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Molecular and Cellular Biology, September 1998, p. 5157-5165, Vol. 18, No. 9
Gladstone Institute of Virology and
Immunology1 and
Departments of Medicine,
Microbiology, and Immunology4 University of
California, San Francisco, San Francisco, California 94141-9100;
Department of Biochemistry and Cell Biology, State
University of New York at Stony Brook, Stony Brook, New York
11794-52153; and
Central Research
Division, Pfizer Inc., Groton, Connecticut
063402
Received 20 January 1998/Returned for modification 7 March
1998/Accepted 2 June 1998
Tax corresponds to a 40-kDa transforming protein from the
pathogenic retrovirus human T-cell leukemia virus type 1 (HTLV-1) that
activates nuclear expression of the NF- Human T-cell leukemia virus type 1 (HTLV-1) represents the first pathogenic human retrovirus identified
(49, 78). After a long latency period, approximately 0.05 to
0.1% of individuals infected with this retrovirus develop an
often aggressive and fatal proliferation of activated
CD3+ CD4+ T cells, termed adult
T-cell leukemia (ATL) (66). HTLV-1 has also been
etiologically linked with a progressive neurodegenerative syndrome termed HTLV-1-associated myelopathy/tropical
spastic paraparesis (13, 46).
HTLV-1 encodes a 40-kDa regulatory protein termed Tax that appears to
play a central role in cell transformation (18, 36, 50, 58, 64,
74). Although Tax does not bind directly to DNA, it alters the
activity of several host transcription factors, notably cyclic AMP
responsive element (CRE) binding protein (CREB)/activating transcription factor (ATF), serum response factor (SRF), and
NF- Tax also modulates transcription of various cellular genes, some of
which may be involved in T-cell transformation (19, 57, 77,
79). Many of these genes are regulated by members of the
NF- The prototypical NF- The molecular basis for Tax induction of NF- However, a convincing series of studies has shown that Tax promotes the
phosphorylation and degradation of both I Three cellular kinases involved in TNF- The studies described in this report were aimed at identifying the
cellular signaling intermediates involved in HTLV-1 Tax induction of
nuclear NF- Expression vectors, biological reagents, and cell lines.
Plasmids pCMV4-HA-I
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human T-Cell Leukemia Virus Type 1 Tax Induction of
NF-
B Involves Activation of the I
B Kinase
(IKK
) and
IKK
Cellular Kinases
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ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B/Rel family of transcription factors by an unknown mechanism. Tax expression promotes N-terminal phosphorylation and degradation of I
B
, a principal
cytoplasmic inhibitor of NF-
B. Our studies now demonstrate that
HTLV-1 Tax activates the recently identified cellular kinases I
B
kinase
(IKK
) and IKK
, which normally phosphorylate I
B
on both of its N-terminal regulatory serines in response to tumor
necrosis factor alpha (TNF-
) and interleukin-1 (IL-1) stimulation.
In contrast, a mutant of Tax termed M22, which does not induce NF-
B, fails to activate either IKK
or IKK
. Furthermore, endogenous IKK
enzymatic activity was significantly elevated in
HTLV-1-infected and Tax-expressing T-cell lines. Transfection
of kinase-deficient mutants of IKK
and IKK
into either
human Jurkat T or 293 cells also inhibits NF-
B-dependent reporter
gene expression induced by Tax. Similarly, a kinase-deficient
mutant of NIK (NF-
B-inducing kinase), which represents an upstream
kinase in the TNF-
and IL-1 signaling pathways leading to IKK
and
IKK
activation, blocks Tax induction of NF-
B. However, plasma
membrane-proximal elements in these proinflammatory cytokine pathways
are apparently not involved since dominant negative mutants of the
TRAF2 and TRAF6 adaptors, which effectively block signaling through the
cytoplasmic tails of the TNF-
and IL-1 receptors, respectively, do
not inhibit Tax induction of NF-
B. Together, these studies
demonstrate that HTLV-1 Tax exploits a distal part of the
proinflammatory cytokine signaling cascade leading to induction of
NF-
B. The pathological alteration of this cytokine pathway
leading to NF-
B activation by Tax may play a central role in
HTLV-1-mediated transformation of human T cells, clinically manifested
as the adult T-cell leukemia.
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B/Rel (79). Tax-induced transactivation of the HTLV-1
long terminal repeat (LTR) is mediated through the CREB/ATF pathway
(5, 15, 63). In this response, Tax participates in the
formation of a ternary complex that includes the viral CREs and CREB,
appears to enhance CREB dimerization, and stabilizes CREB binding to
the CREs (3, 6, 48, 63, 69, 81). Additionally, the
indirectly tethered Tax protein facilitates recruitment of the
transcriptional coactivator CREB binding protein (CBP), through an
interplay with the KIX domain of CBP (16, 28, 76).
B/Rel family of transcription factors. For example, Tax induces
various cytokines and the interleukin-2 (IL-2) receptor
-chain gene
via the NF-
B pathway (19, 57, 77). Tax also induces the
c-fos oncogene through SRF (11, 12). Of note, Tax
induction of the CREB/ATF, SRF, and NF-
B/Rel pathways is selectively
impaired by the introduction of site directed mutations into Tax
(36, 55, 56, 74). For example, the M22 mutant of Tax (T130S
L131A) retains CREB/ATF-stimulatory activity but is strongly impaired
in its ability to activate NF-
B (56). These findings
raise the possibility that different subregion domains of Tax mediate
the activation of these various host transcription factor pathways. The
precise role of each of these pathways in Tax-induced cellular
transformation remains unresolved (36, 58, 74).
B complex corresponds to a heterodimer of
the p50 (NFKB1) and RelA (p65) members of the NF-
B/Rel
family of transcription factors (1, 2, 68). Other factors in this family include RelB, c-Rel, and p52 (NFKB2). Prior to
activation, NF-
B is sequestered in the cytoplasm by its physical
interaction with a set of inhibitors termed the I
Bs (I
B
,
I
B
, and I
B
), p105 (NFKB1), and p100
(NFKB2) (1, 2, 68). Stimulation of the cell with
proinflammatory cytokines (e.g., tumor necrosis factor alpha
[TNF-
] and interleukin-1 [IL-1]), phorbol esters, lipopolysaccharide, and several other activators leads to the phosphorylation of I
B
on two N-terminal serine residues located at positions 32 and 36. This posttranslational modification targets the
inhibitor for rapid ubiquitination and degradation within the 26S
proteasome. The proteolysis of cytoplasmic I
B
allows nuclear
translocation of NF-
B (1, 2, 68). How cytoplasmic sequestration of NF-
B by the p100 and p105 inhibitors is relieved remains largely unknown.
B has been a topic of
extensive investigation. Tax has been reported to interact physically
with many members of the NF-
B/Rel family (2, 21, 79) but
appears to display highest affinity for the p100 product of the
NFKB2 gene (4, 26, 31, 44). The amino terminus of
p100, which contains p52 and its Rel homology domain, appears to
mediate this interaction (4, 26, 44). Expression of Tax has
also been reported to overcome the cytoplasmic sequestration property
of p100 and p105, permitting the release and nuclear translocation of
NF-
B (26, 43, 44).
B
and I
B
, suggesting that this HTLV-1 regulatory protein may induce nuclear translocation of NF-
B by acting prior to or at the level of I
B phosphorylation (7, 17, 24, 29, 39, 62). This notion is
further strengthened by the finding that degradation-resistant I
B
mutants lacking the two N-terminal serine phosphorylation sites
effectively block Tax-induced
B-dependent transcription (7,
25). Together, these findings suggest that Tax may coopt a
signaling pathway that leads to I
B phosphorylation.
and IL-1 induction of
NF-
B have recently been identified. These include the
NF-
B-inducing kinase (NIK), a mitogen-activated protein kinase
kinase kinase (MAP3K)-related kinase that associates with various
TNF receptor (TNFR)-associated factors (TRAFs) and corresponds to
a strong inducer of NF-
B. When rendered catalytically inactive, NIK
functions as a dominant-negative inhibitor of TNF-
- and IL-1-induced
NF-
B activation (35, 59). However, NIK does not directly
phosphorylate the I
Bs but instead associates with and activates
(51) the recently recognized I
B kinase
(IKK
)
(10, 40, 51), previously designated the conserved
helix-loop-helix ubiquitous kinase (9). IKK
has been
shown to phosphorylate both N-terminal serines of I
B
and
preferentially serine 23 of I
B
(10, 39, 50). A second
related I
B kinase, termed IKK
, has also been isolated and
characterized (40, 72, 80). This enzyme phosphorylates the
regulatory serines of both I
B
and I
B
. These
and
IKKs can form hetero- and homodimers (40, 72, 80) via their
leucine zipper domains, although heterodimers appear to be favored in vivo.
B expression. We demonstrate that Tax activates the
enzymatic activity of ectopically expressed IKK
and IKK
and that
the enzymatic activity of endogenous IKKs is elevated in
HTLV-1-infected and Tax-expressing T-cell lines. In addition, we report
that kinase-deficient versions of NIK, IKK
, and IKK
effectively block Tax action. In contrast, based on the use of dominant
negative mutants, Tax induction of NF-
B does not appear to involve
the TRAF2 and TRAF6 adaptor proteins which represent more proximal
components of the TNF
and IL-1 signaling pathways. These findings
suggest that distal components of the proinflammatory cytokine
signaling pathway are selectively coopted by Tax, leading to
the activation of NF-
B.
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
B
(SS32/36AA),
B-TATA-luciferase, pCMV4Tax,
and pCMV4TaxM22 have been previously described (4, 61). The
HTLV-1 LTR luciferase reporter (pLuc HTLV-1 LTR) was generated by
introducing the full-length HTLV-1 LTR into the pGL2-Basic vector
(Promega, Madison, Wis.). The Rous sarcoma virus LTR-driven LacZ
reporter construct (6RZ) was obtained from D. Pearce (University of
California, San Francisco) and has been previously described (47). The wild-type FLAG-TRAF2 and dominant negative
FLAG-TRAF2(87-501) expression plasmids were provided by D. V. Goeddel (Tularik Inc., South San Francisco, Calif.). The expression
vector for the N-terminal truncated constitutively active MEKK1 was
provided by G. L. Johnson (National Jewish Medical and Research
Center, Denver, Colo.). The Myc-PAK1 K299R expression plasmid was
provided by G. M. Bokoch and U. Knaus (Scripps Research Institute,
La Jolla, Calif.). The FLAG-JNK1 expression vector was provided by M. Karin (University of California, San Diego).
was amplified by PCR from a previously described vector
(9) and subcloned either into pEV3S (37),
in frame with a C-terminal T7 tag to generate
pEV-IKK
-T7, or into pcDNA 3.1 (Invitrogen, Carlsbad, Calif.), in
frame with a C-terminal hemagglutinin (HA) epitope tag to generate
pcDNA-IKK
-HA. The human IKK
cDNA was amplified by PCR using
a 3' oligonucleotide encoding a FLAG epitope tag and subcloned into
pcDNA3.1 to generate pcDNA-IKK
-FLAG. The TRAF6(289-522) and
NIK cDNAs were generated by reverse transcription-PCR using Jurkat E6-1
mRNA; the TRAF2 cDNA was amplified by PCR from the vector described
above. Each of these cDNAs was then subcloned in frame with an
N-terminal Myc epitope tag in the pRK6 vector. The kinase-deficient
K44M IKK
, K44A IKK
, and KK429/430AA NIK mutants were generated by PCR.
and IL-1
were purchased from Endogen
(Cambridge, Mass.). The 293 human embryonic kidney and HeLa cell lines
were maintained in Dulbecco modified Eagle medium supplemented with
10% heat-inactivated fetal bovine serum and antibiotics. Jurkat E6-1
cells as well as the HTLV-1-infected T-cell lines C8166 and HUT102 were
maintained in RPMI 1640 medium supplemented with 10% heat-inactivated
fetal bovine serum and antibiotics. The previously described
(70) Jurkat-Tax and Jurkat-anti-Tax cell lines were
maintained in the same medium.
Transfections and reporter assays.
293 cells (7 × 105 cells per well) or HeLa cells (2 × 105 cells per well) were seeded into six-well
(35-mm-diameter) plates and transfected the following day with 4 µg
of DNA by the calcium phosphate precipitation method (53).
Transfection of Jurkat E6-1 cells (2 × 106) was
performed as follows. Cells were incubated with 4.25 µg of DNA and 8 µl of DMRIE-C (Gibco-BRL Life Technologies, Gaithersburg, Md.) for
4 h in serum-free medium (OPTIMEM; Gibco-BRL Life Technologies) in
35-mm-diameter wells. Cells were then suspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum. Luciferase activity was determined 18 to 24 h later, using an enhanced
luciferase assay kit and a Monolight 2010 luminometer (Analytical
Luminescence Laboratory, Ann Arbor, Mich.). All transfections included
the 6RZ plasmid to allow normalization for differences in gene transfer efficiency by assay of
-galactosidase activity.
Immune complex kinase assays.
For the IKK assays, HeLa cells
were transfected by the calcium phosphate precipitation method and
lysed 24 to 48 h posttransfection in a buffer containing 1%
Nonidet P-40, 250 mM NaCl, 50 mM HEPES (pH 7.4), 1 mM EDTA, and the
protease inhibitors phenylmethylsulfonyl fluoride (1 mM), antipain
(5 µg/ml), aprotinin (5 µg/ml), leupeptin (5 µg/ml),
pepstatin (0.5 µg/ml), bestatin (7.5 µg/ml),
phosphoroamidon (4 µg/ml), and trypsin inhibitor (5 µg/ml).
Lysates were immunoprecipitated with either anti-HA monoclonal antibody
covalently linked to Sepharose (BABCO, Richmond, Calif.) or anti-FLAG
M2 antibody covalently attached to agarose (Eastman Kodak Company, New
Haven, Conn.). Immunoprecipitates were washed three times in lysis
buffer and then once in kinase buffer containing 10 mM HEPES (pH 7.4),
1 mM MnCl2, 5 mM MgCl2, 12.5 mM
-glycero-2-phosphate, 50 µM Na3VO4, 2 mM
NaF, 50 µM dithiothreitol, and 10 µM ATP. To assess endogenous IKK
/
enzymatic activity, 25 × 106 cells were
lysed and immunoprecipitated with the anti-IKK
antibody H744 (Santa
Cruz Biotechnology, Santa Cruz, Calif.), which cross-reacts with
IKK
. After suspension in 20 µl of kinase buffer, the
immunoprecipitates were incubated with 5 µCi of
[
-32P]ATP (6,000 Ci/mmol) and 1 µg of
recombinant glutathione S-transferase (GST)-I
B
(1-62) as an exogenous substrate for 30 min at
30°C. The kinase reactions were terminated by the addition of sodium dodecyl sulfate (SDS) sample buffer. The samples were analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) followed by transfer to
nitrocellulose membranes and exposure to Hyperfilm MP (Amersham Life
Sciences). The membranes were subsequently probed with either anti-HA,
anti-FLAG M2, or anti-IKK
H744 antibody to determine the amount of
immunoprecipitated kinase. Cell lysates were immunoblotted with
peptide-specific anti-Tax antibodies to assess the levels of Tax
protein expression. The JNK1 kinase assays were performed in a similar
fashion except that (i) 293 cells were transfected with FLAG-JNK1 and
(ii) GST-c-Jun(1-79) (Santa Cruz Biotechnology) was used as an
exogenous substrate in the kinase reaction.
| |
RESULTS |
|---|
|
|
|---|
Signaling pathways leading to JNK and NF-
B activation.
A
summary of the recognized signaling pathways leading to NF-
B and
c-Jun activation is schematically presented in Fig.
1. TRAF2 and TRAF6 represent proximal
signaling intermediates that are recruited to the cytoplasmic tail of
the type 1 TNFR (TNFR1) and the type 1 IL-1 receptor (IL-1R1),
respectively, following ligand binding (8, 22). TRAF2 is
recruited through an interaction with TRADD, while TRAF6 appears to
associate with MyD88 and IRAK (IL-1R-associated kinase) (8, 22,
45, 71). Both of these TRAFs are capable of engaging NIK, a
MAP3K-related kinase, which thus forms a nexus in these two
proinflammatory cytokine signaling pathways leading to NF-
B
activation (35, 59). In turn, NIK associates with and
activates IKK
and -
(51, 72). The IKKs phosphorylate
the I
Bs on two N-terminal serine residues (10, 40, 51, 72,
80), leading to the ubiquitination and degradation of these
inhibitors in the 26S proteasome (1, 2, 68). MEKK1
corresponds to a second MAP3K that forms a key signaling intermediate
in the TNFR1 pathway leading to JNK activation. MEKK1 is also capable
of activating NF-
B when ectopically expressed, but its role in
TNF-
induction of this transcription factor remains controversial
(20, 32, 34, 59). PAK1 corresponds to a kinase that is
activated by small GTPases of the Rho family that has also been
shown to activate JNK (27). The precise involvement of PAK1
in these signaling pathways is not yet clear. Finally, pp90rsk is a phorbol ester-activated kinase,
positioned downstream of the extracellular response kinases (ERKs)
(60), that has been shown to directly phosphorylate I
B
on serine 32 in vitro and to induce degradation of this inhibitor
(14, 54). We have explored the participation of several of
these signaling components in the HTLV-1 Tax response leading to
NF-
B induction.
|
The I
B
SS32/36AA mutant blocks HTLV-1 Tax induction of
NF-
B in 293 cells.
As a starting point in our studies, we
verified that a constitutive repressor mutant of I
B
(the
SS32/36AA mutant), in which serines 32 and 36 were substituted with
alanines, effectively blocked Tax induction of
B-dependent
luciferase activity in human 293 cells. These cells were cotransfected
with a Tax expression vector and increasing amounts of the I
B
SS32/36AA mutant (25 to 400 ng of expression plasmid [Fig.
2A]). In these cells, the I
B
SS32/36AA mutant functioned as a potent inhibitor of Tax-induced NF-
B expression. This finding confirms for 293 cells the results of
prior studies performed with different cell types (7, 25) and suggests that Tax activates NF-
B by entering a signaling pathway
prior to or at the level of I
B
.
|
Kinase-deficient mutants of IKK
and IKK
block HTLV-1 Tax
induction of NF-
B in 293 cells.
The effects of both wild-type
and kinase-deficient mutants of IKK
(K44M) and IKK
(K44A) on
Tax-induced NF-
B expression were next assessed in 293 cells. Since
the wild-type IKKs were included as controls for their kinase-deficient
mutant counterparts, the functional effects of wild-type IKK
and
IKK
were first evaluated (Fig. 2B). IKK
produced only a twofold
increase in
B-luciferase activity at the highest dose of expression
vector (400 ng), while IKK
mediated a sevenfold increase in
luciferase activity at the same dose of expression vector (400 ng).
(K44M) or IKK
(K44A) kinase-deficient expression vector produced a dose-dependent suppression of the Tax response (Fig. 2C). In contrast, addition of increasing amounts of the wild-type IKK
and IKK
expression vectors did not inhibit the Tax response and in some cases
produced a slight amplification of the response. Immunoblotting of the
cell lysates confirmed the presence of comparable levels of
Tax protein expression in each of the transfection conditions (Fig. 2C,
inset), eliminating suppressive effects of the IKK
and IKK
kinase-deficient mutants on Tax expression as the cause of the observed
inhibition.
Kinase-deficient IKK
and IKK
mutants inhibit HTLV-1
Tax-mediated activation of NF-
B in Jurkat T cells.
Since
HTLV-1 preferentially infects human T cells, which in some
cases leads to ATL, the effects of the kinase-deficient mutants of
IKK
and IKK
on Tax induction of
B-luciferase activity were evaluated in human Jurkat T cells (Fig.
3). As a control for these experiments,
the effects of these IKK mutants on Tax-mediated activation of the
HTLV-1 LTR, which involves the CREB/ATF rather than NF-
B/Rel family
of transcription factors, was evaluated in parallel. Transfection of
escalating doses (0.1, 0.3, and 1 µg) of the expression vectors
encoding either IKK
(K44M) or IKK
(K44A) resulted in
dose-dependent inhibition of Tax activation of
B-luciferase activity
(Fig. 3). In contrast, over these same doses, the kinase-deficient IKK
mutants produced no significant inhibition of Tax-induced HTLV-1
LTR luciferase activity. These results suggest that Tax induction of
NF-
B in human Jurkat T cells, as in 293 cells, involves the
participation of IKK
and IKK
.
|
HTLV-1 Tax activates IKK
and IKK
enzymatic activity.
To
independently assess whether HTLV-1 Tax induction of NF-
B
proceeds through IKK
and IKK
, we examined whether
Tax, like TNF-
, stimulates the enzymatic activity of IKK
and
IKK
(Fig. 4). We chose to perform
these experiments with HeLa cells because of a failure to observe
induction of IKK enzymatic activity by either Tax or TNF-
in 293 cells. This result most likely reflects a high constitutive enzymatic
activity due to overexpression of these kinases. Thus, HeLa cells were
cotransfected with expression vectors encoding either IKK
or IKK
and wild-type Tax or the M22 mutant of Tax. M22 Tax fails to induce
NF-
B expression; however, it continues to activate the HTLV-1 LTR
via the CREB/ATF pathway, indicating that it is not completely
misfolded (55). As an additional control, selected cell
aliquots were stimulated with TNF-
for 10 min. Kinase activity for
IKK
(Fig. 4A) and IKK
(Fig. 4B) was assessed in in vitro kinase
assays involving immunoprecipitation of the kinases and the addition of
recombinant GST-I
B
(1-62) as an exogenous substrate. As shown
in Fig. 4A and B, wild-type Tax, like TNF-
, but not M22 Tax
activated the enzymatic activity of the IKK
and IKK
proteins. The
levels of immunoprecipitated IKK
and IKK
, as well as the levels
of Tax in the cell lysates, in each transfection condition are also
shown. Of note, the M22 Tax mutant was consistently expressed at lower
levels than wild-type Tax; however, when comparable levels of protein
expression were examined (for example, wild-type Tax at 0.1 µg and
M22 Tax at 1 µg), wild-type Tax significantly activated the enzymatic
activity of IKK
compared with the M22 Tax mutant (Fig. 4A).
Similarly, when 1.8 µg of wild-type Tax was compared to 5.4 µg of
M22 Tax, a significant induction in IKK
enzymatic activity was
observed in response to Tax expression (Fig. 4B). Utilization of a
GST-I
B
substrate containing alanine substitutions for serine at
positions 32 and 36 demonstrated that the measured Tax response
involved phosphorylation at the two N-terminal serine residues of
I
B
(data not shown). Together, these findings demonstrate that
like the proinflammatory cytokine TNF-
, HTLV-1 Tax, but not M22 Tax, augments the enzymatic activity of both IKK
and IKK
.
|
Endogenous IKKs are enzymatically more active in HTLV-1-infected
T-cell lines and Jurkat T cells expressing Tax.
To assess whether
Tax also activates the endogenous IKK
and IKK
enzymes, we
analyzed the HTLV-1-infected HUT102 or C8166 T-cell lines and Jurkat T
cells engineered to stably express Tax or antisense Tax
(70). Endogenous IKK
and IKK
were immunoprecipitated from these cells with antibody H744, which cross-reacts with both enzymes, and analyzed in an in vitro kinase assay (Fig. 4C). Although somewhat less IKK
/
was immunoprecipitated from HUT102 and C8166 cells than from Jurkat E6-1 cells, both HTLV-1-infected cell lines displayed significantly higher levels of endogenous IKK
/
enzymatic activity. Indeed these levels of activity exceeded that
obtained in Jurkat E6-1 cells stimulated with TNF-
. While the HUT102
and C8166 cells expressed large amounts of Tax (Fig. 4C), it was
possible that other differences in these cells from Jurkat T cells were responsible for the altered IKK
/
activity. Accordingly, we
compared Jurkat-Tax and Jurkat-anti-Tax cell lines, which are identical except for the presence of Tax (70). Although expressing
comparable amounts of IKK
/
, the Jurkat-Tax cells displayed
moderately increased IKK
/
enzymatic activity relative to the
Jurkat-anti-Tax cell line (Fig. 4D). The small increase in IKK activity
paralleled the relatively low level of Tax expression observed in the
Jurkat-Tax cells (Fig. 4D). These results demonstrate that IKK
/
enzymatic activity is constitutively elevated in T-cell lines
expressing HTLV-1 Tax.
Kinase-deficient mutants of NIK, but not PAK1, inhibit HTLV-1
Tax induction of NF-
B.
The potential involvement of NIK in Tax
induction of NF-
B was next evaluated. As noted, NIK represents the
next upstream component of the TNF-
and IL-1 signaling pathways
(Fig. 1). When introduced into 293 cells alone, NIK functioned as a
potent activator of
B-luciferase activity (Fig.
5A) as previously reported
(35). Cotransfection of 293 cells with Tax and increasing
amounts of the kinase-deficient NIK mutant (KK429/430AA) produced a
dose-dependent inhibition of
B-luciferase reporter activity in these
cells (Fig. 5B). In contrast, cotransfection of matching amounts of the
wild-type NIK expression vector led to enhancement rather than
inhibition of the Tax response. Immunoblotting of the cell lysates
verified the presence of comparable levels of Tax in the presence of
the NIK KK429/430AA mutant (Fig. 5B, inset).
|
B activation is unknown. Cotransfection of increasing
amounts of a kinase-deficient mutant (K299R) of PAK1 had no effect on
Tax induction of NF-
B (Fig. 5D). Blotting of the cellular lysates confirmed dose-related expression of PAK1 and comparable levels of Tax
expression (Fig. 5D, inset).
The effects of the kinase-deficient NIK and PAK1 mutants on Tax induced
NF-
B expression were also assessed in Jurkat T cells. The NIK
KK429/430AA mutant produced dose-dependent inhibition of Tax-induced
B-luciferase activity but had essentially no effect on Tax induction
of HTLV-1 LTR luciferase activity, which occurs independently of
NF-
B (Fig. 5C). As observed in 293 cells, the kinase-deficient PAK1
K299R mutant did not inhibit
B-luciferase activity induced by Tax,
nor did it alter the Tax induction of the HTLV-1 LTR (Fig. 5E).
The lack of effect of the kinase-deficient PAK1 mutant further supports
a specific effect for the kinase-deficient NIK mutant. Together, these
findings raise the possibility that Tax action proceeds through the
activation of NIK enzymatic activity. However, the observed inhibitory
effects of the NIK KK429/430AA mutant observed in both 293 and
Jurkat T cells could also result from its binding to and inactivation
of IKKs, making these latter kinases unresponsive to Tax. These two
possibilities could be distinguished by assessing the effects of Tax on
NIK enzymatic activity. However, our studies indicate that ectopically
expressed NIK is constitutively active and not induced further by
TNF-
, TRAF2, or Tax (data not shown). Resolution of this issue thus
awaits the development of an immunoprecipitating anti-NIK antibody
that will allow assessment of endogenous NIK activity in the presence
and absence of Tax.
Dominant negative mutants of TRAF2 and TRAF6 fail to inhibit
HTLV-1 Tax induction of NF-
B.
TRAF2 interacts with TRADD
and activates NF-
B presumably through its interaction with NIK
(35, 52). An amino-terminal deletion mutant of TRAF2
[TRAF2(87-501)], lacking its RING finger domain, functions as a
dominant negative inhibitor in the TNF-
/TNFR1 signaling pathway,
blocking both NF-
B and JNK activation (22, 34, 59). When
cotransfected with Tax, the dominant negative TRAF2 mutant did not
alter Tax activation of NF-
B (Fig. 6B). However, in agreement with
prior studies (22), this same mutant significantly inhibited
the TNF-
-induced NF-
B response (Fig. 6A). This result suggests that Tax enters
the TNF-
signaling pathway downstream of TRAF2 or perhaps through a
different TRAF. In this regard, TRAF6 is recruited to IL-1R1 by the
MyD88 adaptor and a serine/threonine kinase termed IRAK (8, 45,
71). To rule out that Tax activates NF-
B via TRAF6, the
amino-terminal deletion mutant TRAF6(289-522) was cotransfected with
Tax. While the TRAF6 dominant negative mutant effectively blocked
IL-1 signaling (Fig. 6C) (8), no significant
inhibitory effects were obtained for Tax induction of NF-
B (Fig.
6D). These results indicate that Tax induction of NF-
B involves
neither TRAF2 nor TRAF6 and suggest that Tax enters this
proinflammatory cytokine signaling pathway downstream of the site of
action of these TRAFs.
|
HTLV-1 Tax is a weak inducer of JNK1 enzymatic activity in 293 cells.
MEKK1 represents another kinase of the MAP3K level that
acts immediately downstream of TRAF2 (34, 59). MEKK1 acting
through the stimulation of MKK4 is a critical signaling intermediate
for TNF-
activation of JNK, which in turn phosphorylates the
transcription factor c-Jun (41, 75). MEKK1 has also been
reported to activate NF-
B when ectopically expressed, but its role
in the NF-
B arm of the TNF-
signaling pathway (Fig. 1) is
controversial (20, 32, 34, 59). Having established that Tax
enters the TNF-
/TNFR1 pathway downstream of TRAF2 and perhaps at the
level of NIK, we next explored whether the MEKK1-MKK4-JNK axis (Fig. 1)
was activated by Tax. For these studies, we examined whether
HTLV-1 Tax activated JNK1 enzymatic activity. 293 cells were
transfected with both
B-luciferase and FLAG-JNK. Cotransfection of
an N-terminally truncated MEKK1 mutant (30) which is
constitutively active led to substantial activation of JNK1 as assessed
by phosphorylation of GST-c-Jun(1-79) substrate in vitro (Fig.
7A). At the highest dose of MEKK1
expression vector (2 µg), NF-
B was also activated (Fig. 7B).
Cotransfection of Myc-TRAF2 or treatment of cells with TNF-
for
30 min also led to enhanced JNK1 activity (Fig. 7A). Myc-TRAF2
expression as well as treatment of cells with TNF-
for 6 h also
led to NF-
B activation (Fig. 7B). In contrast, cotransfection of Tax
caused only a modest induction of JNK1 activation compared to the
other agonists despite causing substantial NF-
B activation in these
same transfected cells (Fig. 7). This finding is consistent with a
previous report demonstrating a similar modest induction of JNK1
enzymatic activity by Tax in other cell types (23). These
results suggest that in contrast to its ability to activate NF-
B,
Tax does not significantly activate the MEKK1-MKK4-JNK1 pathway.
|
| |
DISCUSSION |
|---|
|
|
|---|
To identify the molecular mechanism(s) underlying HTLV-1
Tax induction of NF-
B, we have explored the potential
involvement of several components of the TNF-
and IL-1
signaling pathways. These studies have revealed that
kinase-deficient forms of IKK
, IKK
, and NIK, but not PAK1,
effectively inhibit Tax induction of NF-
B in both human 293 and Jurkat T cells. Further, these investigations have demonstrated
that wild-type Tax enhances both IKK
and IKK
enzymatic
activities. In contrast, the M22 Tax mutant, which manifests a specific
defect in NF-
B activation, does not significantly stimulate these
kinases. Additionally, we have found that the endogenous IKK
and
IKK
enzymes present in HTLV-1-infected and Tax-expressing T-cell
lines are quite active. Further, we have detected Tax activation of
endogenous IKK
/
in Jurkat T cells stably expressing Tax compared
with matched Jurkat anti-Tax controls. These results thus significantly
extend our prior study in which we demonstrated the involvement of
pp90rsk in phorbol ester- but not Tax-induced
phosphorylation and degradation of I
B
(14). These
findings indicate that the HTLV-1 transactivator protein activates
only a subset of the cellular kinases capable of phosphorylating
I
B
. The ability of Tax to activate the IKKs appears to be quite
specific since a parallel pathway which is initiated by TRAF2 and
involves MEKK1-MKK4 activation of JNK1 does not appear to be
significantly induced by Tax. Thus, although MEKK1 has been proposed as
an inducer of NF-
B, it seems unlikely that Tax activation of NF-
B
proceeds through MEKK1. For this to occur, Tax would have to activate
MEKK1 in a manner not leading to standard JNK1 activation.
The ability of a kinase-deficient form of NIK to block Tax induction of
NF-
B raised the distinct possibility that Tax action involved
this component of the TNF-
and IL-1 signaling cascade. However,
the high constitutive activity of this enzyme obtained following its
ectopic expression coupled with a lack of response of the kinase to
TNF-
, TRAF2, or Tax precluded firm assignment of this enzyme to the
intracellular pathway utilized by Tax. The involvement of NIK in this
reaction could be evaluated if an active inducible form of endogenous
NIK could be immunoprecipitated from cells expressing Tax.
Unfortunately, reagents to perform such an experiment are not available
at the present time. In addition, we cannot completely exclude the
involvement of yet another MAP3K in the Tax response whose activity is
inhibited by expression of kinase-deficient NIK. However, it is
interesting that both Tax and NIK possess similar properties with
regard to activation of IKKs and JNK. Specifically, Tax (Fig. 4), like
NIK (51, 72), activates both IKKs, while neither of these
proteins significantly activates JNK1 (23, 59, 73) (Fig. 7).
While Tax utilizes the IKK
, IKK
, and perhaps NIK components of
the TNF-
and IL-1 signaling pathways, more proximal components of these pathways, specifically TRAF2 and TRAF6, do not appear to be
involved in Tax induction of NF-
B. This conclusion is based on
the fact that dominant negative mutants of TRAF2 and TRAF6, which
effectively block TNF-
and IL-1 signaling, respectively, fail to
inhibit Tax induction of NF-
B. These findings further suggest that
Tax does not activate NF-
B via the induced transcription and
autocrine action of TNF-
or IL-1. However, our results do not
formally exclude the possibility that Tax activates the expression of
another cytokine that is secreted and binds to a receptor that functions independently of TRAF2 and TRAF6, leading to the stimulation of NIK and IKKs.
It remains unknown whether Tax subverts the TNF-
/IL-1 signaling
pathway by directly interacting with and activating NIK and/or IKK
or IKK
or by indirectly altering the expression levels of various
components of this pathway. However, enhanced levels of IKKs in
HTLV-1 Tax-expressing cell lines is unlikely to be the cause of
increased NF-
B activity since the levels of endogenous IKKs were
comparable to levels in cells that did not express Tax (Fig. 4C and D).
A prior report indicating that Tax induces nuclear NF-
B
expression in the absence of de novo protein synthesis further argues
against an indirect effect involving new gene transcription (33). Although Tax has been reported to be principally a
nuclear protein, it is also present in the cytoplasm (4, 26,
43). Indeed, Tax avidly binds to the p100 product of the
NF-
B2 gene, an exclusively cytoplasmic member of the
I
B family of proteins (4, 26, 31, 44). This interaction
may thus ensure a cytoplasmic pool of Tax to mediate subsequent
activation of IKK
, IKK
, and perhaps NIK.
If Tax does in fact act in a direct manner at the level of NIK, our
findings raise the intriguing possibility that Tax functions as a
TRAF-like molecule. The TRAFs may act by producing oligomerization of
the downstream kinase, leading to altered enzymatic activity. In
this regard, the M22 mutant of Tax, which fails to activate IKK
and IKK
catalytic activities, also fails to dimerize
(65). Alternatively, Tax may function through an as yet
unidentified TRAF to promote aggregation of NIK. Indeed, biological
precedence exists for such a mechanism. Specifically, the cytoplasmic
domain of the transforming LMP1 protein encoded by Epstein-Barr virus has been shown to associate with TRAFs and to induce NF-
B
(42). It has been suggested that LMP1 causes aggregation of
TRAFs and may mimic the effects of TNF binding to its trimeric receptor (42).
It is also possible that Tax induces nuclear NF-
B expression by
altering the levels of endogenous NIK. In this regard, NIK expression
levels have been shown to correlate with NF-
B activation (35) (Fig. 5A). Finally, it is possible that Tax induces the expression of an unknown activator of the NIK or IKK enzymes or perhaps
down-regulates the expression or activity of an inhibitor of these
kinases such as protein phosphatase 2A (10). Future studies
will help delineate which of these mechanisms of action is utilized by
Tax.
In terms of the biology of HTLV-1 infection, Tax induction of
NF-
B in virally infected T cells may play a central role in cellular
transformation leading to the ATL. One possible role for Tax-induced
NF-
B expression would be to inhibit apoptosis in infected T cells.
In this regard, it has recently been shown that NF-
B expression is
induced by transforming Ras mutants and that NF-
B production
inhibits a p53-independent pathway of apoptosis in these cells
(38).
In conclusion, our studies reveal how HTLV-1 Tax subverts specific
plasma membrane-distal components of the physiological proinflammatory
signaling pathway leading to the pathologically sustained induction of
NF-
B. These findings thus provide new insights into the molecular
basis of Tax action, emphasizing its ability to directly or indirectly
alter the activity of cytoplasmic kinases pivotally involved in
signal-coupled degradation of I
B. Should the abnormal expression of
NF-
B play a central role in HTLV-1-associated leukemogenesis,
the development of inhibitors of NIK or IKK
and IKK
might serve
not only as anti-inflammatory agents but also as novel
chemotherapeutics potentially active against HTLV-1-induced ATL.
| |
ACKNOWLEDGMENTS |
|---|
R.G. is supported by a Centennial Fellowship from the Medical Research Council of Canada. E.T.C. is supported by NIH grant KO8-EY00352. This work was supported by grants from the UCSF Center for AIDS Research (P30A127763) and from Pfizer.
R.G., S.F., and X.L. contributed equally to this work.
We thank G. Bokoch and U. Knaus for providing the PAK1 K299R expression vector, D. Goeddel for the TRAF2 and TRAF2(87-501) vectors, G. Johnson for the MEKK1 vector, D. Pearce for the 6RZ reporter construct, M. Karin for the FLAG-JNK1 construct, Christophe Beraud for helpful discussions, Weiduan Xu for technical support, and John Carroll, Neile Shea, Stephen Gonzales, and Chris Goodfellow for preparation of the figures.
| |
FOOTNOTES |
|---|
* Corresponding author: Mailing address: Gladstone Institute of Virology and Immunology, P.O. Box 419100, San Francisco, CA 94141-9100. Phone: (415) 695-3825. Fax: (415) 826-1514. E-mail: Romas_Geleziunas{at}quickmail.ucsf.edu.
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