From the Department of Microbiology and Immunology,
Uniformed Services University of the Health Sciences, Bethesda,
Maryland 20814 and § Molecular Virology Section, Laboratory
of Molecular Microbiology, NIAID, National Institutes of Health,
Bethesda, Maryland 20892-0460
Received for publication, October 17, 2002
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ABSTRACT |
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I- NF- The phosphorylation of I- The transactivator/oncoprotein of HTLV-I, Tax, has been shown to
activate IKK constitutively (11-16). Tax-mediated IKK activation is
due in part to a direct interaction between Tax and IKK- PP2A is a major serine/threonine protein phosphatase in all eukaryotic
cells (for comprehensive reviews, see Refs. 19-23). It is crucial for
the negative regulation of multiple cellular processes. The holo-PP2A
enzyme is a heterotrimer that consists of a core enzyme formed by the
highly conserved A subunit (60 kDa, 2 human isoforms) and C (catalytic)
subunit (36 kDa, 2 human isoforms) together with many different
regulatory B-subunits that derive from multiple genes (three distinct
families: B/B55/PR55 (3 genes), B'/B56/PR61 (>3 genes), and
B''/PR72/PR130 (1 gene)), and their alternatively spliced mRNAs
(19-21, 23, 24). The crystal structure of the A-subunit shows it to
contain 15 tandem helical repeats (HEAT motifs) that assume a shape
that resembles a horseshoe (25). It serves as a scaffold to which C-
and B-subunits are attached (19-21, 23). The heterodimer formed by A-
and C-subunits and the heterotrimer containing all three subunits are
termed core enzyme and holoenzyme, respectively (19-21, 23). A role of
PP2A in controlling the signal transduction pathway that leads to
IKK/NF- Reagents and Cell Culture--
The anti-Tax monoclonal antibody
4C5 is of IgG2a subtype and reacts with amino acid residues 333-353 of
Tax.3 The anti-PP2A C-subunit
monoclonal antibody (the COOH-terminal amino acid residues 295-309 of
the catalytic subunit of human PP2A) and anti-IKK Plasmid Construction and Recombinant Protein
Purification--
The glutathione S-transferase (GST)
fusion of wild-type and mutant forms of Tax, GST-Tax, GST-H43Q,
GST-K85N, GST-M22, GST-M47, were constructed as reported previously
(28). GST-I Immunoprecipitation and Western
Blotting--
Immunoprecipitations were carried out using extracts
prepared from Jurkat and HTLV-I-transformed MT-4 cells. Ten million
cells were harvested, washed 3 times, each with 10 ml of
phosphate-buffer saline, and lysed by repeated passage through a
27.5-gauge syringe in 1 ml of lysis buffer (100 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 2 mM
EDTA, 5 mM NaF, 1 mM
Na3VO4, 0.2 mM phenylmethylsulfonyl fluoride) containing 50 µg/ml each of the protease inhibitors pepstatin, leupeptin, bestatin, and aprotinin (Roche Molecular Biochemicals). Cell debris was removed by centrifugation at 12,000 rpm
in a microcentrifuge for 10 min at 4 °C. Immunoprecipitation was carried out with 200 µg of proteins in 500 µl of cell lysates. Briefly, cell lysates were precleared by incubation with 100 µl of a
50% slurry of protein G-agarose (Invitrogen) and 2 µg of normal
mouse antiserum in 5 µl for 30 min at 4 °C followed by centrifugation at 12,000 rpm for 5 min. After preclearing, 2 µg of a
Tax monoclonal antibody (4C5), PP2A C-subunit monoclonal antibody, or
IKK Glutathione S-Transferase Pull-down Assay--
For GST pull-down
experiments, ~500 ng each of purified GST, GST-Tax, or GST-IKK In Vitro I- IKK
To determine whether IKK, PP2A, and Tax form a ternary complex in
vivo, cell extracts were prepared from a Tax-expressing HTLV-I-transformed human T-cell line, MT-4, and a control human T-cell
line, Jurkat, and subjected to immunoprecipitation using antibodies
against Tax, the catalytic subunit of PP2A (PP2Ac), and IKK IKK Tax-PP2A Interaction Is Necessary for NF- Tax Prevents I- In this study, we have demonstrated that the IKK Although interaction between IKK and PP2A can be detected in Tax-null
Jurkat cells, their interaction appears weaker than that seen in
Tax-positive MT-4 cells (Fig. 1). This is most likely due to the
tripartite IKK-PP2A-Tax interaction and is supported by the increased
stability of IKK and PP2A interaction in vitro when Tax is
present (Fig. 2). Whether PP2A may also be recruited specifically to
the activated form of IKK is not clear. Although our results indicate
that Tax can prevent active IKK from dephosphorylation and inactivation
by PP2A, they do not rule out the possibility that Tax may also prevent
phospho-I- Two Tax mutants, H41Q and H43Q, are of particular interest. As
previously reported by Semmes and Jeang (41) and confirmed here, H41Q
and H43Q transactivate HTLV-I LTR reporter at a level comparable with
that of the wild-type Tax but is considerably impaired in activating
the E-selectin enhancer/promoter, whose activity is driven by two
NF- The functional diversity of PP2A is mediated by the regulatory subunits
that control its subcellular localization and substrate specificity.
Our data indicate that IKK Our recent data indicate that Tax binds the catalytic subunit of PP2A
and acts as a noncompetitive inhibitor of PP2A. In reactions where
32P-labeled glycogen phosphorylase a is used as
a substrate, the ki (inhibitory constant) of Tax for
PP2A is estimated to be ~300 nM. This contrasts with the
most common inhibitor of PP2A, okadaic acid, a compound isolated from
marine plankton, which has an IC50 of 1 nM or
lower for PP2A. How can the weaker interaction/inhibition of PP2A by
Tax explain the potent activation of IKK and NF-B kinase (IKK) is a serine/threonine kinase
that phosphorylates I-
B
and I-
B
and targets them for
polyubiquitination and proteasome-mediated degradation. IKK consists of
two highly related catalytic subunits,
and
, and a regulatory
subunit, which becomes activated after serine phosphorylation of
the activation loops of the catalytic domains. The human T-lymphotropic
retrovirus type-I trans-activator, Tax, has been shown to interact
directly with IKK
and activates IKK via a mechanism not fully
understood. Here we demonstrate that IKK binds serine/threonine protein
phosphatase 2A (PP2A), and via a tripartite protein-protein
interaction, Tax, IKK
, and PP2A form a stable ternary complex.
In vitro, PP2A down-regulates active IKK prepared from
Tax-producing MT4 cells. In the presence of Tax, however, the ability
of PP2A to inactivate IKK is diminished. Despite their interaction with
IKK
, PP2A-interaction-defective Tax mutants failed to activate
NF-
B. Our data support the notion that IKK
-associated PP2A is
responsible for the rapid deactivation of IKK, and inhibition of PP2A
by Tax in the context of IKK·PP2A·Tax ternary complex leads
to constitutive IKK and NF-
B activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B/Rel family of transcription factors are controlled by
inhibitory I-
B proteins I-
B
and I-
B
and the I-
B-like
domains in NF-
B1 and NF-
B2 that sequester NF-
B/Rel in the
cytoplasm as multiprotein complexes (for reviews, see Ref. 1-3). Upon
activation by extracellular stimuli such as interleukin-1, tumor
necrosis factor-
, bacterial lipopolysaccharide, or by human
T-lymphotropic virus type I
(HTLV-I)1 Tax, I-
B
and
I-
B
become serine-phosphorylated and polyubiquinated and are
rapidly degraded via proteasome-mediated proteolysis, resulting in
heightened nuclear levels of NF-
B and increased expression of a
plethora of cellular genes under NF-
B regulation, including the
genes of many cytokines and their receptors, adhesion molecules, and
immune modulators (1-3). Dysregulation and/or hyperactivation of the
NF-
B/I-
B regulatory pathway as caused by chromosomal
translocation (4), oncogene transduction (5), viral infection,
or targeted gene disruption (6, 7) leads to cancers of the
hematopoietic cells or chronic inflammatory diseases.
B
and I-
B
is mediated by a kinase
called I-
B kinase (IKK) (8, 9). The core IKK enzyme consists of two
highly homologous catalytic subunits
and
of 85 and 87 kDa in
sizes, respectively, and a 48-kDa regulatory subunit, IKK-
/NEMO
(referred to as IKK-
herein) (9, 10). Both IKK-
and IKK-
contain NH2-terminal kinase domains followed by leucine
zippers and helix-loop-helix domains that mediate protein-protein interactions important for IKK oligomerization and kinase activity (8,
9). Likewise, IKK-
also contains extensive helical regions and
leucine zipper domains that engage in protein-protein interaction (9,
10). In vivo the IKK holoenzyme exists as a large protein
complex of at least 700-900 kDa in size (8, 9). It is not clear what
other protein components are present in the holo-IKK enzyme complex in
addition to IKK-
, IKK-
, and IKK-
. Several members of the
I-
B and NF-
B/Rel families of proteins and mitogen-activated
protein kinase phosphatase-1 (MKP), MEK kinase, and NF-
B inducing
kinase (NIK) have been reported to interact with IKK, although
these proteins have not been found to co-elute with the 900-kDa IKK
complex chromatographically (9).
(14, 16,
18). The molecular mechanism via which Tax affects IKK activation after
its association with IKK-
remains incompletely understood, however.
We have found recently that Tax can interact directly with the
catalytic subunit of the major serine/threonine protein phosphatase 2A
(PP2Ac) in vivo and in
vitro.2 Furthermore, Tax
acts as a noncompetitive inhibitor of PP2A in in vitro
assays where 32P-labeled glycogen phosphorylase
a is used as a PP2A substrate.2 Consistent with
the notion that Tax inhibits the activity of PP2A, MEK phosphorylation
and to a lesser extent cAMP-response element-binding protein and ATF-1
phosphorylation in Tax-expressing HTLV-I-transformed cells and in
Tax-transfected human embryonic kidney 293 cells are greatly
elevated.2 Most interestingly, PP2A interaction-defective
mutants of Tax fail to stimulate MEK phosphorylation and are unable to
activate NF-
B, thus suggesting a link between the inhibition of
PP2A by Tax and NF-
B activation.2
B activation has been reported previously (8). Okadaic acid,
an inhibitor of PP2A, has been shown to activate NF-
B in vivo (26, 27). Furthermore, PP2A can inactivate the kinase activity of IKK in vitro (8). Here we provide evidence to
show that via a tripartite interaction, Tax, PP2A, and IKK
form a ternary complex. In in vitro IKK assays, Tax reduces the
ability of PP2A to inactivate IKK. Consistent with the notion that
activation of IKK requires that Tax bind to both IKK
and PP2A,
several Tax mutants that are abrogated or attenuated for PP2A binding
fail to activate NF-
B even though they continue to bind IKK
.
These results indicate that, in the context of the IKK·PP2A·Tax
complex, PP2A activity is inhibited or diminished. In essence, IKK is
activated by serine phosphorylation of its activation loop upon
extracellular stimulation. In normal cells, phospho-IKK becomes rapidly
inactivated by IKK
-associated PP2A, returning IKK to a resting
state. In HTLV-I infected or transformed cells, PP2A inhibition by
IKK
-bound Tax maintains IKK in the phosphorylated and active form,
causing constitutive phosphorylation and degradation of I-
B, which
in turn leads to nuclear presence of NF-
B/Rel and potent activation of genes under NF-
B/Rel control.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(amino acids
1-419, representing full-length human IKK
) monoclonal antibody were
from Upstate Biotechnology, Inc. and Santa Cruz Biotechnology,
Inc., respectively. The purified PP2A was derived from human red blood
cells and consists of both A and C subunits (Upstate Biotechnology).
Jurkat and the HTLV-I-transformed T-cell line MT-4 were cultured in
RPMI medium supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and 100 units/ml penicillin (Invitrogen) in
the presence of 10% CO2.
B
-(1-54) and Ser-32 and Ser-36 phosphorylation
sites mutant GST-I
B
-(1-54)AA were generous gifts of Drs.
DiDonato and Karin (8). GST-IKK
C was constructed by replacing in
GST-Tax an NcoI, SmaI fragment that contains the
coding sequence for wild-type Tax with an NcoI, SmaI fragment that encodes the NH2-terminal 306 amino acid residues of IKK
. GST fusion proteins were expressed and
prepared by standard protocols and stored frozen in buffer D (20 mM Hepes (pH 7.9), 100 mM KCl, 0.2% (v/v)
2-mercaptoethanol, 1 µM phenylmethylsulfonyl fluoride,
and 20% glycerol) at
80 °C.
monoclonal antibody were added to each sample. The reactions
were preincubated at 4 °C for 1 h. After the addition of 30 µl of protein G-agarose to each reaction, the mixtures were incubated
overnight. On the following day, immune complexes were pelleted by
centrifugation at 3000 rpm for 5 min at 4 °C, washed 3 times with
0.8 ml of lysis buffer, resuspended in 30 µl of SDS-PAGE loading
buffer, heated at 95 °C for 3 min, and centrifuged, and then 15 µl
of sample were loaded and resolved on a 12% SDS-polyacrylamide gel
containing a 4% stacking gel. Proteins resolved in the gels were
transferred to nitrocellulose membranes (Schleicher & Schuell). For
Western blotting, membranes were incubated with blocking buffer (50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.05%
Tween, 0.02% sodium azide, 5% nonfat dry milk) for 3 h and then
probed in the same buffer for 2 h with mouse monoclonal antibodies
against PP2Ac, IKK
, or Tax. After washing, the blots were incubated
for 1 h in the same buffer containing an anti-mouse horseradish
peroxidase-conjugated, secondary antibody (diluted 1:1000, Santa Cruz
Biotechnology), washed three times, developed using a chemiluminescent
substrate (SuperSignal; Pierce), and exposed to x-ray films.
was
incubated with 300 ng of purified PP2A or PP2A and Tax in 30 µl of 1× binding buffer (25 mM HEPES (pH 7.9), 5 mM KCl, 0.5 mM MgCl2, 0.5 mM EDTA, 1 mg/ml bovine serum albumin, 10% glycerol,
0.15% Nonidet P-40, 0.25 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride) for 30 min at 30 °C. Forty microliters of a 50% slurry of prewashed glutathione-Sepharose 4B (Amersham Biosciences) were added to each binding reaction, and
samples were incubated for 1 h with agitation at 4 °C. After incubation, the protein-bound Sepharose beads were washed 3 times with
800 µl of 1× binding buffer, pelleted by centrifugation at 1200 rpm
for 5 min, resuspended in 30 µl of SDS-PAGE loading buffer, and
heated to 95 °C, and 15 µl from each reaction were resolved by
12% SDS-PAGE followed by immunoblotting, as described. To remove the
GST moiety from GST-IKK
, the fusion protein was proteolysed with 0.2 unit of thrombin (Sigma) overnight at 4 °C, and the treated protein
was then incubated with GST-Tax as described above. Under these
conditions, GST-IKK
was converted completely to IKK
by thrombin,
and the residual thrombin in the reaction mixture did not interfere
significantly with the binding reactions.
B Kinase Assay--
To measure IKK activity,
107 MT-4 cells were harvested by centrifugation and
extracted in 1 ml of a lysis buffer containing 20 mM
Tris-HCl (pH 7.6), 20 mM glycerol phosphate, 250 mM NaCl, 3 mM EGTA, 3 mM EDTA,
0.5% Nonidet P-40, 0.1 mM sodium vanadate, 10 µg/ml
aprotinin, 2 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml of leupeptin. After a
brief centrifugation, extracts were incubated for 2 h with
agitation at 4 °C with 0.5 µg of an antibody against IKK
followed by protein G-Sepharose (Invitrogen) precipitation. Each IKK
immune complex reaction was carried out using 1 µg of recombinant GST
protein fused to amino acid residues 1-54 of I
B
(GST-
I
B
-(1-54)) as the substrate and 10 µM
[
-32P]ATP in a final volume of 30 µl of
a reaction buffer containing 30 mM HEPES (pH 7.4), 10 mM MgCl2, and 1 mM dithiothreitol
at 25 °C for 30 min. The reactions were terminated by the addition of SDS-PAGE loading buffer. IKK activity as measured by the
phosphorylation level of I
B
was evaluated by SDS-PAGE followed by
autoradiography. When applicable, 5 ng of PP2A and 0.1-0.6 µg of Tax
were added into a 30-µl reaction.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Interacts with PP2A and Tax in Vivo--
Several cellular
kinases and regulatory proteins whose activities are regulated by
serine/threonine phosphorylation form protein complexes with PP2A
(29-34). In this manner, PP2A is localized to its targets at once and
can affect rapid on/off control of regulatory processes immediately
after signaling events are initiated. Because Tax interacts with both
PP2A and IKK
, we wondered if PP2A also forms a complex with IKK.
This might explain the rapidity with which the IKK/NF-
B pathway is
returned to the inactive state soon after its activation. Furthermore,
because Tax constitutively activates IKK, we wondered if, in the
context of the IKK-PP2A-Tax complex, the phosphatase activity of PP2A
may be inhibited by Tax. Because IKK is activated through serine
phosphorylation of its activation loop by upstream kinases, a block in
its de-phosphorylation by Tax would maintain phospho-IKK in an active
state and can explain the constitutive activation of IKK by Tax.
,
respectively. As expected, mouse monoclonal antibody against IKK
precipitated IKK
easily (Fig. 1,
lanes 3 and 4, upper panel). From
Tax-positive MT-4 cell extracts, both the PP2A c-subunit (PP2Ac) and
Tax were readily co-precipitated (lane 4, middle
and lower panels, respectively). In Jurkat extracts, a low
but detectable trace of PP2Ac was also present (lane 3,
middle panel). Likewise, Tax monoclonal antibody 4C5
co-precipitated Tax, PP2Ac, and IKK
(lane 6,
lower, middle, and upper panels). The
4C5 antibody is highly specific for Tax. It did not react with either
PP2Ac or IKK
, as indicated by immunoprecipitations done with
Tax-null Jurkat extracts (lane 5). Finally, a mouse monoclonal antibody against the COOH-terminal region of PP2Ac co-precipitated IKK
from both Jurkat and MT-4 extracts.
Interestingly and in agreement with the immunoprecipitation performed
with anti- IKK
(lanes 3 and 4), the amount of
IKK
that co-precipitated with PP2Ac is lower when Jurkat extracts
were used (compare lanes 7 and 8, upper
panel). The PP2Ac antibody failed to co-immunoprecipitate Tax,
however (lanes 7 and 8, bottom panel).
We think this is due to a disruption of the protein-protein interaction
between Tax and PP2Ac by the PP2Ac antibody. The COOH-terminal region
of PP2Ac is involved in extensive protein-protein interaction; the
carboxyl leucine residue undergoes a methyl esterification (35-37)
critical for C subunit assembly with A and B subunits (36, 38, 39) to
form the core and holoenzymes. It is likely that this region is also
important for binding Tax. Together, these data indicate that PP2A
interacts with IKK directly. Furthermore, Tax interacts with IKK
and
PP2A in vivo in a stable ternary complex.
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Fig. 1.
IKK interacts with
the catalytic subunit of PP2A and Tax in vivo.
Immunoprecipitations were carried out as detailed in "Experimental
Procedures" using extracts prepared from Jurkat and the
HTLV-I-transformed cell line MT-4. One-half of 1 ml of cell extracts
were immunoprecipitated with 2 µg of nonspecific mouse IgG
(CTRL) and antibodies against IKK
, Tax, and the
catalytic subunit of PP2A (PP2Ac) as labeled at the top. The
immunoprecipitates (IP) were resolved on a SDS, 12% PAGE
and analyzed by Western blotting with IKK
, Tax, and PP2Ac monoclonal
antibodies (WB: IKK
, WB: PP2Ac, and
WB: Tax), respectively. The data shown are representative of
three independent experiments.
, Tax, and PP2A Form a Stable Multiprotein Complex in
Vitro--
To characterize further the interaction among Tax, IKK
,
and PP2A, GST pull-down experiments were performed using purified PP2A,
consisting principally of both the catalytic C-subunit (PP2Ac) and the
regulatory A-subunit, derived from human red blood cells (Upstate
Biotechnology) in the presence or absence of Tax protein purified from an Escherichia coli expression system. Care
was taken to ensure comparable levels of purified GST, GST-Tax, and GST-IKK
fusion proteins (Fig.
2A) were used. In binding
reactions containing PP2A only, GST-Tax, GST-IKK
, and
GST-IKK
C, the GST fusion of an IKK
mutant deleted for the
COOH-terminal Tax binding domain (amino acid residues 307-419), all
displayed efficient binding to PP2Ac (Fig. 2B, lower
panel). Under these experimental conditions, ~2 and 4% of input
PP2Ac became bound to GST-Tax and GST- IKK
, respectively. The
interactions appear specific since under the same binding condition,
GST control did not bind PP2Ac. Interestingly, in reactions where both
PP2A and Tax were added, the binding of PP2A to GST-IKK
became
significantly enhanced (compare Fig. 2, B and C,
GST-IKK
lanes, lower panel). By
contrast, Tax did not increase PP2A binding to GST-IKK
C (Fig.
2C, compare the GST-IKK
and
GST-IKK
C lanes in the
lower panel) whose Tax binding site is deleted and is unable
to bind Tax (upper panel), suggesting that when Tax is bound
to IKK
, it can further stabilize or enhance IKK
-PP2A interaction.
As anticipated, GST-IKK
interacted with Tax (Fig. 2C,
GST-IKK
lane, upper panel). In
agreement with earlier results, GST-Tax interacted with PP2A
(GST-Tax lane, lower panel). Because Tax exists
as homodimer, GST-Tax and Tax interaction was also seen (GST-Tax
lane, upper panel). The exogenously added Tax (300 ng)
did not significantly compete for PP2Ac binding to GST-Tax (500 ng),
most likely because the interaction between Tax and PP2Ac was not
saturating (only 2% input PP2Ac was pulled down by GST-Tax). No
binding of Tax or PP2A to GST was detected. Together, these data
support the immunoprecipitation results shown above and indicate that
PP2A is associated with IKK. Furthermore, in the presence of Tax, a
stable IKK·PP2A·Tax ternary complex can form. Finally, even though
IKK
C mutant is defective in binding Tax (18, 40), it is still
able to associate with PP2A, much like the full-length IKK
,
suggesting that PP2A binds to a region in IKK
upstream of the
COOH-terminal Tax binding site.
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Fig. 2.
A, profile of purified Tax and
GST fusion proteins. GST, GST-Tax, GST-IKK , GST-IKK
c, GST-M22,
GST-M47, GST- I
B
-(1-54) were expressed and purified as
previously reported (28). Procedures for the expression and
purification of hexahistidine-tagged Tax protein are as published (45).
After electrophoresis in a SDS, 12% polyacrylamide gel, the proteins
were stained with Coomassie Brilliant Blue R-250 (Sigma). Molecular
mass markers are as shown. B and C, IKK
, Tax,
and PP2A form a ternary complex in vitro. Purified GST,
GST-Tax, GST-IKK
, or GST-IKK
C (500 ng each) was incubated with
300 ng of purified PP2A (PP2A panel) (B) or both
300 ng each of purified PP2A and Tax (PP2A+Tax panel)
(C) as described under "Experimental Procedures." The
proteins bound to the glutathione-Sepharose were resolved in SDS-12%
PAGE and Western blotted (WB) with either 4C5 Tax-monoclonal
antibody (WB: Tax, upper panels) or PP2Ac
monoclonal antibody (WB: PP2Ac, lower panels).
The data shown are representative of two independent
experiments.
B Activation--
If
indeed the inhibition of PP2A by Tax in the context of the
IKK·PP2A·Tax complex leads to a constitutive activation of IKK, then one might expect that both Tax-PP2A and Tax-IKK
interactions would be necessary for Tax-mediated activation of IKK and NF-
B. Using yeast 2-hybrid analysis, we have previously mapped the domain critical for PP2Ac binding to the NH2-terminal 100-amino
acid residues of Tax (Fig.
3A).2 This region
is distinct from the domain responsible for IKK
binding previously
localized to approximately amino acid residues 100-150 previously
(Fig. 3A) (40). Two Tax mutants, H43Q and K85N, shown to be
defective in binding PP2A earlier,2 and another mutant,
H41Q (41), were examined for their interactions with PP2A and IKK
.
As reported previously and confirmed here, H43Q together with H41Q
transactivate HTLV-I LTR reporter at a level comparable with that of
the wild-type Tax but are considerably attenuated in activating the
E-selectin enhancer/promoter driven by two NF-
B binding motifs (Fig.
3B). K85N is a much more severe mutation that abrogated both
LTR and NF-
B activation of Tax (Fig. 3B). As shown in
Fig. 3C, all three
mutants are indeed defective in PP2A binding. Notably, H41Q and H43Q
bound IKK
efficiently. Under the same conditions, K85N also showed
reduced but detectable interaction with IKK
. These results suggest
that for IKK activation, Tax-IKK
interaction is necessary but not
sufficient. Most likely, the binding and inhibition of PP2A by Tax is
also needed. We also examined two other Tax mutants, M22 (T130A,L131S)
and M47 (L319R,L320S), which have been used widely in HTLV-I Tax
studies (42). M22 is a mutant impaired in dimerization (43) and IKK
binding (14, 16, 18). It is unable to activate NF-
B but can
transactivate HTLV-I LTR (Fig. 3B). By contrast, M47 is
defective in LTR activation (because of a defect in P/CAF
binding) (42, 44) but shows considerable activity in promoting NF-
B
activation (Fig. 3B). Consistent with the notion that Tax
interacts with both IKK and PP2A to activate IKK and NF-
B, M22
showed little binding to either IKK or PP2A; by contrast, M47, like
wild-type Tax, interacted with both IKK and PP2A (Fig. 3D).
Together, these data support the functional importance of PP2A
binding/inhibition by Tax in activating IKK and NF-
B. Finally,
although we think distinct domains of Tax interact with PP2A and
IKK
, M22 and K85N mutants appeared to be impaired in both IKK
binding and PP2A binding. This may be due to conformational alterations
of Tax as a result of the amino acid substitutions. Indeed, K85N also
failed to trans-activate HTLV-I LTR (Fig. 4B), and M22 has
been shown previously to be impaired in dimerization (43).
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Fig. 3.
Tax binding to both PP2A and
IKK is required for
NF-
B activation. A, domain
organization of Tax. Arrows mark major trypsin sensitive
sites of Tax at Lys-88 and Lys-346, or Arg-349. H41Q, H43Q, and K85N
denote three mutants attenuated or abrogated for PP2A binding. M22 is
defective in dimer formation, IKK
binding, and NF-
B activation.
M47 is defective in P/CAF binding and LTR transactivation.
B, transactivation properties of Tax mutants. DNA
transfection and luciferase assays were as previously reported (17).
Typically, 0.5 µg of CMV-Tax (or mutants) plasmid and 0.5 µg of the
reporter were used to transfect 3 × 105 human
embryonic kidney 293 cells in each well of a 12-well plate. DNA
transfections were carried out in triplicate. The means of luminescence
units from each set of three luciferase assays were then used to
compute fold trans-activation of each Tax construct for the HTLV-I
LTR-Luc (solid bar) or the E-selectin-Luc (open
bar) reporter. C, in vitro interaction of
PP2A and IKK
with Tax mutants H41Q, H43Q, and K85N. One-half of 1 µg each of GST, GST-Tax, GST-H41Q, GST-H43Q, or GST-K85N was
incubated with 0.3 µg each of purified PP2A and IKK
as described
under "Experimental Procedures." Proteins bound to the
glutathione-Sepharose 4B were resolved by SDS, 12% PAGE and
Western-blotted with PP2Ac, IKK, and Tax (4C5) antibodies (WB:
PP2Ac, WB: IKK
, WB: Tax), respectively.
The data shown are representative of three independent experiments.
D, in vitro interaction of PP2A and IKK
with
M22 and M47 mutants. Pull-down experiments were performed as in
C. The data shown are representative of three independent
experiments. WT, wild type.
View larger version (25K):
[in a new window]
Fig. 4.
Tax prevents PP2A from inactivating IKK
in vitro. A, MT4 cell extracts were
prepared as described under "Experimental Procedures" and
immunoprecipitated with nonspecific mouse sera (CTRL,
lane 1) or IKK antibody (lanes 2-10). IKK
immune complex kinase assays contained 1 µg of recombinant GST fusion
containing amino acid residues 1-54 of I
B
(GST-
I
B
-(1-54), denoted as wild type (WT)) or its
phosphorylation-defective mutant (with S32A-S36A amino acid
substitutions, denoted as AA), and 10 µM
[
-32P]ATP in a final volume of 30 µl. When
applicable, 5 ng of PP2A were added (lanes 3-5 and
8-10). Lanes 4, 8, 9, and
10 contained 0.1, 0.6, 0.3, and 0.1 µg of Tax,
respectively. Lane 5 contained OA at a concentration of 10 nM. The reactions were carried out at 25 °C for 30 min
and terminated by the addition of 6 µl of 6 × SDS gel-loading
buffer, and the phosphorylation level of I
B
was evaluated by SDS,
12% PAGE followed by autoradiography. B, the IKK kinase
reactions were carried out as in A except that the IKK
immunoprecipitates were incubated with PP2A for 1 h at 25 °C
for 1 h (lanes 1-7) with or without OA, GST-Tax, or
GST-H43Q. In lane 2, OA was added together with the
substrates after the PP2A preincubation. In lane 8, no PP2A
was added. One of three repeats of the experiment is shown.
IP, immunoprecipitates.
B Kinase Down-regulation by PP2A--
To test
the idea that Tax inhibits IKK-associated PP2A and by so doing
maintains IKK in an active state, we performed in vitro IKK
assays using IKK immunoprecipitated from MT-4 cell extracts and a GST
fusion containing amino acid residues 1-54 of I
B
, GST-I
B
-(1-54), as a substrate (8). The form of IKK derived from
MT-4 cells is activated (by Tax). As expected, it phosphorylated GST-I
B
-(1-54) efficiently but not the AA mutant in which the Ser-32 and Ser-36 of I
B
-(1-54) were substituted by alanine
residues (compare Fig. 4A, lanes 6 and
7). Consistent with the notion that IKK activity is
negatively regulated by PP2A, phosphorylation of GST-I
B
-(1-54)
in vitro became significantly reduced in the presence of
exogenously added PP2A (compare Fig. 4A, lanes 2 and 3). The PP2A inhibitor, okadaic acid (OA),
effectively blocked this effect of PP2A (Fig. 4A, lane
5). Likewise, Tax also dampened the decrease in IKK activity
caused by PP2A (Fig. 4A, lane 4) in a
dose-dependent manner (Fig. 4A, lanes
8-10). To show that IKK is inactivated by PP2A directly, we
treated the IKK immunoprecipitate with PP2A in the presence or absence
of OA for 1 h. The PP2A-pretreated IKK was then incubated with the
reaction substrates GST-I
B
-(1-54) and [
-32P]ATP
for 30 min. As expected, IKK pre-incubated with PP2A lost its kinase
activity, as reflected by a significant reduction in 32P
incorporation into GST-I
B
-(1-54) (Fig. 4B,
lanes 1, 2, and 4). IKK inactivated by
PP2A pretreatment lost its activity irreversibly; the addition of OA or
Tax after the PP2A preincubation could not reverse the inhibitory
effect of PP2A (Fig. 4B, compare lanes 1 and
2). By contrast, in the reaction where PP2A is inhibited by
the simultaneous addition of OA (Fig. 4B, lane 3)
or GST-Tax (Fig. 4B, lanes 6) during the
pretreatment, IKK retained significant activity. The addition of PP2A
binding-defective GST-H43Q, however, could not effectively prevent IKK
inactivation by PP2A (Fig. 4B, lane 7). Finally,
the addition of GST-Tax in the absence of PP2A did not affect IKK
kinase activity significantly (Fig. 4B, lane 8),
consistent with the notion that Tax does not activate IKK directly. In
aggregates, these data support the idea that IKK activation by Tax is
most likely mediated by its binding and inhibition of PP2A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
regulatory
subunit, PP2A, and Tax form a stable multiprotein complex in HTLV-I-transformed MT-4 cells and in vitro. Furthermore, we
show that Tax mutants defective in PP2A binding failed to activate NF-
B despite wild-type levels of binding to IKK, indicating that interaction with both IKK
and PP2A is necessary for Tax to activate IKK-NF-
B. Finally, evidence is provided to indicate that Tax prevents the active form of IKK isolated from MT-4 cells from inactivation by PP2A in vitro. Together, these results
provide a mechanistic explanation for the constitutive activation of
IKK by Tax; IKK-associated PP2A is responsible for rapid deactivation of IKK; in the context of the IKK·PP2A·Tax complex, PP2A is
inhibited by Tax, causing constitutive phosphorylation/activation of
IKK; the activated IKK, in turn, promotes phosphorylation and
degradation of I-
B, and potent NF-
B activation.
B
from dephosphorylation by PP2A. Further experiments
are needed to determine whether the latter mechanism contributes to the
potent NF-
B activation brought on by Tax.
B binding motifs, suggesting that these two amino acid
substitutions impact on a very specific protein-protein interaction
critical for NF-
B activation. Our data indicate that the specific
defect of H43Q and H41Q lies in an inability to interact with and, most
likely, a failure to inhibit PP2A. We have also examined two other Tax
mutants, M22 and M47. M22, a mutant that fails to activate NF-
B but
can transactivate via HTLV-I LTR, is defective in both IKK and PP2A
binding. As expected, M47, a mutant that cannot transactivate LTR
because of a defect in P/CAF binding but continues to promote NF-
B
activation binds both IKK and PP2A. These data add to the IKK
interaction with M47 and the lack thereof with M22 previously reported
and support the notion that for NF-
B activation, Tax needs to
interact with both IKK and PP2A.
binds both PP2A and Tax. Yeast 2-hybrid
analysis had shown that Tax and PP2A C-subunit interact
directly.2 Whether other PP2A or IKK subunits also
participate in the tripartite interaction remains to be determined.
Finally, IKK
C, which is deleted for the COOH-terminal Tax binding
region, interact with PP2A like full-length IKK
, indicating that Tax
and PP2A binds to distinct domains of IKK
.
B? We think the
interaction between Tax and IKK is particularly relevant. Even though
Tax is a weaker inhibitor for PP2A, the interaction between Tax and IKK
would locate Tax in the immediate environment where its interaction
with PP2A would be the most effective. In essence, through Tax-IKK
interaction, the effective concentration of Tax around IKK is high,
thus allowing strong inhibition of PP2A to occur. Indeed, we think Tax
does not target all forms of PP2A. Rather, via additional
protein-protein interactions such as binding to IKK, it interacts and
inhibits sub-populations of PP2A involved in regulating specific
signaling pathways. The fact that ~2-4% of PP2Ac
co-immunoprecipitated with Tax2 is consistent with this
notion. Finally, it should be pointed out that IKK down-regulation
involves not only phosphatases such as PP2A. The highly active IKK
phosphorylated in the activation loop undergoes autophosphorylation at
multiple sites in the COOH termini (9), which down-modulates its
activity. Although it is not clear which form(s) of IKK and which
phosphoamino acid residue(s) is the target of PP2A, based on the
okadaic acid results and Tax-related studies it may be inferred that
the phosphoserine residues in the activation loop are most likely the
targets. In summary, the data reported here provide a novel,
mechanistic explanation for the potent activation of
IKK/NF-
B-signaling pathway by Tax.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. M. Di Donato and M. Karin for
the plasmids for GST-IB
-(1-54) and it mutant,
GST-I
B
-(1-54)AA and other members of the Giam lab for helpful
discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants RO1 CA48709 and RO1 CA/GM 75688.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, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814. Tel.: 301-295-9624; Fax: 301-295-1545; E-mail: giam@bob.usuf2.usuhs.mil.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M210631200
2 Y.-L. Kuo, L.-C. Wang, M.-H. Liang, Y. Tang, D.-X. Fu, B.-Y. Liu, R. Harrod, H.-J. Kung, H.-M. Shih, and C.-Z. Gram, submitted for publication.
3 D.-X. Fu, Y.-L. Kuo, B.-Y. Liu, K.-T. Jeang, and C.-Z. Giam, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HTLV, human
T-lymphotropic virus type I;
IKK, I-B kinase;
PP2A, protein
phosphatase 2A;
GST, glutathione S-transferase;
LTR, long
terminal repeat;
OA, okadaic acid;
MEK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase;
CREB, cAMP-response element-binding protein;
p/CAF, p300 CREB-binding
protein-associated factor.
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