skin inflammation or humoral immunodeficiencies in humans (15). In terms of structural organization, sequences within the NH2-terminal half of IKK
mediate its interaction with IKK
(11). In contrast, the COOH-terminal half of IKK
is required for signal-dependent regulation of I
B kinase activity, suggesting that IKK
links IKK
to upstream activators (11, 12). Despite all of these findings, the mechanism of IKK
action remains elusive (16). In this regard, we and others (9, 17, 18, 19) have recently shown that IKK
is phosphorylated in response to NF-
B agonists such as TNF and the Tax oncoprotein of HTLV1. Considering the key role that phosphorylation plays in the mechanism for IKK
activation, these findings suggest that IKK
subunit phosphorylation is important for proper regulation of the NF-
B signaling pathway.
To extend these fundamental observations, we conducted new experiments that address the mechanism of IKK phosphorylation and the relevant phosphoacceptor sites. In this report, we demonstrate that endogenous IKK
but not IKK
is required for signal-dependent phosphorylation of IKK
in vivo. Using a combination of site-directed mutagenesis and phosphopeptide mapping, we have also monitored changes in the phosphorylation status of IKK
in metabolically radiolabeled cells. Results from these biochemical experiments indicate that human IKK
is phosphorylated at Ser-31, Ser-43, and Ser-376 in response to cellular stimulation with either TNF or the HTLV1 Tax oncoprotein. Minimal deletion of the zinc finger domain of IKK
attenuates this inducible response. We conclude that IKK
mediates phosphorylation of IKK
at both amino-terminal and carboxyl-terminal sites via a zinc finger-dependent mechanism. The observed in vivo pattern of signal-dependent phosphorylation at distal sites in the protein may reflect post-translational control of IKK
at two distinct levels.
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EXPERIMENTAL PROCEDURES |
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Metabolic Radiolabeling and Subcellular FractionationMurine embryonic fibroblasts (MEFs) derived from mice lacking either IKK or IKK
have been described previously (24, 25). MEFs and human 293T cells (26) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. Human embryonic kidney 293T cells were transfected using the calcium phosphate method (27). At 36 h post-transfection, 293T cells were labeled for 4 h with [32P]orthophosphate (1 mCi/ml, ICN) in phosphate-free Dulbecco's modified Eagle's medium (Mediatech). Cytoplasmic extracts were prepared from recipient cells by detergent lysis (28) in the presence of phosphatase and protease inhibitors (5). Extracts were subjected to immunoprecipitation with anti-FLAG M2 antibodies coupled to agarose in ELB buffer (250 mM NaCl, 50 mM HEPES, 5 mM EDTA, 0.1% Nonidet P-40) (5). MEFs were incubated overnight in Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum and then labeled in phosphate-free medium for 4 h with [32P]orthophosphate (1 mCi/ml) prior to agonist treatment. Cytoplasmic extracts were prepared as described above, precleared with polyclonal anti-HA antibodies bound to protein A, and immunoprecipitated with polyclonal anti-IKK
antibodies. Resultant complexes were washed sequentially with ELB buffer containing 2 M urea and then with 150 mM NaCl, 10 mM sodium phosphate pH 7.2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40. Immunoprecipitates were fractionated by SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membranes. Phosphoproteins were identified by autoradiography and analyzed by immunoblotting using an enhanced chemiluminescence system (Pierce) (5). Resolved phosphoproteins were quantitatively analyzed with a Storm 860 PhosphorImager (Amersham Biosciences).
Phosphopeptide Mapping and Phosphoamino Acid AnalysisPhosphoproteins were separated by SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography. Membrane sections containing radiolabeled IKK were excised, incubated with methanol, and then blocked with 50 mM NH4HCO3 containing 0.1% Tween 20 (Bio-Rad) for 30 min at room temperature. Phosphoproteins were digested in situ with N-tosyl-L-lysine chloromethyl ketone (TLCK) treated chymotrypsin (8 µg, Worthington) in 125 µl of NH4HCO3 (37 oC, 12 h) (29). Released peptides were subjected to two-dimensional phosphopeptide mapping as described by Boyle et al. (30) excluding the performic acid oxidation. Phosphoproteins were first separated by electrophoresis on TLC plates at pH 1.9 (30) using the Hunter thin-layer electrophoresis system (model HTLE-7000, CBS Scientific). Separation in the second dimension was performed by ascending chromatography in n-butanol (37.5%), pyridine (25%), and acetic acid (7.5%) (30). Resolved phosphopeptides were visualized by autoradiography using Biomax MS high speed film (Eastman Kodak Co.). Phosphoaminoacid analysis was performed by hydrolysis of membrane-bound IKK
with 6 N HCl as described previously (30).
IB Kinase AssaysCytoplasmic extracts were immunoprecipitated with anti-T7 antibodies in the presence of ELB buffer. I
B kinase activity was measured as described previously (5, 31) in a reaction mixture containing ATP (10 µM), [
-32P]ATP (5 µCi), and recombinant glutathione S-transferase protein fused to amino acids 154 of I
B
. Radiolabeled products were fractionated by SDS-PAGE, transferred to PVDF membranes, and visualized by autoradiography.
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RESULTS |
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To extend our findings with Tax, we monitored IKK subunit phosphorylation in MEFs following treatment with proinflammatory mediators. In initial experiments, wild type MEFs were metabolically radiolabeled with [32P]orthophosphate and then stimulated with either TNF, IL-1, or LPS. Endogenous IKK
complexes were purified by immunoprecipitation, fractionated by SDS-PAGE, and analyzed by sequential autoradiography and immunoblotting. As shown in Fig. 1A (top panel), TNF induced significant phosphorylation of IKK
as compared with untreated controls (top panel, lanes 1 and 2). Similar results were obtained with IL-1 and LPS (lanes 3 and 4). Changes in the phosphorylation status of IKK
could not be attributed to differences in either IKK
or IKK
protein content (lower panels). As expected, IKK
was also phosphorylated and activated under these stimulatory conditions (top panel and data not shown). We conclude that phosphorylation of endogenous IKK
is induced not only by Tax but also by multiple proinflammatory agonists of NF-
B in murine fibroblasts.
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In prior studies, we found that IKK has the capacity to phosphorylate a recombinant IKK
substrate in vitro (19). This finding raised the possibility that in vivo phosphorylation of IKK
is mediated by IKK
or the structurally related IKK
catalytic subunit. To test this hypothesis, MEFs deficient for either IKK
or IKK
were cultured in the presence of [32P]orthophosphate and stimulated with TNF under conditions leading to optimal I
B kinase activity (data not shown). We then prepared IKK immunocomplexes and monitored the core subunits for phosphoprotein content. As shown in Fig. 1B (top panel), treatment of wild type MEFs with TNF led to a 13.6-fold increase in IKK
phosphorylation (lanes 1 and 2). This inducible response was readily detected in MEFs lacking IKK
(lanes 3 and 4), whereas IKK
phosphorylation was almost completely blocked in MEFs lacking IKK
(lanes 5 and 6). Coupled with our prior in vitro results (19), we conclude that IKK
mediates phosphorylation of IKK
under physiologic signaling conditions. However, we cannot exclude the possibility that IKK
plays a secondary role in IKK
phosphorylation.
IKK Is Phosphorylated on Multiple SerinesA prerequisite for understanding the functional consequences of IKK
subunit phosphorylation is to identify the relevant IKK
-responsive acceptor sites. To address this important question, expression vectors for IKK
, IKK
, and HTLV1 Tax were transfected into 293T cells. After metabolic radiolabeling with 32Pi, ectopic IKK
complexes were immunopurified and analyzed by SDS-PAGE. As shown in Fig. 2A, phosphoryl group transfer to IKK
was significantly increased in the presence of Tax relative to the basal level of IKK
phosphorylation in Tax-deficient cells. Importantly, IKK
was chronically phosphorylated and activated by Tax under these experimental conditions, permitting us to capture sufficient quantities of stably modified IKK
for subsequent phosphoamino acid and phosphopeptide-mapping analyses. As shown in Fig. 2B, these biochemical studies revealed the presence of phosphoserine in IKK
but no evidence for signal-dependent phosphorylation of either threonine or tyrosine residues.
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Identification of Inducible Phosphoacceptors in IKKTo assign specific phosphoacceptors in IKK
, we next used site-directed mutagenesis to replace individual serine residues with alanine in the full-length protein. In this regard, sequences in the NH2-terminal region of IKK
(amino acids 1120) are necessary for its interaction with the IKK
catalytic subunit (11, 32). To determine whether the IKK
binding domain of IKK
is subject to signal-dependent phosphorylation, 293T cells were programmed with expression vectors for Tax, IKK
, and human IKK
containing alanine replacements at serine residues 17, 31, 43, 68, and 85. All five of these serines are conserved between mouse and human IKK
(10, 11, 12, 13). After labeling recipient cells with 32Pi, IKK
proteins were isolated, digested with chymotrypsin, and analyzed by two-dimensional phosphopeptide mapping. As shown in Fig. 3 (upper panels), point mutations at Ser-31 and Ser-43 in IKK
eliminated two of the four major phosphopeptides identified in control-mapping experiments with wild type IKK
(PP-1 and PP-2, respectively). In sharp contrast, replacement of either Ser-17, Ser-68, or Ser-85 with alanine yielded a phosphopeptide fingerprint that was indistinguishable from the pattern for wild type IKK
following chymotrypsin digestion (Fig. 3, lower panels). These in vivo results indicate that IKK
mediates inducible phosphorylation of IKK
at Ser-31 and Ser-43, whereas other local serine residues in the IKK
binding domain of IKK
are spared from modification.
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In contrast to the NH2-terminal region of IKK, sequences in the COOH-terminal half of the protein (amino acids 320419) appear to couple IKK to upstream signal (11, 12). In the case of human IKK
, this region contains potential serine phosphoacceptors at positions 341, 346, 350, 364, 376, 377, 383, and 387. Based on their corresponding phosphopeptide fingerprints, mutations affecting the first four members of this set did not significantly change the phosphorylation status of IKK
in Tax-expressing cells (data not shown). Accordingly, we engineered alanine replacements into IKK
at the cluster of serines positioned between amino acids 370 and 390 (denoted as S376A, S377A, S383A, and S387A). As shown in Fig. 4 (top panels), point mutations at either Ser-383 or Ser-387 had no significant effect on distribution of the four major phosphopeptides identified in studies with wild type IKK
. However, in vivo phosphorylation of S376A in Tax-expressing cells yielded a pattern of chymotryptic peptides that was clearly distinct from the wild type IKK
fingerprint, specifically with respect to PP-3 and PP-4 (Fig. 4, bottom panels). Of particular interest, the mutation of Ser-376 was associated with the appearance of two new phosphopeptides (designated PP-3' and PP-4'), suggesting the presence of compensatory phosphoacceptors in the same chymotryptic fragments. Consistent with this hypothesis, alanine replacement of Ser-377 in mutant S376A eliminated the appearance of PP-3' and PP-4' (mutant S376/377A), whereas the disruption of Ser-377 alone was without affect (mutant S377A). These data indicate that Ser-376 is the primary target for phosphorylation in the COOH-terminal region of IKK
and the vicinal serine at position 377 serves a compensatory role. Taken together with data shown in Fig. 3, our mapping experiments strongly suggest that Ser-31, Ser-43, and Ser-376 are the major phosphoacceptor sites in human IKK
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To confirm this interpretation, we next engineered expression vectors for IKK containing serial mutations at the identified serine phosphoacceptors and introduced them into 293T cell transfectants along with Tax and IKK
effector plasmids. IKK
immunocomplexes were then isolated from 32Pi-labeled recipients and analyzed for phosphoprotein content. As shown in Fig. 5, simultaneous disruption of Ser-31 and Ser-43 in the NH2-terminal region of IKK
led to a significant reduction in subunit-specific phosphorylation (mutant S31/43A, lane 3). Simultaneous disruption of Ser-376 and compensatory Ser-377 in the COOH-terminal half of IKK
also attenuated 32Pi incorporation into the protein (mutant S376/377A, lane 4). Consistent with the biochemical phenotype of these double point mutants, the defect in substrate-radiolabeling efficiency was further evident (
19% wild type control) when all four of the identified serine targets in IKK
were disrupted in combination by site-directed mutagenesis (lane 5). Observed changes in the phosphorylation status of IKK
were not attributed to fluctuations in ectopic protein expression, because each of the phosphorylation-defective mutants of IKK
were comparably expressed in metabolically radiolabeled cells (Fig. 5, bottom panels). Moreover, these results could not be attributed to changes in the overall structural integrity of IKK
, because each of the phosphorylation-defective mutants retained the capacity to form stable complexes with IKK
(Fig. 5, bottom panels). Coupled with the two-dimensional phosphopeptide mapping data (Figs. 3 and 4), we conclude that Ser-31, Ser-43, and Ser-377 in human IKK
are the three major targets for signal-dependent phosphorylation in Tax-expressing cells.
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Tax and TNF Converge on the Same Set of IKK PhosphoacceptorsSimilar to the Tax oncoprotein of HTLV1, cellular stimulation with the proinflammatory cytokine TNF leads to rapid phosphorylation of both IKK
and IKK
(Fig. 1A) (9, 17, 18, 19). We have previously shown that Tax mediates these IKK subunit modifications by interacting directly with IKK
, which functions as an essential molecular adaptor for Tax in HTLV1-infected cells (19, 33). In contrast, TNF acts indirectly on IKK via binding to TNF-R1, its cognate cell-surface receptor. In turn, IKK and other accessory proteins are recruited to the cytoplasmic tail of TNF-R1, resulting in catalytic activation of IKK
(34, 35). Given these divergent signaling mechanisms, we reasoned that Tax and TNF might target distinct phosphoacceptors in IKK
.
To explore this possibility, we transfected 293T cells with expression vectors for IKK, IKK
, and either TNF-R1 or Tax. We then conducted comparative phosphopeptide mapping studies on IKK
derived from metabolically radiolabeled recipients. As shown in Fig. 6 (left panel), each of the four major phosphopeptides characteristics of wild type IKK
(PP-1, PP-2, PP-3, and PP-4) were identified in control mapping experiments with Tax-expressing cells. Despite the distinct mechanism by which TNF activates IKK signaling, the pattern of IKK
subunit phosphorylation induced by TNF-R1 was indistinguishable from the phosphopeptide fingerprint of IKK
derived from Tax-expressing cells (Fig. 6, right panel). The complete overlap between IKK
phosphoacceptors targeted in the Tax versus TNF-R1 backgrounds was confirmed in subsequent mixing experiments (data not shown). These biochemical data along with results shown in Fig. 1 strongly suggest that IKK
-directed phosphorylation of IKK
at Ser-31, Ser-43, and Ser-376 is an integral step in the TNF/NF-
B signaling axis.
Phosphorylation of IKK Is Dependent on Its Zinc Finger DomainDespite the apparent capacity of IKK
to phosphorylate both I
B
and IKK
, these two substrates share no obvious signature sequences that might underlie a common mechanism for IKK
targeting specificity. However, recent studies (36, 37) have indicated that the COOH-terminal zinc finger (ZF) domain of IKK
is required for NF-
B activation by a subset of IKK-inducing agents. This ZF motif lies within the final 30 residues of the full-length protein (419 amino acids) and contains no serines, which serve as the primary phosphoacceptors sites in IKK
(Fig. 2B).
To determine whether the ZF motif is important for IKK-directed phosphorylation of IKK
, we metabolically radiolabeled 293T cells following transfection with expression vectors for Tax, IKK
, and either wild type IKK
(IKK
.WT, amino acids 1419) or a minimal deletion mutant of IKK
lacking the ZF motif (IKK
.ZF, amino acids 1396). Ectopic IKK complexes were immunopurified from recipient cells, fractionated by SDS-PAGE, and then monitored for phosphoprotein content. As shown in Fig. 7A (upper panel), Tax readily stimulated phosphorylation of IKK
.WT in the presence of IKK
(lanes 1 and 2). Under the same Tax-inducing conditions, the phosphorylation of IKK
.ZF was significantly attenuated (lanes 3 and 4). The ZF mutation had no detectable effect on Tax-induced phosphorylation of IKK
(Fig. 7A, upper panel), I
B kinase activity (Fig. 7A, lower panel), IKK protein expression (Fig. 7B, upper panel), or the formation of Tax·IKK complexes (Fig. 7B, lower panel). These metabolic radiolabeling experiments indicate that the ZF motif is required for efficient signal-dependent phosphorylation of IKK
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DISCUSSION |
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Emerging evidence indicates that IKK is the target for a fourth integral phosphorylation step in this process (9, 18, 19, 20). In our studies, we have found that IKK
is chronically phosphorylated in cells expressing the HTLV1 Tax oncoprotein, which interfaces directly with the I
B kinase complex (Fig. 2A) (20). As well, this mechanism is operative in Tax-deficient cells following exposure to three different mediators of inflammation, including TNF, IL-1, and bacterial LPS (Fig. 1A). We have also found that IKK
is phosphorylated in vitro in the presence of activated IKK
, which is the key enzyme subunit responsible for I
B
phosphorylation and degradation (19). Consistent with these in vitro results, metabolic radiolabeling experiments with mutant MEFs indicate that IKK
is required for IKK
subunit phosphorylation (Fig. 1B). Taken together, these data indicate an important biochemical interplay between IKK
and IKK
that may contribute to the control of NF-
B signaling during the cellular response to immune and inflammatory mediators.
A prerequisite for understanding the functional consequences of IKK subunit phosphorylation is to identify the relevant acceptor sites. To achieve this experimental objective, we first conducted phosphopeptide mapping studies of IKK
in metabolically radiolabeled cells expressing the Tax oncoprotein of HTLV1. Unlike the transient action of TNF and other cytokines, Tax stimulates chronic phosphorylation of IKK
, enabeling us to capture a stably modified form of IKK
(Fig. 2A). These biochemical studies indicated that Tax induces the phosphorylation of human IKK
at Ser-31, Ser-43, and Ser-376 (Figs. 3, 4, 5). All three of these serine phosphoacceptors are found in murine IKK
as well (10). Similar results were obtained in phosphopeptide mapping experiments conducted with the type 1 TNF receptor (Fig. 6), which is known to activate IKK and NF-
B when expressed ectopically in mammalian cell transfectants (40). In this regard, Prajapati et al. (17) have reported in vitro evidence for the phosphorylation of murine IKK
at Ser-369, which corresponds to Ser-376 of human IKK
. However, results obtained from our in vivo mapping studies did not correlate with other putative phosphoacceptors identified by Prajapati et al. (17), presumably reflecting significant in vitro versus in vivo differences in IKK
substrate targeting by IKK
.
The three major phosphoacceptors identified in our studies reside in distal regions of IKK. Whereas Ser-31 and Ser-43 are positioned in the IKK
binding domain of human IKK
, Ser-376 lies downstream in the COOH-terminal domain of IKK
that is required for signal-dependent activation of IKK
(11, 12). Thus far, we have no compelling evidence that inducible phosphorylation of these IKK
domains is important for their assigned functions. Specifically, immunoprecipitation experiments indicated that phosphorylation-defective mutants of IKK
lacking Ser-31 and Ser-43 retain the capacity to form stable complexes with IKK
(Fig. 5). Moreover, IKK
is efficiently phosphorylated in cells expressing mutants of IKK
that lack Ser-376 and Ser-377 (Fig. 5), suggesting that IKK
activity is unaffected as well. Regarding this latter observation, prior transfection studies (18) have revealed modest increases in I
B kinase activity when IKK
is coexpressed with murine IKK
containing alanine substitutions at Ser-369 and Ser-375 (equivalent to human Ser-376 and Ser-383). However, we have been unable to detect significant changes in the wild type pattern of I
B kinase activity in preliminary studies with the phosphorylation-defect mutants of human IKK
described in Fig. 5 (data not shown). Accordingly, it may be difficult to ascribe a functional phenotype to these phosphorylation-defective mutants of IKK
solely on the basis of in vitro kinase assays.
An unexpected finding to emerge from our studies is that in vivo phosphorylation of human IKK is contingent upon the presence of its COOH-terminal ZF domain (Fig. 7A). We suspect a role for the ZF domain in substrate recognition, because its removal from IKK
had no detectable effect on IKK
-mediated phosphorylation of I
B
(Fig. 7A). Consistent with this proposal, ZF-deleted forms of IKK
are impaired for phosphorylation by a constitutively active mutant of IKK
(data not shown). The observed mechanistic relationship between the ZF motif and IKK
phosphorylation may help explain why mutations in the ZF motif of IKK
interfere with the regulated action of NF-
B under certain stimulatory conditions (36, 37). This relationship may also have clinical implications. Specifically, mutations in the X-linked gene encoding IKK
can cause anhidrotic ectodermal dysplasia with severe immunodeficiency (15). In the majority of affected patients, the IKK
gene has missense mutations or small deletions that selectively target the ZF domain of the protein (amino acids 390419) (15). Although the underlying signaling defects remain unclear, our data raise the intriguing possibility that some of these ZF mutations will interfere with inducible phosphorylation of IKK
in the disease state.
In summary, we demonstrate here that IKK is required for in vivo phosphorylation of IKK
in intact cells. Taken together with our prior in vitro data (19), we conclude that IKK
mediates IKK
phosphorylation under physiologic signaling conditions. We have also conducted phosphopeptide mapping studies to monitor signal-dependent changes in the phosphorylation status of IKK
in metabolically radiolabeled cells. These biochemical experiments indicate that both Tax and TNF induce phosphorylation of human IKK
at Ser-31, Ser-43, and Ser-376, which are fully conserved in murine IKK
. Signal-dependent phosphorylation of these target sites in IKK
is contingent upon the presence of its COOH-terminal zinc finger domain, a frequent target for mutations in anhidrotic ectodermal dysplasia with severe immunodeficiency disease. This requirement suggests an important regulatory role for IKK
-mediated phosphorylation of IKK
in the immune system. Resolution of this key question awaits more detailed gene-targeting studies with the phosphorylation-defective mutants of IKK
identified in the present report.
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FOOTNOTES |
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Supported by Training Grant T32 CA09385.
To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Vanderbilt University School of Medicine, A4301 Medical Center North, 1161 21st Ave., S., Nashville, TN 37232-2363. Tel.: 615-343-1918; Fax: 615-343-8648; E-mail: dean.ballard{at}vanderbilt.edu.
1 The abbreviations used are: TNF, tumor necrosis factor ; HTLV1, human T-cell leukemia virus type 1; IKK, I
B kinase; IL-1, interleukin 1; LPS, lipopolysaccharide; TNF-R1, tumor necrosis factor receptor-1; MEF, murine embryonic fibroblasts; WT, wild type; ZF, zinc-finger.
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ACKNOWLEDGMENTS |
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REFERENCES |
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