ACCELERATED PUBLICATION
Persistent Activation of NF-kappa B by the Tax Transforming Protein Involves Chronic Phosphorylation of Ikappa B Kinase Subunits IKKbeta and IKKgamma *

Robert S. Carter, Brian C. Geyer, Minhui Xie, Carlos A. Acevedo-Suárez, and Dean W. BallardDagger

From the Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0295

Received for publication, October 31, 2000, and in revised form, March 23, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The Tax transforming protein encoded by human T-cell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-kappa B and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of Ikappa B kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the Ikappa B inhibitory subunit of NF-kappa B. We now demonstrate that the IKKbeta catalytic subunit and IKKgamma regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKKbeta protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of Ikappa B kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKKbeta that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKKgamma substrate in vitro. We conclude that Tax-induced phosphorylation of IKKbeta is required for IKKbeta activation, phosphoryl group transfer to IKKgamma , and acquisition of the deregulated IKK phenotype.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To initiate an adaptive immune response, T lymphocytes execute a signal transduction program that triggers the transient induction of transcription factor NF-kappa B, activation of downstream growth-related genes, and cell cycle entry (1). Part of this program is regulated from the cytoplasm by Ikappa Balpha , an inhibitory subunit of NF-kappa B, and an inducible Ikappa B kinase called IKK1 (2). In response to immune system cues such as the cytokine tumor necrosis factor-alpha (TNF), IKK phosphorylates Ikappa Balpha at Ser-32 and Ser-36 (2). In turn, phosphorylated Ikappa Balpha is degraded and NF-kappa B translocates to the nucleus (2).

The most well-characterized form of IKK contains two catalytic subunits, termed IKKalpha and IKKbeta , and a regulatory subunit called IKKgamma (NEMO) (2). In response to TNF, IKKbeta is rapidly phosphorylated, activated, and down-regulated within 30 min (3). The relevant phosphoacceptors in IKKbeta have been mapped to a region in its catalytic domain that shares strong homology with "T loop" regulatory sequences found in members of the mitogen-activated protein kinase kinase (MAP2K) family of enzymes (3). Consistent with this structural link, members of the MAP2K kinase (MAP3K) family of enzymes have been implicated in TNF-induced activation of IKK (4).

In contrast to their transient action in TNF-treated cells, IKK and NF-kappa B are constitutively activated in T lymphocytes infected with human T-cell leukemia virus type 1 (HTLV1) (5). This process is mediated by the Tax transforming protein of HTLV1 and appears to play an essential role in the pathogenesis of HTLV1-associated disease (5). Chronic stimulation of IKK catalytic activity by Tax is dependent on IKKgamma , which directs the assembly of Tax·IKK complexes (5). Tax also binds to and activates MEKK1, a MAP3K that phosphorylates IKK in vitro (6). The dual specificity of Tax for these two enzymes may promote chronic phosphorylation of IKK and acquisition of the deregulated IKK phenotype. However, the phosphorylation status of IKK in Tax-expressing cells has not been examined.

We now demonstrate that IKKbeta and IKKgamma are chronically phosphorylated in Tax-expressing cells and HTLV1-infected T lymphocytes. Alanine replacements at Ser-177 and Ser-181 in the T loop of IKKbeta inhibit Tax-directed phosphorylation of both subunits and block the chronic stimulatory effects of Tax on Ikappa B kinase activity. Moreover, Tax-induced phosphorylation of IKKbeta and IKKgamma is antagonized by the bacterial protein YopJ, a potent in vivo inhibitor of T loop phosphorylation and IKKbeta catalytic activity (7). The finding that Tax-induced phosphorylation of IKKgamma is contingent upon Ikappa B kinase activity may reflect IKKbeta -mediated phosphorylation of IKKgamma within individual Tax·IKK signaling complexes. Consistent with this finding, constitutively active forms of IKKbeta have the capacity to phosphorylate a recombinant IKKgamma substrate in vitro.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Reagents-- Polyclonal anti-IKK antibodies (H-470, FL-419) and monoclonal anti-HA antibodies (F-7) were purchased from Santa Cruz, Inc. Monoclonal (M2) and polyclonal anti-FLAG antibodies were purchased from Sigma. Rabbit antisera specific for Tax have been described (8). Monoclonal anti-Tax antibodies (LT4) were kindly provided by Dr. Yuetsu Tanaka (Okinawa-Asia Research Center, Japan). Expression vectors for Tax, YopJ, and IKK have been described (8-12). HA-tagged IKKbeta (13) was subcloned into pCMV4 (14). The luciferase reporter plasmid NF-kappa B-Luc was obtained from Stratagene. The reporter plasmid HTLV1 LTR-Luc was engineered by subcloning the firefly luciferase gene into HTLV1 LTR-CAT (15).

Cell Culture, Transfections, and Reporter Assays-- HeLa and 293T cells (16) were maintained in DMEM with 10% fetal bovine serum, 2 mM glutamine, and antibiotics. HeLa cells (1 × 106) were transfected using Effectene (Qiagen), whereas 293T cells were transfected using calcium phosphate (17). Jurkat and MT-2 T cells were cultured in RPMI containing 55 µM beta -mercaptoethanol and the supplements listed above. Reporter gene activity was determined using a Promega Luciferase Assay Kit and a Turner Designs luminometer. All data were normalized to the activity of a cotransfected beta -galactosidase expression vector.

Subcellular Fractionation and Biochemical Analyzes-- Cytoplasmic extracts were prepared as described (18). Microcystin (1 µM; Alexis Biochemical) was included in the lysis buffer for isolation of endogenous IKKs. Immunoprecipitations were performed in the presence of ELB buffer (18). Kinase activity was measured (18) in reaction mixtures containing ATP (10 µM), [gamma -32P]ATP (5 µCi), and recombinant glutathione S-transferase protein fused to either a fragment of Ikappa Balpha (amino acids 1-54; GST·Ikappa Balpha ) or full-length IKKgamma (GST·IKKgamma ) (19). To measure IKK phosphorylation in vitro, reaction mixtures contained 2.5 µM ATP and lacked recombinant substrate. Radiolabeled products were washed with RIPA buffer (150 mM NaCl, 10 mM sodium phosphate pH 7.2, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40), fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride (PVDF) membranes. Proteins were analyzed by immunoblotting using an enhanced chemiluminescence system (Pierce) (18).

Metabolic Radiolabeling-- Following transfection (18 h), HeLa and 293T cells were labeled for 8 h with [32P]orthophosphate (1 mCi/ml; ICN) in phosphate-free DMEM (Mediatech). Immunocomplexes were washed with ELB buffer containing 0.5 M NaCl and 1 M urea, followed by RIPA buffer. Jurkat and MT-2 T cells were labeled in phosphate-free DMEM for 8 h with [32P]orthophosphate (2 mCi/ml). Cytoplasmic extracts were precleared with anti-FLAG M2 antibodies prior to immunoprecipitation with anti-IKKgamma antibodies. Resultant complexes were washed with ELB buffer containing 2 M urea followed by RIPA buffer. Phosphoproteins were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by autoradiography and immunoblotting.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Tax-dependent Phosphorylation of IKK in Vitro-- Prior studies have established that IKKgamma directs the assembly of Tax·IKK complexes (5). However, the biochemical mechanism by which Tax stimulates IKKbeta catalytic activity in these complexes remains unknown. To explore the role of phosphorylation in Tax·IKK signaling, HeLa cells were transfected with combinations of expression vectors for IKKbeta , IKKgamma , and wild-type Tax. Parallel transfections were performed with mutants of Tax that are selectively defective for either CREB/ATF (Tax-M47) or NF-kappa B (Tax-M22) activation (8). We then isolated IKKbeta complexes from recipient cell extracts by immunoprecipitation and subjected them to in vitro kinase assays in the presence of [gamma -32P]ATP. As shown in Fig. 1A (top panel), the phosphorylation status of IKKbeta was essentially unaffected by Tax in the absence of ectopic IKKgamma (lanes 1 and 2). Programming cells with this Tax docking subunit resulted in significant phosphorylation of both IKKbeta and IKKgamma (lane 4). Similar results were obtained in experiments with Tax-M47 (lane 6), but not Tax-M22 (lane 5), consistent with their differing capacities to engage and activate IKK (18). This pattern of phosphorylation was not attributable to inefficient or variable protein expression (lower panels).


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Fig. 1.   Tax-dependent phosphorylation of IKK in vitro. A, HeLa cells (2 × 106) were transfected with vectors for FLAG-tagged IKKbeta (50 ng), human IKKgamma (T7 epitope-tagged, 200 ng), and either wild-type Tax (200 ng), Tax-M22 (600 ng), or Tax-M47 (200 ng). Cytoplasmic extracts were immunoprecipitated with either anti-FLAG M2-agarose beads (top and middle panels) or monoclonal anti-Tax antibodies (bottom panel). IKKbeta complexes were subjected to in vitro kinase assays in the presence of [gamma -32P]ATP. Radiolabeled proteins were resolved by SDS-PAGE and analyzed by autoradiography (top panel) and immunoblotting with IKKbeta -specific antibodies (middle panel). Tax was detected by immunoblotting with polyclonal anti-Tax antibodies (bottom panel). B, HeLa cells (1 × 106) were transfected with vectors for Tax (100 ng), human IKKgamma (T7-tagged, 100 ng), and FLAG-tagged wild-type IKKbeta (WT), IKKbeta .SA, or IKKbeta .KM (25 ng each). Ectopic IKKbeta was isolated with anti-FLAG M2-agarose beads and subjected to in vitro kinase assays as described in A (top panel). IKK protein levels were determined by immunoblotting with subunit-specific antibodies (middle and bottom panel). C, HeLa cells were transfected as described in B. Tax complexes were prepared with monoclonal anti-Tax antibodies and subjected to in vitro kinase assays as described in A (top panel). Relative protein levels were determined by immunoblotting (lower panels).

Phosphorylation of Ser-177 and Ser-181 in the T loop of IKKbeta is required for its activation by TNF (3). To explore the role of Ser-177 and Ser-181 in Tax-dependent phosphorylation of IKKbeta , HeLa cells were transfected with vectors for Tax, IKKgamma , and either wild-type IKKbeta or a mutant containing alanine replacements at these two sites (IKKbeta .SA). We then prepared IKKbeta (Fig. 1B) or Tax (Fig. 1C) immunoprecipitates for in vitro kinase assays. As shown in Fig. 1B, top panel, mutations affecting Ser-177 and Ser-181 in IKKbeta completely blocked its phosphorylation in the presence of Tax (lanes 3 and 6). These mutations also prevented Tax-induced phosphorylation of IKKgamma . Similar results were obtained with a kinase-dead mutant of IKKbeta that is defective for ATP binding (IKKbeta .KM, lanes 7-9) (10). These mutations had no significant effect on IKKbeta protein levels (middle panel). However, we detected a significant shift in the electrophoretic mobility of IKKgamma when coexpressed with Tax and wild-type IKKbeta , consistent with a change in its phosphorylation status (bottom panel). All of these findings were recapitulated with Tax immunoprecipitates (Fig. 1C), indicating that the kinase activity responsible for IKKbeta and IKKgamma subunit phosphorylation is stably associated with Tax. We conclude that Ser-177 and/or Ser-181 in the T loop of IKKbeta are required for Tax-directed phosphorylation of IKKbeta and IKKgamma in vitro.

Tax-dependent Phosphorylation of IKK in Vivo-- We next used [32P]orthophosphate to metabolically label endogenous IKK in MT-2 cells. This transformed T lymphocyte line is chronically infected with HTLV1 and expresses high constitutive levels of Ikappa B kinase activity (18). Parallel experiments were conducted with Jurkat T lymphocytes, which are transformed by an HTLV1-independent mechanism. Endogenous IKK was immunoprecipitated from the corresponding extracts, fractionated by SDS-PAGE, and analyzed by autoradiography. As shown in Fig. 2A, IKKbeta and IKKgamma were both hyperphosphorylated in MT-2 cells as compared with Jurkat cells (top panel, lanes 3 and 4). The observed pattern of IKK phosphorylation could not be attributed to cell type-specific differences in the steady-state level of IKK protein expression (lower panels, lanes 3 and 4). These findings demonstrate that endogenous IKKbeta and IKKgamma are chronically phosphorylated in the physiologically relevant setting of HTLV1-infected T cells.


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Fig. 2.   Tax induces phosphorylation of IKKbeta and IKKgamma in vivo. A, Jurkat and MT-2 T cells were radiolabeled with [32P]orthophosphate for 8 h. Cytoplasmic extracts (400 µg) were subjected to immunoprecipitation with the indicated monoclonal antibodies. Resultant complexes were fractionated by SDS-PAGE and analyzed for 32P incorporation (top panel). IKK protein content was determined by immunoblotting with subunit-specific antibodies (middle and lower panels). B, HeLa cells (1 × 106) were transfected with vectors for Tax (100 ng), murine IKKgamma (Myc epitope-tagged, 100 ng), and FLAG-tagged forms of either wild-type IKKbeta (WT), IKKbeta .SA, or IKKbeta .KM (50 ng each). Cells were radiolabeled with [32P]orthophosphate for 8 h. Ectopic IKKbeta complexes were isolated from cytoplasmic extracts using anti-FLAG M2-agarose beads and fractionated by SDS-PAGE. Resolved proteins were subjected to autoradiography (top panel) and immunoblotting with IKK subunit-specific antibodies (middle and bottom panels). C, HeLa cells were transfected as described in B. Ectopic IKKbeta was isolated from cytoplasmic extracts with anti-FLAG M2-agarose beads and assayed for Ikappa B kinase activity in the presence of GST-Ikappa Balpha (1 µg) and [gamma -32P]ATP. Phosphoproteins were resolved by SDS-PAGE and visualized by autoradiography (top panel). Relative levels of IKKbeta protein were determined by immunoblotting with IKKbeta -specific antibodies (bottom panel).

To extend these findings, HeLa cells were transfected with various combinations of Tax and IKK expression vectors and then metabolically labeled with [32P]orthophosphate. Ectopic IKKbeta was isolated by immunoprecipitation, fractionated by SDS-PAGE, and analyzed by autoradiography. As shown in Fig. 2B, top panel, Tax induced significant phosphorylation of IKKbeta and IKKgamma in cells programmed with the wild-type catalytic subunit (lanes 1 and 2). In contrast, Tax-directed phosphorylation of both subunits was blocked in cells expressing either IKKbeta .SA (lanes 3 and 4), which has alanine replacements at Ser-177/Ser-181, or the kinase-dead mutant IKKbeta .KM (lanes 5 and 6). Comparable amounts of IKKbeta and IKKgamma protein were detected in all of the samples analyzed (Fig. 2B, middle and lower panels). These in vivo results correlated strongly with the in vitro phosphorylation data shown in Fig. 1B.

To explore the functional consequences of IKKbeta phosphorylation, IKKbeta complexes were immunopurified from HeLa cell transfectants expressing Tax, IKKgamma , and either wild-type IKKbeta or IKKbeta .SA. These complexes were then monitored for Ikappa B kinase activity in vitro using a recombinant Ikappa Balpha substrate (GST-Ikappa Balpha ). As shown in Fig. 2C, upper panel, Tax potently induced the catalytic activity of wild-type IKKbeta via an IKKgamma -dependent mechanism (lanes 1-3). In contrast, we were unable to detect Ikappa B kinase activity in IKKbeta .SA immunoprecipitates (lanes 4-6). These differences in Tax responsiveness were significant, because wild-type IKKbeta and IKKbeta .SA were comparably expressed at the protein level (lower panel). Given that IKKbeta .SA escapes from phosphorylation in Tax-expressing cells (Fig. 2B), we conclude that Tax-induced phosphorylation of the IKKbeta catalytic subunit is required for acquisition of constitutive Ikappa B kinase activity.

YopJ Interferes with Tax·IKK Signaling-- The bacterial virulence factor YopJ binds to multiple MAP2K proteins and interferes with T loop phosphorylation (7). In keeping with the structural link between MAP2K and IKK proteins, YopJ also forms complexes with IKKbeta (7). To determine whether YopJ affects the Tax/NF-kappa B signaling axis, 293T cells were transfected with an NF-kappa B reporter plasmid (NF-kappa B-Luc) along with expression vectors for Tax and YopJ. As shown in Fig. 3A, left panel, Tax potently stimulated NF-kappa B-directed transcription in the absence of YopJ. However, coexpression with YopJ blocked this Tax response in a dose-dependent fashion. In contrast, YopJ failed to inhibit Tax-induced activation of the HTLV1 LTR (right panel), which involves the transcriptional action of CREB/ATF rather than NF-kappa B (8).


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Fig. 3.   YopJ prevents Tax-induced activation of IKK and NF-kappa B. A, 293T cells (5 × 105) were transfected with a Tax expression vector (1 µg), the indicated amounts of an effector plasmid encoding FLAG-tagged YopJ, and either the NF-kappa B-Luc or HTLV1 LTR-Luc reporter plasmid (100 ng). Whole cell extracts were prepared after 24 h and assayed for luciferase activity. Average values obtained from four replicates are reported as the mean -fold induction (±S.E.) of luciferase activity by Tax relative to basal expression of the reporter gene in Tax-deficient cells. In the absence of Tax, YopJ-induced changes in the basal activity of either reporter plasmid was essentially negligible (<2-fold). B, 293T cells (1 × 106) were transfected with expression plasmids for Tax (2 µg) and FLAG-tagged YopJ (0.5 µg) as indicated. Endogenous IKK complexes were immunoprecipitated with monoclonal anti-IKKgamma antibodies (PharMingen) and assayed for Ikappa B kinase activity as described in the Fig. 2 legend (top panel). IKKbeta protein levels were monitored by immunoblotting with polyclonal anti-IKKbeta antibodies (second panel). Levels of Tax and YopJ protein expression were determined by immunoblotting cytoplasmic extracts with anti-Tax or anti-FLAG antibodies (lower two panels). C, 293T cells (1 × 106) were transfected with vectors for HA-tagged IKK (25 ng), Tax (0.5 µg), human IKKgamma (T7-tagged, 25 ng), and YopJ (100 ng). Cells were radiolabeled with [32P]orthophosphate for 8 h. Ectopic IKKbeta complexes were isolated using anti-HA antibodies, washed at high stringency, and fractionated by SDS-PAGE. Resolved proteins were subjected to sequential autoradiography (top panel) and immunoblotting (middle and bottom panels).

To examine the mechanism of YopJ action on the Tax/NF-kappa B axis, 293T cells were transfected with vectors for Tax and YopJ, either alone or in combination. We then purified endogenous IKK complexes from recipient cells and monitored them for Ikappa B kinase activity. As shown in Fig. 3B, top panel, Tax potently induced the catalytic activity of IKK in the absence of YopJ (lanes 1 and 2). In contrast, endogenous IKK complexes isolated from cells coexpressing Tax and YopJ failed to affect GST·Ikappa Balpha phosphorylation (lane 4). Immunoblotting experiments confirmed that comparable amounts of endogenous IKKbeta were co-immunoprecipitated under each condition and that YopJ and Tax were both expressed efficiently (lower two panels).

To determine whether YopJ interferes with Tax-induced phosphorylation of IKK, 293T cells expressing ectopic IKK, Tax, and YopJ were radiolabeled with [32P]orthophosphate. We then isolated IKKbeta complexes and analyzed their phosphoprotein content. As shown in Fig. 3C, top panel, phosphorylation of IKKbeta and IKKgamma was significantly increased in the presence of Tax relative to the level of subunit radiolabeling detected in Tax-deficient cells (lanes 3 and 4). Coexpression with YopJ completely blocked this Tax-dependent increase in IKK phosphorylation (lane 5). Given the capacity of YopJ to prevent T loop phosphorylation (7), these findings provide further evidence indicating that Ser-177 and Ser-181 in the T loop of IKKbeta function as Tax-responsive phosphoacceptors.

Phosphorylation of IKKgamma by IKKbeta -- The finding that IKKgamma phosphorylation induced by Tax is dependent on the catalytic activity of IKKbeta (Figs. 1-3) led us to hypothesize that IKKgamma is a substrate of IKKbeta . To test this possibility, 293T cells were transfected with an expression vector encoding a constitutively active mutant of IKKbeta that contained glutamic acid substitutions at Ser-177 and Ser-181 (IKKbeta .SE) (10). IKKbeta .SE immunoprecipitates derived from these transfectants were incubated with [gamma -32P]ATP and a substrate containing GST fused to full-length IKKgamma (GST·IKKgamma ). As shown in Fig. 4 (top, lane 2), GST·IKKgamma phosphorylating activity was readily detected in IKKbeta .SE immunocomplexes. Removal of the IKKgamma sequences from the GST·IKKgamma fusion protein eliminated phosphoryl group transfer, thus confirming specificity (middle, lane 2). IKKgamma kinase activity was not detected in immunoprecipitates derived from cells expressing a kinase-deficient mutant of IKKbeta (IKKbeta .SA) (top, lane 3), indicating that phosphorylation of IKKgamma is dependent on the catalytic function of IKKbeta . Moreover, the IKKgamma kinase activity detected in IKKbeta .SE immunocomplexes was retained after high stringency washing with 3 M urea (top, lane 5), excluding the involvement of a kinase that associates loosely with IKKbeta .


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Fig. 4.   Phosphorylation of IKKgamma by IKKbeta . 293T cells (0.5 × 106) were transfected with expression plasmids for FLAG-tagged IKKbeta .SE (25 ng) or IKKbeta .SA (50 ng). Ectopic IKKbeta was isolated with anti-FLAG M2-agarose beads and washed under low (250 mM NaCl) or high (250 mM NaCl, 3 M urea) stringency conditions. Resultant immunocomplexes were incubated with either GST·IKKgamma (top panel) or GST (middle panel) for 30 min and then assayed for kinase activity in the presence of [gamma -32P]ATP. Relative levels of IKKbeta protein were determined by immunoblotting (bottom panel).

In summary, our data indicate that the assembly of Tax·IKK complexes leads to chronic phosphorylation of IKKbeta and IKKgamma . Activation of IKKbeta by Tax appears to involve a kinase that phosphorylates the T loop of IKKbeta at Ser-177 and Ser-181. Given that kinase-dead mutants of IKKbeta are defective for Tax-induced phosphorylation, this kinase may be IKKbeta . Point mutations in IKKbeta that disrupt its catalytic function also prevent Tax-induced phosphorylation of IKKgamma , suggesting that IKKgamma is phosphorylated by IKKbeta within the same complex. In keeping with this proposal, IKKbeta has the capacity to phosphorylate a recombinant IKKgamma substrate in vitro. As such, further studies are warranted to define whether the phosphorylation status of IKKgamma affects the temporal regulation of Ikappa B kinase activity.

    FOOTNOTES

* This study was supported by National Institutes of Health Grant RO1 CA82556 (to D. W. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Vanderbilt University School of Medicine, A4301 Medical Center N., Nashville, TN 37232-0295. Tel.: 615-343-1548; Fax: 615-343-5743; E-mail: dean.ballard@mcmail.vanderbilt.edu.

Published, JBC Papers in Press, April 26, 2001, DOI 10.1074/jbc.C000777200

    ABBREVIATIONS

The abbreviations used are: IKK, Ikappa B kinase; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; HTLV1, human T-cell leukemia virus type 1; LTR, long terminal repeat; MAP2K, mitogen-activated protein kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; TNF, tumor necrosis factor-alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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