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
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ABSTRACT |
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The Tax transforming protein encoded by human
T-cell leukemia virus type 1 (HTLV1) persistently activates
transcription factor NF- To initiate an adaptive immune response, T lymphocytes execute a
signal transduction program that triggers the transient induction of
transcription factor NF- The most well-characterized form of IKK contains two catalytic
subunits, termed IKK In contrast to their transient action in TNF-treated cells, IKK and
NF- We now demonstrate that IKK 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 IKK 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
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), [ 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-IKK Tax-dependent Phosphorylation of IKK in
Vitro--
Prior studies have established that IKK
Phosphorylation of Ser-177 and Ser-181 in the T loop of IKK 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 I
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 IKK
To explore the functional consequences of IKK 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 IKK
To examine the mechanism of YopJ action on the Tax/NF-
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
IKK Phosphorylation of IKK
In summary, our data indicate that the assembly of Tax·IKK
complexes leads to chronic phosphorylation of IKKB 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 I
B kinase (IKK), a cellular enzyme complex that phosphorylates
and inactivates the I
B inhibitory subunit of NF-
B. We now
demonstrate that the IKK
catalytic subunit and IKK
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 IKK
protect both of these IKK subunits from
Tax-directed phosphorylation and prevent the induction of I
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 IKK
that contain glutamic acid substitutions at
Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant
IKK
substrate in vitro. We conclude that Tax-induced
phosphorylation of IKK
is required for IKK
activation, phosphoryl
group transfer to IKK
, and acquisition of the deregulated IKK phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B, activation of downstream growth-related genes, and cell cycle entry (1). Part of this program is regulated from
the cytoplasm by I
B
, an inhibitory subunit of NF-
B, and an
inducible I
B kinase called
IKK1 (2). In response to
immune system cues such as the cytokine tumor necrosis factor-
(TNF), IKK phosphorylates I
B
at Ser-32 and Ser-36 (2). In turn,
phosphorylated I
B
is degraded and NF-
B translocates to the
nucleus (2).
and IKK
, and a regulatory subunit called IKK
(NEMO) (2). In response to TNF, IKK
is rapidly
phosphorylated, activated, and down-regulated within 30 min (3). The
relevant phosphoacceptors in IKK
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).
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
IKK
, 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.
and IKK
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 IKK
inhibit Tax-directed phosphorylation of both subunits and
block the chronic stimulatory effects of Tax on I
B kinase activity. Moreover, Tax-induced phosphorylation of IKK
and IKK
is
antagonized by the bacterial protein YopJ, a potent in vivo
inhibitor of T loop phosphorylation and IKK
catalytic activity (7).
The finding that Tax-induced phosphorylation of IKK
is contingent
upon I
B kinase activity may reflect IKK
-mediated phosphorylation
of IKK
within individual Tax·IKK signaling complexes. Consistent
with this finding, constitutively active forms of IKK
have
the capacity to phosphorylate a recombinant IKK
substrate in
vitro.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(13) was subcloned into pCMV4 (14). The luciferase reporter plasmid
NF-
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).
-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
-galactosidase expression vector.
-32P]ATP (5 µCi), and
recombinant glutathione S-transferase protein fused to
either a fragment of I
B
(amino acids 1-54; GST·I
B
) or
full-length IKK
(GST·IKK
) (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).
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
directs the
assembly of Tax·IKK complexes (5). However, the biochemical mechanism by which Tax stimulates IKK
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 IKK
, IKK
, and wild-type Tax. Parallel transfections were performed with mutants of Tax that are selectively defective for
either CREB/ATF (Tax-M47) or NF-
B (Tax-M22) activation (8). We then isolated IKK
complexes from recipient cell extracts by immunoprecipitation and subjected them to in vitro kinase
assays in the presence of [
-32P]ATP. As shown in Fig.
1A (top panel), the
phosphorylation status of IKK
was essentially unaffected by Tax in
the absence of ectopic IKK
(lanes 1 and 2).
Programming cells with this Tax docking subunit resulted in significant
phosphorylation of both IKK
and IKK
(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 IKK
(50 ng), human IKK
(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). IKK
complexes were subjected
to in vitro kinase assays in the presence of
[
-32P]ATP. Radiolabeled proteins were resolved by
SDS-PAGE and analyzed by autoradiography (top panel) and
immunoblotting with IKK
-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 IKK
(T7-tagged, 100 ng), and FLAG-tagged wild-type IKK
(WT), IKK
.SA, or IKK
.KM (25 ng each). Ectopic IKK
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).
is
required for its activation by TNF (3). To explore the role of Ser-177
and Ser-181 in Tax-dependent phosphorylation of IKK
,
HeLa cells were transfected with vectors for Tax, IKK
, and either
wild-type IKK
or a mutant containing alanine replacements at these
two sites (IKK
.SA). We then prepared IKK
(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 IKK
completely blocked its
phosphorylation in the presence of Tax (lanes 3 and
6). These mutations also prevented Tax-induced
phosphorylation of IKK
. Similar results were obtained with a
kinase-dead mutant of IKK
that is defective for ATP binding (IKK
.KM, lanes 7-9) (10). These mutations had no
significant effect on IKK
protein levels (middle panel).
However, we detected a significant shift in the electrophoretic
mobility of IKK
when coexpressed with Tax and wild-type IKK
,
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 IKK
and IKK
subunit phosphorylation is
stably associated with Tax. We conclude that Ser-177 and/or Ser-181 in
the T loop of IKK
are required for Tax-directed phosphorylation of
IKK
and IKK
in vitro.
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, IKK
and
IKK
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 IKK
and IKK
are chronically phosphorylated in the physiologically
relevant setting of HTLV1-infected T cells.
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Fig. 2.
Tax induces phosphorylation of
IKK and IKK
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
IKK
(Myc epitope-tagged, 100 ng), and FLAG-tagged forms of
either wild-type IKK
(WT), IKK
.SA, or IKK
.KM (50 ng
each). Cells were radiolabeled with [32P]orthophosphate
for 8 h. Ectopic IKK
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 IKK
was
isolated from cytoplasmic extracts with anti-FLAG M2-agarose beads and
assayed for I
B kinase activity in the presence of GST-I
B
(1 µg) and [
-32P]ATP. Phosphoproteins were resolved by
SDS-PAGE and visualized by autoradiography (top panel).
Relative levels of IKK
protein were determined by immunoblotting
with IKK
-specific antibodies (bottom panel).
was
isolated by immunoprecipitation, fractionated by SDS-PAGE, and analyzed
by autoradiography. As shown in Fig. 2B, top panel, Tax
induced significant phosphorylation of IKK
and IKK
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 IKK
.SA (lanes
3 and 4), which has alanine replacements at
Ser-177/Ser-181, or the kinase-dead mutant IKK
.KM (lanes
5 and 6). Comparable amounts of IKK
and IKK
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.
phosphorylation,
IKK
complexes were immunopurified from HeLa cell transfectants expressing Tax, IKK
, and either wild-type IKK
or IKK
.SA. These complexes were then monitored for I
B kinase activity in
vitro using a recombinant I
B
substrate (GST-I
B
). As
shown in Fig. 2C, upper panel, Tax potently induced the
catalytic activity of wild-type IKK
via an
IKK
-dependent mechanism (lanes 1-3). In contrast, we were unable to detect I
B kinase activity in IKK
.SA immunoprecipitates (lanes 4-6). These differences in Tax
responsiveness were significant, because wild-type IKK
and IKK
.SA
were comparably expressed at the protein level (lower
panel). Given that IKK
.SA escapes from phosphorylation in
Tax-expressing cells (Fig. 2B), we conclude that Tax-induced
phosphorylation of the IKK
catalytic subunit is required for
acquisition of constitutive I
B kinase activity.
(7). To determine whether YopJ affects the Tax/NF-
B signaling axis,
293T cells were transfected with an NF-
B reporter plasmid
(NF-
B-Luc) along with expression vectors for Tax and YopJ. As shown
in Fig. 3A, left panel, Tax potently stimulated NF-
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-
B (8).
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Fig. 3.
YopJ prevents Tax-induced activation of IKK
and NF- 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-
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-IKK
antibodies (PharMingen) and assayed for I
B kinase activity as
described in the Fig. 2 legend (top panel). IKK
protein
levels were monitored by immunoblotting with polyclonal anti-IKK
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 IKK
(T7-tagged, 25 ng),
and YopJ (100 ng). Cells were radiolabeled with
[32P]orthophosphate for 8 h. Ectopic IKK
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).
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 I
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·I
B
phosphorylation (lane 4). Immunoblotting experiments
confirmed that comparable amounts of endogenous IKK
were
co-immunoprecipitated under each condition and that YopJ and Tax were
both expressed efficiently (lower two panels).
complexes and analyzed their phosphoprotein content. As shown in
Fig. 3C, top panel, phosphorylation of IKK
and IKK
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 IKK
function as
Tax-responsive phosphoacceptors.
by IKK
--
The finding that IKK
phosphorylation induced by Tax is dependent on the catalytic activity
of IKK
(Figs. 1-3) led us to hypothesize that IKK
is a substrate
of IKK
. To test this possibility, 293T cells were transfected with
an expression vector encoding a constitutively active mutant of IKK
that contained glutamic acid substitutions at Ser-177 and Ser-181
(IKK
.SE) (10). IKK
.SE immunoprecipitates derived from these
transfectants were incubated with [
-32P]ATP and a
substrate containing GST fused to full-length IKK
(GST·IKK
). As
shown in Fig. 4 (top,
lane 2), GST·IKK
phosphorylating activity was readily
detected in IKK
.SE immunocomplexes. Removal of the IKK
sequences
from the GST·IKK
fusion protein eliminated phosphoryl group
transfer, thus confirming specificity (middle, lane
2). IKK
kinase activity was not detected in immunoprecipitates derived from cells expressing a kinase-deficient mutant of IKK
(IKK
.SA) (top, lane 3), indicating that
phosphorylation of IKK
is dependent on the catalytic function of
IKK
. Moreover, the IKK
kinase activity detected in IKK
.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 IKK
.
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Fig. 4.
Phosphorylation of IKK
by IKK
. 293T cells (0.5 × 106) were transfected with expression plasmids for
FLAG-tagged IKK
.SE (25 ng) or IKK
.SA (50 ng). Ectopic IKK
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·IKK
(top panel) or GST (middle
panel) for 30 min and then assayed for kinase activity in the
presence of [
-32P]ATP. Relative levels of IKK
protein were determined by immunoblotting (bottom
panel).
and IKK
.
Activation of IKK
by Tax appears to involve a kinase that
phosphorylates the T loop of IKK
at Ser-177 and Ser-181. Given that
kinase-dead mutants of IKK
are defective for Tax-induced
phosphorylation, this kinase may be IKK
. Point mutations in IKK
that disrupt its catalytic function also prevent Tax-induced
phosphorylation of IKK
, suggesting that IKK
is phosphorylated by
IKK
within the same complex. In keeping with this proposal, IKK
has the capacity to phosphorylate a recombinant IKK
substrate
in vitro. As such, further studies are warranted to define
whether the phosphorylation status of IKK
affects the temporal
regulation of I
B kinase activity.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviations used are:
IKK, IB 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-
.
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