From the Department of Microbiology and Immunology,
Pennsylvania State University College of Medicine, Hershey,
Pennsylvania 17033 and the ** Department of Pharmacology, Laboratory of
Gene Regulation and Signal Transduction, University of California at
San Diego, La Jolla, California 92093-0636
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ABSTRACT |
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The tax gene product of human T-cell
leukemia virus I induces aberrant expression of various cellular genes,
which contributes to transformation of host cells. Induction of many
Tax target genes is mediated through transcription factor NF-B. Here
we show that Tax triggers activation of cellular protein kinases, I
B
kinase
(IKK
) and IKK
, which phosphorylate the NF-
B
inhibitory protein I
B
, resulting in its degradation and NF-
B
activation. Constitutive IKK activation occurs in both Tax-transfected
and human T-cell leukemia virus I-infected T cells. We further
demonstrate that Tax-mediated NF-
B signaling also requires the
NF-
B-inducing kinase (NIK). Consistently, inactive forms of either
IKKs or NIK attenuate Tax-mediated NF-
B activation. Therefore, Tax
activates NF-
B by targeting cellular signaling molecules, including
both IKKs and NIK.
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INTRODUCTION |
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The type I human T-cell leukemia virus (HTLV-I)1 is the etiologic agent of an acute T-cell malignancy, termed adult T-cell leukemia (1-3). HTLV-I encodes a regulatory protein, Tax, which plays a central role in HTLV-I-induced host cell transformation (4-6). Tax not only serves as a transactivator of the HTLV-I viral long terminal repeat (LTR) but also alters the expression of a large number of cellular genes (7). Deregulation of cellular genes appears to contribute to HTLV-I-induced host cell transformation (7, 8).
Lacking DNA binding activity, Tax induces the target genes indirectly
by modulating the activity of specific host transcription factors (7,
9). Tax activates HTLV-I LTR by physical interaction with the CREB/ATF
family of proteins, which specifically bind to the cAMP response
element-like sequences present in HTLV-I LTR (10-12). Tax interacts
with the basic leucine zipper region of the CREB/ATF factors, enhancing
their dimerization and DNA binding activities (13-15). Induction of
many cellular genes by Tax is mediated through the cellular
transcription factor NF-B (7), a key regulator of genes involved in
cell activation and proliferation (16). NF-
B activity is normally
regulated through its cytoplasmic retention by specific inhibitors,
including I
B
and related proteins (17). Activation of NF-
B by
cytokines is mediated by signal transduction cascades, leading to
activation of the I
B kinases, IKK
and IKK
(18-22). These
kinases phosphorylate the I
Bs, a modification that triggers their
ubiquitination and proteolysis, allowing the released NF-
B dimers to
enter the nucleus and activate target genes (23). In HTLV-I-infected
cells or cells transfected with Tax cDNA expression vector, NF-
B
is constitutively nuclear (24). The deregulated nuclear expression of
NF-
B is essential for Tax-induced host cell transformation (25).
Although precisely how Tax induces the nuclear translocation of NF-
B
is not fully understood, recent studies have shown that Tax induces the
phosphorylation and degradation of I
Bs (26-33).
In this paper, we show that Tax triggers activation of the IKKs, and
this action of Tax requires the recently identified NF-B-inducing kinase (NIK) (34).
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MATERIALS AND METHODS |
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Cell Cultures--
Jurkat T cells (ATCC) and Jurkat cells
expressing the SV40 large T antigen (Jurkat-Tag; Ref. 35) were
maintained in RPMI 1640 medium supplemented with 10% fetal bovine
serum, 2 mM L-glutamine, and antibiotics
(complete RPMI). C8166 (36), HUT102 (37), MT-2 (38), and SLB-1 (39) are
IL-2-independent HTLV-I-transformed T-cell lines. E55, N1185, and N1186
are HTLV-I-infected human T cells that require IL-2 for growth (40).
Tax1 is a human T-cell clone expressing Tax in the context of a herpes
saimiri vector (6). Human peripheral blood T cells were prepared as
described (41). In vitro infection of T cells with HTLV-I
was performed by cocultivating the target cells with -irradiated
HTLV-I donor cells (MT-2) (42, 43).
Luciferase Assay-- Jurkat T cells (5 × 106) were transfected using DEAE-dextran (44) with the indicated luciferase reporters and cDNA expression vectors. After 40 h, luciferase activity was determined as described (45).
Immunoblotting Assays--
Human 293 kidney carcinoma cells were
seeded in 0.1% gelatin-treated 24-well plates (2.5 × 104 cells/well) and transfected using DEAE-dextran (44)
with 0.12 µg of HA-IB
and other indicated expression vectors.
Jurkat-Tag cells (5 × 106) were transfected using the
same protocol with 1.6 µg of HA-I
B
and other indicated
expression vectors. After 40 h, whole-cell extracts were prepared
and analyzed by Western blotting (46).
Immune Complex Kinase Assays (KA)--
Jurkat-Tag cells were
transfected with HA-IKKs together with either the wild type (WT) or
mutant forms of Tax. After 40 h, cells were lysed in a lysis
buffer, containing 20 mM Hepes (pH 7.6), 250 mM
NaCl, 0.5% Nonidet P-40, 20 mM -glycerophosphate, 1 mM EDTA, 20 mM p-nitrophenyl
phosphate, 0.1 mM Na3VO4, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, and a previously described protease inhibitor mixture (47).
HA-IKK
and HA-IKK
were isolated by IP using anti-HA antibody.
Immune complexes were washed three times with lysis buffer, once with
lysis buffer supplemented with 1 M urea, and twice with a
kinase buffer (20 mM Hepes, pH 7.6, 20 mM
MgCl2, 20 mM
-glycerophosphate, 1 mM EDTA, 2 mM p-nitrophenyl phosphate, and 2 mM dithiothreitol). KA were performed as
described (20) using GST-I
B
-(1-54) or the mutant
GST-I
B
-(1-54)32A/36A as substrate. IKK proteins in the immune
complexes were detected by immunoblotting. Endogenous IKK activity in
Tax-expressing and HTLV-I-infected T cells was determined by a similar
strategy but using anti-IKK
antibody (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) in the immune precipitation.
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RESULTS |
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Catalytically Inactive IKKs Inhibit Tax-mediated NF-B
Activation--
To determine whether the recently cloned IKKs are
required for activation of NF-
B by Tax, we examined the effects of
catalytically inactive forms of IKK
(IKK
(K44M)) and IKK
(IKK
(K44A)) (20, 21) on Tax-mediated transactivation of a
B-driven luciferase reporter gene (
B-TATA-luc). The
B-TATA-luc
reporter gene was transfected into Jurkat T cells either alone or
together with a Tax expression vector along with increasing amounts of
IKK
(K44M) or IKK
(K44A). As expected, Tax potently activated the
B enhancer, resulting in marked induction of luciferase activity
(Fig. 1A, column
2). More importantly, expression of increasing amounts of
catalytically inactive IKK
or IKK
led to a
dose-dependent inhibition of reporter induction
(columns 3-6). At the concentrations used, the
inactive IKKs only modestly affected RelA-mediated
B-TATA-luc transactivation (Fig. 1B). Thus, the IKKs appeared to be
required for Tax-mediated NF-
B activation but not the nuclear
function of NF-
B. Parallel experiments performed with the WT forms
of IKK
and IKK
revealed that IKK
potentiated Tax-mediated
induction of
B-dependent transcription in a
dose-dependent manner (Fig. 1C,
columns 2-4). At low concentrations, IKK
also
significantly enhanced Tax-mediated transactivation (columns
5 and 6), although expression of higher amounts
of IKK
caused a reduction in reporter gene expression
(column 7). At the amounts transfected, neither IKK
nor IKK
significantly affected reporter expression in Tax's absence (columns 8 and 9). These
results further suggested that these I
B kinases participate in
Tax-mediated NF-
B activation. The different dose requirement for
IKK
and IKK
to potentiate Tax activity may be due to differential
expression of the endogenous IKKs in Jurkat cells. However, it may also
be due to their different properties (19, 21, 22).
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Inactive IKKs Interfere with Tax-induced IB
Phosphorylation--
To determine whether the effect of the
transdominant IKK mutants on Tax-mediated NF-
B activation was due to
the inhibition of I
B phosphorylation, immunoblotting was used to
examine phosphorylation of I
B
in transiently transfected cells.
When coexpressed with I
B
, Tax induced the appearance of a slower
migrating form of I
B
(Fig. 2,
lane 2), which upon incubation with alkaline
phosphatase regained faster electrophoretic mobility (data not shown).
As demonstrated (29, 48), this electrophoretic mobility change of
I
B
resulted from its phosphorylation at serines 32 and 36, since
mutation of these two serines within I
B
abolished this change
(data not shown). We then examined the effect of the inactive IKKs on
Tax-induced I
B
phosphorylation. Expression of either IKK
(K44M)
or IKK
(K44A) led to a dose-dependent inhibition of the
Tax-induced appearance of the phosphorylated form of I
B
, I
B
-P (Fig. 2, lanes 3-9, middle
panel). Under these conditions, the level of Tax expression
was not significantly influenced (lower panel),
suggesting that the dominant negative effect of the inactive IKKs was
specifically on Tax-induced signal transductions. These findings
strongly suggest that IKK
and IKK
are key components of
Tax-mediated induction of both I
B
phosphorylation and subsequent NF-
B activation.
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Tax Activates IKKs in both Transfected and HTLV-I-infected Human T
Cells--
To examine whether Tax causes IKK activation, we
transfected Jurkat T cells with cDNA expression vectors encoding
hemagglutinin (HA)-tagged IKK or IKK
in the presence or absence
of the Tax expression vector. The transiently expressed IKK
and
IKK
were immunoprecipitated using an anti-HA monoclonal antibody,
and their kinase activity was measured by an in vitro kinase
assay (20, 21) using a substrate containing the N-terminal portion of
I
B
fused to glutathione S-transferase
(GST-I
B
-(1-54)). When expressed alone, IKK
exhibited low
basal kinase activity (Fig.
3A, lane 1, top). However, Tax coexpression led to marked
enhancement in IKK
catalytic activity (lane 2,
top). Additionally, Tax enhanced autophosphorylation of
IKK
(lane 2, top). We similarly
examined whether IKK
also serves as a target of Tax. As previously
reported (21, 22), IKK
exhibited higher levels of basal kinase
activity than IKK
(Fig. 3A, lane 3,
top). However, this isoform of IKK appeared to be less
responsive to Tax than IKK
. Nevertheless, expression of Tax in the
cells did result in significant enhancement of IKK
catalytic
activity (lane 4).
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Activation of IKKs by Tax Correlates with Its Ability to Activate
NF-B--
Prior studies led to identification of Tax mutants that
are either defective or effective in NF-
B activation (49). To
correlate Tax-mediated activation of IKK
with induction of NF-
B,
we examined the effect of two well characterized Tax mutants, M22 and
M47, on IKK
activity. Consistent with its deficiency in NF-
B
activation (49), M22 did not activate IKK
(Fig.
4, lane 3). In
contrast, M47, which is fully effective in NF-
B activation (49),
significantly stimulated IKK
activity and autophosphorylation
(lane 4). Of note, neither the
autophosphorylation nor phosphorylation of I
B
-(1-54) was
detected with a catalytically inactive IKK
mutant, IKK
(K44M), even when it was coexpressed with the wild type Tax (lane
5), suggesting the requirement of IKK
catalytic activity
in these phosphorylation events. Furthermore, Tax-induced IKK
activity was specific for serine 32 and serine 36 of I
B
, since
substitution of these two serines with alanines in the substrate
abolished 32P incorporation (Fig. 4, lane
6, middle). Similar results were obtained with
IKK
, whose catalytic activity was enhanced by both WT Tax and TaxM47
but not influenced by TaxM22 (lanes 8-10). We noticed that TaxM47 consistently induced stronger IKK activity than WT
Tax (compare the middle part of lanes
2 and 8 and lanes 4 and
10). While the underlying mechanism remains to be further investigated, this result is consistent with the finding that M47 is a
stronger NF-
B activator compared with WT Tax (49). Parallel
immunoblotting assays revealed that the kinase activity of IKKs was
well correlated with phosphorylation of I
B
in intact cells,
indicated by its retarded electrophoretic mobility (data not
shown).
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NIK Participates in Tax-induced IB
Phosphorylation and
NF-
B Activation--
Recent studies suggested that a MAP3K-related
kinase, NIK, participates in NF-
B activation induced by cytokines
(34). NIK was suggested to physically interact and activate the IKKs
(18, 19). To investigate the role of NIK in Tax-mediated NF-
B
signaling, we examined the effect of a catalytically inactive form of
NIK (NIK(K429A/K430A)) (34) on Tax-induced I
B
phosphorylation (Fig. 5A). At moderate doses
(0.2-0.5 µg), NIK(K429A/K430A) significantly inhibited Tax-induced
generation of the slower migrating phosphorylated I
B
(I
B
-P)
(Fig. 5A, lanes 3 and 4).
Reporter gene assays demonstrated that NIK(K429A/K430A) also inhibited
Tax-mediated
B-TATA-luc activation (Fig. 5B). Thus, NIK
appears to play an important role in Tax-induced NF-
B
activation.
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DISCUSSION |
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We have shown that Tax activation of NF-B is mediated by an
indirect mechanism, which requires cellular signaling machinery. This
mechanism differs from the one that mediates Tax activation of the
CREB/ATF factors, which involves direct interaction between Tax and
these basic leucine zipper-containing cellular DNA binding proteins
(13-15). Our data demonstrate that the recently cloned I
B kinases,
IKK
and IKK
, are among the cellular signaling molecules participating in Tax-induced NF-
B activation. Catalytically inactive IKKs interfere with both the phosphorylation of I
B
and subsequent NF-
B activation induced by Tax (Figs. 1 and 2). Consistently, Tax
triggers the activation of both IKK
and IKK
, and this is tightly
associated with the site-specific phosphorylation of I
B
in intact
cells. The importance of IKK for Tax-mediated NF-
B induction is
further supported by experiments performed with Tax mutants. A mutant
form of Tax (M22), defective in NF-
B activation, is unable to
trigger IKK activation. On the other hand, M47, which harbors a
mutation selectively affecting the CREB/ATF pathway, is fully active
upon IKK activation (Fig. 4). Thus, Tax-mediated IKK activation is well
correlated with its ability to activate NF-
B. Notably, IKK
activation occurs not only in Tax-transfected cells but also in various
HTLV-I-infected human T-cell lines as well as in human peripheral blood
T cells freshly infected with HTLV-I (Fig. 3), thus confirming the
physiological relevance of our findings. Our findings support the
previous reports that Tax may induce the phosphorylation and
degradation of I
Bs (26-31). We believe that this is a major pathway
by which Tax activates NF-
B.
Tax has also been suggested to directly bind to NF-B proteins and
enhance their transactivation function in the nucleus (50). Although
this proposal does not explain how Tax induces the nuclear translocation of NF-
B, such a mechanism may exist to amplify the
cytoplasmic action of Tax, i.e. activation of IKKs and
induction of NF-
B nuclear expression. We have noticed that the
catalytically inactive IKKs and NIK have more dramatic inhibitory
effects on Tax-induced I
B
phosphorylation (Figs. 2 and
5A) than on Tax-mediated
B reporter activation (Figs.
1A and 5B). Although this difference could be due
to the higher sensitivity of the luciferase assay compared with
immunoblotting, it may also suggest that Tax has a direct
transactivation function in the nucleus. Nevertheless, our results
indicate that activation of IKKs plays a central and critical role in
Tax-induced nuclear expression of NF-
B in both Tax-transfected and
HTLV-I-infected T cells.
The mechanism by which Tax affects IKK activity remains to be further
investigated. However, it appears that IKKs are not the sole target for
Tax. The dominant-negative inhibition assays suggest that NIK is
involved in Tax-induced IKK signaling functions. Recent studies suggest
that NIK serves as an upstream kinase involved in IKK activation (18,
19, 51, 52). NIK physically interacts with IKKs and stimulates their
kinase activity when these proteins are overexpressed together in 293 cells (19). It remains to be tested whether Tax enhances or stabilizes
the interaction between NIK and IKKs. Nevertheless, the ability of Tax
to induce sustained IKK activation clearly explains the basis for
constitutive IB phosphorylation and NF-
B activation in
Tax-expressing and HTLV-I-infected T cells. Given the critical role of
NF-
B in cell growth and survival (53-57), the activation of these
I
B kinases may be an important mechanism by which HTLV-I transforms
host cells. As such, Tax provides a useful model for how a retroviral
regulatory protein transforms host cells by targeting cellular signal
transduction pathways.
During the revision of this paper, similar findings were reported by
Yin et al. (58). However, unlike our study, this report suggests that IKK, but not IKK
, is required for Tax activation of
NF-
B. This discrepancy is likely due to the different experimental strategies and conditions used in these two studies. For example, Yin
et al. used HIV-1 LTR, instead of the
B enhancer, as an
NF-
B reporter. Since the HIV-1 LTR also contains other enhancers,
such as NF-AT, which may respond to Tax (59), the result obtained with
this reporter may not exactly reflect the activity of NF-
B. Nevertheless, we have performed both reporter gene assays (using the
B reporter, Fig. 1) and in vivo and in vitro
I
B
phosphorylation assays (Figs. 2-4), which clearly demonstrate
that both IKK
and IKK
are involved in Tax activation of
NF-
B.
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ACKNOWLEDGEMENTS |
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We thank W. C. Greene and D. Wallach for cDNA expression vectors, R. Grassmann for Tax1 cells, and G. Franchini for HTLV-I-infected cells. The anti-Tax antiserum was kindly provided by the AIDS Research and Reference Program, NIAID, National Institutes of Health.
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FOOTNOTES |
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* This study was supported by U.S. Public Health Service Grant 1 R01 CA68471-03 (to S.-C. S.) and ES06376-05 (to M. K.).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.
§ These authors contributed equally to this work.
¶ Supported by National Institutes of Health (NIH) postdoctoral fellowship F32 CA 71108-01A1.
Supported by NIH predoctoral training Grant 5 T32 CA
6039-5.
Supported by a postdoctoral fellowship from the American Cancer
Society, California Division.
§§ A scholar of the American Society for Hematology. To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Pennsylvania State University College of Medicine, 500 University Dr., Hershey, PA 17033. Fax: 717-531-6522; Tel.: 717-531-4164; E-mail: sxs70{at}psu.edu.
The abbreviations used are:
HTLV-I, human T-cell
leukemia virus type I; LTR, long terminal repeat; IKK, IB kinase; NIK, NF-
B-inducing kinase; GST, glutathione
S-transferaseCREB, cAMP response element-binding proteinIL, interleukinHA, hemagglutininKA, kinase assay(s)WT, wild
type.
2 E. W. Harhaj and S. C. Sun, unpublished data.
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REFERENCES |
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