1 Ben May Institute for Cancer Research, Committee on Cancer Biology, University of Chicago, Chicago, Illinois 60637; 2 Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel; 4 Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195; and 5 Institute for Virus Research, Kyoto University, Kyoto 606, Japan
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ABSTRACT |
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pX, the hepatitis B virus-encoded
transcription coactivator, is involved in viral infection in vivo. pX
stimulates the activity of several transcription factors including
nuclear factor-B (NF-
B), but the mechanism of activation is
poorly understood. The I
B kinase complex (IKK) mediates activation
of NF-
B in response to various extracellular stimuli, including
inflammatory cytokines like tumor necrosis factor and interleukin 1, human T cell lymphoma virus 1 Tax protein, and tumor promoters like
phorbol esters. It is not known whether IKK also mediates activation of
NF-
B by pX. Here we report that IKK was not essential for activation of NF-
B by pX. Expression of pX resulted in the degradation of I
B
in the absence of its phosphorylation at Ser32 and
Ser36 residues. Although pX stimulated the activity of
cotransfected IKK-
when it was overexpressed, it failed to activate
endogenous IKK. Furthermore, expression of pX stimulated NF-
B
nuclear translocation and transcriptional activity in IKK-
-null
fibroblast 5R cells. Our data indicate that pX stimulates NF-
B
activity through a mechanism that is dependent on I
B
degradation
but not on IKK activation.
nuclear factor-B; inhibitor of nuclear factor-
B; X protein of
hepatitis B virus
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INTRODUCTION |
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HEPATITIS B VIRUS
(HBV) is a small, hepatotrophic DNA virus that is able to cause viral
hepatitis and may be involved in the development of hepatocellular
carcinoma (4, 29, 65). pX (also known as HBx) is a
promiscuous viral protein encoded by HBV. The open reading frame of pX
is conserved among all mammalian members of the
Hepadnaviridae and is essential for woodchuck hepatitis virus infectivity (10, 72). pX functions as a coactivator that transactivates a variety of viral and cellular genes under the
control of activator protein (AP)-1, nuclear factor (NF)-AT, and
NF-B (12, 23, 31, 56).
The mechanism(s) by which pX activates gene expression is not completely understood. One possible mechanism is that pX may interact directly with the transcription machinery. It is reported that pX interacts with several components of the basal transcription complex including the RPB5 unit of RNA Pol II enzyme, TATA-binding protein (TBP), transcription factor II (TFII)H, TFIIB (11, 24, 38, 51, 64), and some upstream transcription activators, such as cAMP response element binding protein (CREB)/activating transcription factor (ATF) (40) and p53 (60, 61). Another possibility is that pX may stimulate gene expression through the cellular signaling network, including Ras/Raf/mitogen-activated protein (MAP) kinase kinase (MEK)/extracellular signal-related kinase (ERK) MAP kinase pathway (6), protein kinase C (PKC) (28), c-Jun NH2-terminal kinase (JNK) (7), and the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway (32).
pX stimulates NF-B activity (17, 43, 56); however, the
mechanism of stimulation is not clear. The dimeric sequence-specific transcription factor NF-
B comprises a family of proteins that contain a common Rel homology domain, and the classic form NF-
B is a
p65/Rel dimer (2). In most resting cells, NF-
B is bound to its cytoplasmic inhibitors, the I
Bs, and remains in the cytoplasm as a latent-form transcription factor (1-3, 63). On
stimulation by most NF-
B stimuli, the I
B proteins become
phosphorylated on specific serine residues (Ser32 and
Ser36 in I
B
; Ser19 and Ser23
in I
B
) (1-3, 8, 9, 63). Phosphorylation of the
I
B proteins triggers their ubiquitination, followed by degradation by the 26S proteasome (58, 59, 67). Proteolysis of the
I
B proteins results in the release of NF-
B, which translocates
into the nucleus and stimulates transcription of its target genes
(58).
The IB kinase (IKK) was first identified as a high-molecular-weight
protein complex (58). Two catalytic subunits of IKK, IKK-
and IKK-
(also known as IKK-1 and IKK-2, respectively), that
specifically phosphorylate I
B proteins have been isolated (15,
42, 52, 59, 67, 68, 71). A regulatory subunit of IKK, IKK-
(also known as NEMO or IKKAP1) (41, 54, 69) has also been
identified. Phosphorylation of IKK-
on its two serine residues
(Ser177 and Ser181) is essential for
IKK activation (42, 68, 71). Two protein kinases in the
MAP kinase kinase kinase (MEKK) family, NIK and MEKK, have been
proposed as immediate upstream kinases of IKK (14, 19, 37, 41,
44, 45, 50, 54, 62, 69, 71, 72). Several other protein kinases
including transforming growth factor-
activated kinase-1 (TAK1)
(46) and Akt (39, 48, 53) may also function
as upstream activators. Activation of IKK is essential for
mediating NF-
B activation by many extracellular stimuli including
tumor necrosis factor (TNF)-
(15, 34, 35, 45, 57),
interleukin (IL)-1 (15, 34-35, 45, 57),
12-O-tetradecanoylphorbol 13-acetate (TPA)
(30), lipopolysaccharide (LPS) (18, 25, 47),
-irradiation (5, 33) and Tax, a viral protein of human
T lymphocyte virus 1 (HTLV-1) (13, 19-20, 27, 62,
70).
Here we report that pX activates NF-B through an IKK-independent
mechanism. Expression of pX triggers the degradation of I
B
without inducing its phosphorylation at Ser32 and
Ser36 residues. Consistently, pX is unable to stimulate the
enzymatic activity of endogenous IKK-
in human embryonic kidney 293 cells. Furthermore, pX stimulates NF-
B transcriptional activity in
IKK-
-null fibroblast 5R cells, in which IKK is refractory to
activation by many NF-
B stimuli such as TNF-
. Thus our data
indicate that pX stimulates NF-
B activity through a mechanism that
does not depend on IKK activation and phosphorylation of I
B
NH2-terminal serines.
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MATERIALS AND METHODS |
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Cell culture and transfection. HeLa, COS-1, Rat-1, and 5R cells were grown in DMEM (Mediatech, Herndon, VA) supplemented with 10% FCS (Atlanta Biol., Atlanta, GA), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Human embryonic kidney 293 cells were grown in DMEM-F-12 (Mediatech) with the supplements. Transfections were described previously (36).
Plasmids, fusion proteins, and reagents.
Hemagglutinin (HA)-tagged IKK-, HA-IKK-
, HA-I
B
,
HA-I
B
(AA), in which Ser32 and Ser36 were
replaced by alanines, M2-pX, or its transcription-inactive mutant
M2-pXm7, were described previously (22, 45).
Glutathione-S-transferase (GST)-I
B
(1-54) and
GST-c-Jun(1-79) were purified on glutathione-agarose as described previously (36, 45). Proteasome inhibitors
AcLLnL (N-Ac-Leu-Leu-norleucinal) and lactacystin
(clasto-lactacystin
-lactone) were purchased from Calbiochem (San
Diego, CA). Rabbit polyclonal anti-p65 was from Zymed (San Francisco,
CA), and mouse monoclonal anti-p65 was from Boehringer Mannheim
(Indianapolis, IN). Rabbit polyclonal
anti-phospho-I
B
(Ser32) antibody was from New England
BioLabs (Beverly, MA). Anti-M2 and anti-actin monoclonal antibodies
were from Sigma (St. Louis, MO).
Protein kinase assays and transcription assays.
Transfected HA-IKK- and HA-IKK-
were immunoprecipitated from HeLa
or COS-1 cell extracts with an anti-HA monoclonal antibody (12CA5,
Santa Cruz, Santa Cruz, CA), and the activity of the immune complex was
assayed at 30°C for 30-60 min in 30 µl of kinase buffer (45) in the presence of 10 µM ATP/10 µCi
[
-32P]ATP (10 Ci/mmol) with
GST-I
B
(1-54) as a substrate. The reactions were
terminated with 4× Laemmli sample buffer. The proteins were resolved
by 13% SDS-PAGE followed by autoradiography. Radioactivity in the
phosphorylated proteins was quantitated by a phospho/chemifluorescence imager (Molecular Dynamics, Sunnyvale, CA). To assay the activity of
endogenous IKK or JNK, the kinase was immunoprecipitated from 293 cell
extracts using a polyclonal anti-IKK-
antibody (Zymed) or monoclonal
anti-JNK antibody (PharMigen, San Diego, CA) and the kinase activity
was measured by immunocomplex kinase assays with
GST-I
B
(1-54) or
GST-c-Jun(1-79) as a substrate, respectively.
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Immunoblotting and immunofluorescence. Immunoblotting was performed as described previously (45), and the expression of proteins was quantitated using a phospho/chemifluorescence imager (Molecular Dynamics). Detection of subcellular localization of p65 and M2-pX was performed as described previously (54, 73).
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RESULTS |
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pX stimulates NF-B nuclear translocation and transcription
activity.
To study activation of NF-
B by pX, we determined the effect of pX on
NF-
B nuclear translocation. 293 Cells grown on coverslips were
transiently transfected with expression vectors encoding M2-pX or its
inactive mutant M2-pXm7 or empty vector. After 40 h,
cells were treated with TNF-
for 15 min or left untreated. Cells
were fixed, and the nuclear translocation of NF-
B was examined by
indirect immunofluorescence analysis using a monoclonal anti-p65
antibody. Treatment with TNF-
induced the accumulation of p65 in the
nucleus (Fig. 1A, b) as
reported previously (69). Expression of M2-pX also induced
the accumulation of p65 in the nucleus as evident by the enhanced
nuclear staining (Fig. 1A, c). Under the same
conditions, pXm7 had no detectable effect (Fig.
1A, d). The pX effect appeared to be specific,
because it was blocked by treatment with the inhibitor AcLLnL, which
inhibits the proteasome-mediated degradation of I
B proteins and
prevents p65 nuclear translocation (data not shown).
pX-induced IB
degradation requires ubiquitination-dependent
proteasome but not Ser32 and Ser36
phosphorylation.
A key step that leads to NF-
B activation in response to many
extracellular stimuli is the degradation of I
B
by the
ubiquitination-dependent proteasome, which is generally triggered by
I
B
phosphorylation at Ser32 and Ser36
(1-3, 8, 9, 63). To understand how pX activates
NF-
B, we determined whether pX induces degradation of I
B
, and,
if so, whether it depends on Ser32 and Ser36 phosphorylation.
The biphasic effect of pX on the activity of HA-IKK-.
The finding that pX can induce I
B
degradation in the absence of
its phosphorylation at Ser32 and Ser36 prompts
us to examine the effect of pX on IKK, the key enzyme that controls
NF-
B activation in response to various extracellular stimuli
(14, 15, 41, 42, 52, 54, 68, 69, 71).
pX does not activate endogenous IKK.
To determine whether pX can stimulate the activity of endogenous IKK,
293 cells were transfected with expression vectors encoding M2-pX or
HA-MEKK1. After 40 h, cells were treated with TNF- for 15 min
or left untreated. Endogenous IKK was immunoprecipitated, and its
activity was measured by immunocomplex kinase assays with GST-I
B
as a substrate. As reported previously, treatment with TNF-
strongly
activated endogenous IKK (Fig.
4A). In contrast, expression
of M2-pX failed to do so (Fig. 4A). The inability of M2-pX
to activate endogenous IKK was not the result of the expression of
M2-pX or the transfection efficiency (data not shown). Under the same
conditions, M2-pX was able to stimulate NF-
B transcription activity
as measured by NF-
B reporter assays (data not shown; see Fig.
1B). Furthermore, expression of M2-pX was able to stimulate the activity of endogenous JNK as measured by immunocomplex kinase assays with GST-c-Jun as a substrate (Fig. 4B). These
results suggest that pX has no detectable stimulatory effect on
endogenous IKK activity.
pX induces NF-B activation in IKK-
-null fibroblast 5R cells.
IKK-
is the regulatory subunit of the IKK complex and is essential
for IKK-mediated NF-
B activation in response to many stimuli
(41, 54, 69). For instance, no NF-
B activity can be
induced by TNF-
, IL-1, or Tax in IKK-
-null fibroblast 5R cells
(69). Because pX was able to stimulate NF-
B activity in
the absence of IKK activation, we examined the effect of pX on NF-
B
activity in IKK-
-null 5R cells.
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DISCUSSION |
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pX, the transcription cofactor encoded by HBV, is a promiscuous
viral protein that is able to transactivate numerous viral and cellular
genes controlled by transcription factors such as AP-1, NF-AT, and
NF-B (12, 23, 31, 56). However, the mechanism of action
of pX is not completely understood. In this study, we examined whether
pX-induced NF-
B activation is mediated by IKK, which is a key enzyme
that controls NF-
B activation in response to various stimuli. Our
results indicate that pX may stimulate NF-
B activity but bypass the
IKK signaling pathway. This conclusion is based on several lines of
evidence. First, pX-induced I
B
degradation is mediated by the
ubiquitination-dependent proteasome. However, the degradation occurs in
the absence of Ser32 and Ser36 phosphorylation
(Fig. 2B). Second, pX is unable to stimulate the enzymatic
activity of IKK unless IKK is overexpressed (Figs. 3 and 4). Third, pX
induces NF-
B activation in IKK-
-null fibroblast 5R cells (Fig.
5).
Phosphorylation of Ser32 and Ser36 residues is
a critical step that triggers IB
degradation by
ubiquitination-dependent proteasome in response to many NF-
B
activators, including TNF-
(15, 34, 35, 45, 57), IL-1
(15, 34, 35, 45, 57), TPA (30), LPS
(18, 25, 47),
-irradiation (5, 33), and
Tax (13, 19, 20, 27, 62, 70). Surprisingly, pX induces
I
B
degradation in the absence of phosphorylation at these
serines, as determined by immunoblotting with the anti-phospho-I
B
antibody that specifically recognizes phosphorylated Ser32
(Fig. 2B). Consistently, pX also induces degradation of the
nonphosphorylatable HA-I
B
(32/36AA) mutant, although to a lesser
extent (Fig. 2B). It is plausible that the basal IKK-
might facilitate the degradation of wild-type I
B
by pX but not
the I
B
(32/36AA) mutant. We also do not detect tyrosine
phosphorylation of I
B
(unpublished results), which is a novel
mechanism by which intact I
B
disassociates from NF-
B in
hypoxic cells on reoxidation (26). Although the known
phosphorylation events are apparently not involved, degradation of
I
B
by pX was still mediated by ubiquitination-dependent
proteasome (Fig. 2B), consistent with previous reports
(56). Thus pX, along with ultraviolet radiation, may form
a unique class of NF-
B activators, which induce degradation of
I
B
in a proteasome-dependent manner but bypass IKK activation and
I
B
phosphorylation. The mechanism by which pX induces degradation
of I
B
is currently under investigation.
These findings contradict an earlier report that expression of pX
induces IB
phosphorylation (56). This discrepancy
may have resulted from different methods used to analyze the status of
I
B
phosphorylation. In the earlier report, phosphorylation of
I
B
was concluded on the basis of the observation that a small portion of I
B
had a slightly slower migration on a SDS gel
(56). However, this slightly slower-migrating form of
I
B
may not have resulted from pX-induced phosphorylation on
Ser32 and Ser36. As shown in Fig.
2B, treatment of cells with the proteasome inhibitor AcLLnL
alone results in a similar slightly slower-migrating form of I
B
.
This slower-migrating form of I
B
can be recognized by the
anti-phospho-I
B
antibody, suggesting that at least low-level phosphorylation of Ser32 may be involved in
the basal turnover of I
B
proteins. Nevertheless, expression of pX
does not enhance phosphorylation of I
B
at these serines. We
cannot, however, formally exclude the possibility that pX may stimulate
other protein kinases that phosphorylate I
B
on other unknown
region(s), leading to its ubiquitination and degradation.
How pX stimulates NF-B activity has yet to be determined. It is
reported that pX may activate NF-
B through inducing the degradation
of I
B
and the NF-
B precursor/inhibitor p105 (56). Our results support this model, although it is not clear how pX induces
I
B
degradation. Alternatively, pX may directly associate with
I
B
and prevent its association with or induce its disassociation from NF-
B. Recently, Israel and colleagues (66) have
elegantly shown that cotransfected I
B
interacted with pX and
transported it into the nucleus. Interestingly, they found that pX
interacts with the newly synthesized endogenous I
B
induced by
TNF-
but not with endogenous I
B
in nonstimulated cells and
that it does not disassociate the preformed NF-
B-I
B
complex
(66). It is proposed that the nuclear localization of the
I
B
-pX complex prevented I
B
from disrupting the NF-
B-DNA
complex, resulting in sustained NF-
B activation (66).
However, we found that transfected pX mainly locates in the nucleus
(Fig. 5A), consistent with previous reports
(24). It is also not clear how pX activates NF-
B in the
first place, i.e., in the absence of cotransfected I
B
or other
stimuli. We propose here that degradation of I
B
induced by pX may
set NF-
B activation in motion. This effect of pX is then reinforced
by retention of newly synthesized I
B
in the nucleus by pX. It is
also plausible that pX may stimulate the activity of the proteasome,
which in turn degrades I
B
. This is not likely, however, because
pX is reported to inhibit, rather than activate, proteasome activity
(21). It is more likely, however, that the interaction
with pX may bring the proteasome near I
B
proteins, resulting in
its degradation. Future studies must test this hypothesis.
The effect of pX on IKK activity is intriguing. In both COS-1 and HeLa
cells expression of pX only stimulates IKK activity when IKK is
overexpressed (Fig. 3B). More importantly, expression of pX
is unable to activate endogenous IKK (Fig. 4A), although it
does activate endogenous JNK (Fig. 4B). IKK must be
incorporated into the IKK complex for its proper regulation
(54). Overexpression of HA-IKK- may result in a portion
of the transfected HA-IKK-
staying as a nonincorporated form and
being activated by pX through a yet to be identified mechanism. It
would be interesting to determine whether expression of IKK is
upregulated in HBV-infected hepatocytes so that pX could activate IKK.
How pX induces expression of the cotransfected IKK-
is not clear. It
may be plausible that pX stimulates yet to be identified transcription
factors that bind to the promoter of the cotransfected IKK-
and
induce its expression. Nevertheless, our data indicate that in the
absence of a functional IKK, pX is still able to induce nuclear
translocation and activation of NF-
B, as demonstrated by the use of
IKK-
-null fibroblast 5R cells (Fig. 5). Thus IKK activation may not
be essential for pX activation of NF-
B. This conclusion is quite
unexpected, considering the fact that the properties of pX are very
similar to those of Tax, a viral transforming protein of HTLV-1 that
activates NF-
B through activation of IKK (13, 19, 20, 27, 62,
70). We cannot, however, rule out the possibility that
activation of NF-
B by pX might be mediated by the atypical
IKK-i/IKK-
(49, 55) or some unknown IKKs. In fact, we
found that activation of NF-
B by pX was blocked by the
dominant-negative mutants of MEKK1, NIK, and Akt, the putative IKK
upstream kinases (unpublished results). This possibility is
currently under investigation in our laboratory. It is possible that
activation of NF-
B by pX may be utilized by HBV for its infection
and may also contribute to the development of hepatic carcinogenesis.
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ACKNOWLEDGEMENTS |
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We thank Tony Hunter, Michael Karin, and David A. Brenner for critical comments and helpful discussions.
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FOOTNOTES |
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* N. H. Purcell, C. Yu, and D. He contributed equally to this work.
This work was supported by National Cancer Institute Grant CA-73740 and American Cancer Society Grant RPG-99-171-01-CCC.
Address for reprint requests and other correspondence: A. Lin, Ben May Institute for Cancer Research, Univ. of Chicago, 5841 S. Maryland, MC 6027, Chicago, IL 60637 (E-mail:alin{at}huggins.bsd.uchicago.edu).
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.
Received 12 July 2000; accepted in final form 10 October 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baeuerle, PA,
and
Baltimore D.
NF-B: ten years after.
Cell
87:
13-20,
1996[ISI][Medline].
2.
Baldwin, AS.
The NF-B and I
B proteins: new discoveries and insights.
Annu Rev Immunol
14:
649-683,
1996[ISI][Medline].
3.
Barnes, PJ,
and
Karin M.
Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases.
N Engl J Med
336:
1066-1071,
1997
4.
Beasley, RP,
Hwang LY,
Lin CC,
and
Chien CS.
Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22,707 men in Taiwan.
Lancet
2:
1129-1133,
1981[ISI][Medline].
5.
Bender, K,
Gottlicher M,
Whiteside S,
Rahmsdorf HJ,
and
Herrlich P.
Sequential DNA damage-independent and -dependent activation of NF-B by UV.
EMBO J
17:
5170-5181,
1998
6.
Benn, J,
and
Schneider RJ.
Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade.
Proc Natl Acad Sci USA
91:
10350-10354,
1994
7.
Benn, J,
Su F,
Doria M,
and
Schneider RJ.
Hepatitis B virus HBx protein induces transcription factor AP-1 by activation of extracellular signal-regulated and c-Jun N-terminal mitogen-activated protein kinases.
J Virol
70:
4978-4985,
1996[Abstract].
8.
Brockman, JA,
Scherer DC,
McKinsey TA,
Hall SM,
Qi X,
Lee WY,
and
Ballard DW.
Coupling of a signal response domain in IB
to multiple pathways for NF-
B activation.
Mol Cell Biol
15:
2809-2818,
1995[Abstract].
9.
Brown, K,
Gerstberger S,
Carlson L,
Franzoso G,
and
Siebenlist U.
Control of IB-
proteolysis by site-specific, signal-induced phosphorylation.
Science
267:
1485-1488,
1995[ISI][Medline].
10.
Chen, HS,
Kaneko S,
Girones R,
Anderson RW,
Hornbuckle WE,
Tennant BC,
Cote PJ,
Gerin JL,
Purcell RH,
and
Miller RH.
The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks.
J Virol
67:
3085-3093,
1993.
11.
Cheong, J,
Yi M,
Lin Y,
and
Murakami S.
Human RPB5, a subunit shared by eukaryotic nuclear RNA polymerases, binds human hepatitis B virus X protein and may play a role in X transactivation.
EMBO J
14:
143-150,
1995[Abstract].
12.
Chirillo, P,
Falco M,
Puri L,
Artini M,
Balsano C,
and
Levrero M.
Hepatitis B virus pX activates NF-B-dependent transcription through a Raf-independent pathway.
J Virol
70:
641-646,
1994[Abstract].
13.
Chu, ZL,
DiDonato JA,
Hawiger J,
and
Ballard DW.
The tax oncoprotein of human T-cell leukemia virus type 1 associates with and persistently activates IB kinases containing IKK
and IKK
.
J Biol Chem
273:
15891-15894,
1998
14.
Cohen, L,
Henzel WJ,
and
Baeuerle PA.
IKAP is a scaffold protein of the IB kinase complex.
Nature
395:
292-296,
1998[ISI][Medline].
15.
DiDonato, JA,
Hayakawa M,
Rothwarf DM,
Zandi E,
and
Karin M.
A cytokine-responsive IB kinase that activates the transcription factor NF-
B.
Nature
388:
548-554,
1997[ISI][Medline].
16.
DiDonato, JA,
Mercurio F,
and
Karin M.
Phosphorylation of IB
precedes but is not sufficient for its dissociation from NF-
B.
Mol Cell Biol
15:
1302-1311,
1995[Abstract].
17.
Doria, M,
Klein N,
Lucito R,
and
Schneider RJ
The hepatitis B virus HBx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors.
EMBO J
14:
4747-4757,
1995[Abstract].
18.
Fischer, C,
Page S,
Weber M,
Eisele T,
Neumeier D,
and
Brand K.
Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IB kinase signalsome activation and I
B proteolysis.
J Biol Chem
274:
24625-24632,
1999
19.
Geleziunas, R,
Ferrell S,
Lin X,
Mu Y,
Cunningham ET, Jr,
Grant M,
Connelly MA,
Hambor JE,
Marcu KB,
and
Greene WC.
Human T-cell leukemia virus type 1 Tax induction of NF-B involves activation of the I
B kinase alpha (IKK
) and IKK
cellular kinases.
Mol Cell Biol
18:
5157-5165,
1998
20.
Harhaj, EW,
and
Sun SC.
IKK serves as a docking subunit of the I
B kinase (IKK) and mediates interaction of IKK with the human T-cell leukemia virus Tax protein.
J Biol Chem
274:
22911-22914,
1999
21.
Huang, J,
Kwong J,
Sun EC,
and
Liang TJ.
Proteosome complex as a potential target of hepatitis B virus X protein.
J Virol
70:
5582-9991,
1996[Abstract].
22.
Haviv, I,
Shamay M,
Doitsh G,
and
Shaul Y.
Hepatitis B virus pX targets TFIIB in transcription coactivation.
Mol Cell Biol
18:
1562-1569,
1998
23.
Haviv, I,
Vaizel D,
and
Shaul Y.
The X protein of hepatitis B virus coactivates potent activation domains.
Mol Cell Biol
15:
1079-1085,
1995[Abstract].
24.
Haviv, I,
Vaizel D,
and
Shaul Y.
pX, the HBV-encoded coactivator, interacts with components of the transcription machinery and stimulates transcription in a TAF-independent manner.
EMBO J
15:
3413-3420,
1996[Abstract].
25.
Hawiger, J,
Veach RA,
Liu XY,
Timmons S,
and
Ballard DW.
IB kinase complex is an intracellular target for endotoxic lipopolysaccharide in human monocytic cells.
Blood
94:
1711-1716,
1999
26.
Imbert, V,
Rupec RA,
Livolsi A,
Pahl HL,
Traenckner EB,
Mueller-Dieckmann C,
Farahifar D,
Rossi B,
Auberger P,
Baeuerle PA,
and
Peyron JF.
Tyrosine phosphorylation of IB-
activates NF-
B without proteolytic degradation of I
B-
.
Cell
86:
787-798,
1996[ISI][Medline].
27.
Jin, DY,
Giordano V,
Kibler KV,
Nakano H,
and
Jeang KT.
Role of adapter function in oncoprotein-mediated activation of NF-B human T-cell interacts directly with I
B kinase
.
J Biol Chem
274:
17402-17405,
1999
28.
Kekule, AS,
Lauer U,
Weiss L,
Luber B,
and
Hofschneider PH.
Hepatitis B virus transactivator HBx uses a tumour promoter signalling pathway.
Nature
361:
742-745,
1993[ISI][Medline].
29.
Kim, C-M,
Koike K,
Saito I,
Miyamura T,
and
Jay G.
HBx gene of hepatitis B virus induces liver cancer in transgenic mice.
Nature
353:
317-320,
1991.
30.
Lallena, MJ,
Diaz-Meco MT,
Bren G,
Paya CV,
and
Moscat J.
Activation of IB kinase
by protein kinase C isoforms.
Mol Cell Biol
19:
2180-2188,
1999
31.
Lara-Pezzi, E,
Armesilla AL,
Majano PL,
Redondo JM,
and
Lopez-Cabrera M.
The hepatitis B virus X protein activates nuclear factor of activated T-cells (NF-AT) by cyclosporin A-sensitive pathway.
EMBO J
17:
7066-7077,
1998
32.
Lee, YH,
and
Yun YD.
HBx protein of hepatitis B virus activates Jak1-STAT signaling.
J Biol Chem
273:
25510-25515,
1998
33.
Li, N,
and
Karin M.
Ionizing radiation and short wavelength UV activate NF-B through two distinct mechanisms.
Proc Natl Acad Sci USA
95:
13012-13017,
1998
34.
Li, Q,
Van Antwerp D,
Mercurio F,
Lee KF,
and
Verma IM.
Severe liver degeneration in mice lacking the IB kinase 2 gene.
Science
284:
321-325,
1999
35.
Li, ZW,
Chu W,
Hu Y,
Delhase M,
Deerinck T,
Ellisman M,
Johnson R,
and
Karin M.
The IKK subunit of I
B kinase (IKK) is essential for nuclear factor
B activation and prevention of apoptosis.
J Exp Med
189:
1839-1845,
1999
36.
Lin, A,
Minden A,
Martinetto H,
Claret FX,
Lange-Carter C,
Mercurio F,
Johnson LJ,
and
Karin M.
Identification of a dual specificity kinase that activates the Jun kinases and p38-Mpk2.
Science
268:
286-290,
1995[ISI][Medline].
37.
Lin, X,
Cunningham ET, Jr.,
Mu Y,
Geleziunas R,
and
Greene WC.
The proto-oncogene Cot kinase participates in CD3/CD28 induction of NF-B acting through the NF-
B-inducing kinase and I
B kinases.
Immunity
10:
271-280,
1999[ISI][Medline].
38.
Lin, Y,
Nomura T,
Cheong J,
Dorjsuren D,
Iida K,
and
Murakami S.
Hepatitis B virus X protein is a transcription modulator that communicates with transcription factor IIB and the RNA polymerase II subunit 5.
J Biol Chem
272:
7132-7139,
1997
39.
Madrid, LV,
Wang CY,
Guttridge DC,
Schottelius AJ,
Baldwin AS,
and
Mayo MW.
Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-B.
Mol Cell Biol
20:
1626-1638,
2000
40.
Maguire, HF,
Hoeffler JP,
and
Siddiqui A.
HBV X protein alters the DNA binding specificity of CREB and ATF-2 by protein-protein interactions.
Science
252:
842-844,
1991[ISI][Medline].
41.
Mercurio, F,
Murray BW,
Shevchenko A,
Bennett BL,
Young DB,
Li JW,
Pascual G,
Motiwala A,
Zhu H,
Mann M,
and
Manning AM.
IB kinase (IKK)-associated protein 1, a common component of the heterogeneous IKK complex.
Mol Cell Biol
19:
1526-1538,
1999
42.
Mercurio, F,
Zhu H,
Murray BW,
Shevchenko A,
Bennett BL,
Li J,
Young DB,
Barbosa M,
Mann M,
Manning A,
and
Rao AF.
IKK-1 and IKK-2: cytokine-activated IB kinases essential for NF-
B activation.
Science
278:
860-866,
1997
43.
Meyer, M,
Caselmann WH,
Schluter V,
Schreck R,
Hofschneider PH,
and
Baeuerle PA.
Hepatitis B virus transactivator MHBst: activation of NF-B, selective inhibition by antioxidants and integral membrane localization.
EMBO J
11:
2991-3001,
1992[Abstract].
44.
Nakano, H,
Shindo M,
Sakon S,
Nishinaka S,
Mihara M,
Yagita H,
and
Okumura K.
Differential regulation of IB kinase
and
by two upstream kinases, NF-
B-inducing kinase and mitogen-activated protein kinase/ERK kinase kinase-1.
Proc Natl Acad Sci USA
95:
3537-3542,
1998
45.
Nemoto, S,
DiDonato JA,
and
Lin A.
Coordinate regulation of IB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-
B-inducing kinase.
Mol Cell Biol
18:
7336-7343,
1998
46.
Ninomiya-Tsuji, J,
Kishimoto K,
Hiyama H,
Inoue J,
Cao Z,
and
Matsumoto K.
The kinase TAK1 can activate the NIK-I B as well as the MAP kinase cascade in the IL-1 signalling pathway.
Nature
398:
252-256,
1999[ISI][Medline].
47.
O'Connell, MA,
Bennett BL,
Mercurio F,
Manning AM,
and
Mackman N.
Role of IKK1 and IKK2 in lipopolysaccharide signaling in human monocytic cells.
J Biol Chem
273:
30410-30414,
1998
48.
Ozes, ON,
Mayo LD,
Gustin JA,
Pfeffer SR,
Pfeffer LM,
and
Donner DB.
NF-B activation by tumour necrosis factor requires the Akt serine-threonine kinase.
Nature
401:
82-85,
1999[ISI][Medline].
49.
Peters, RT,
Liao SM,
and
Maniatis T.
IKK is part of a novel PMA inducible I
B kinase complex.
Mol Cell
5:
513-522,
2000[ISI][Medline].
50.
Pomerantz, JL,
and
Baltimore D.
NF-B activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase.
EMBO J
18:
6694-6704,
1999
51.
Qadri, I,
Maguire HF,
and
Siddiqui A.
Hepatitis B virus transactivator protein X interacts with the TATA-binding protein.
Proc Natl Acad Sci USA
92:
1003-1007,
1995[Abstract].
52.
Regnier, CH,
Song HY,
Gao X,
Goeddel DV,
Cao Z,
and
Rothe M.
Identification and characterization of an IB kinase.
Cell
90:
373-383,
1997[ISI][Medline].
53.
Romashkova, JA,
and
Makarov SS.
NF-B is a target of AKT in anti-apoptotic PDGF signalling.
Nature
401:
86-90,
1999[ISI][Medline].
54.
Rothwarf, DM,
Zandi E,
Natoli G,
and
Karin M.
IKK- is an essential regulatory subunit of the I
B kinase complex.
Nature
395:
297-300,
1998[ISI][Medline].
55.
Shimada, T,
Kawai T,
Takeda K,
Matsumoto M,
Inoue J,
Tatsumi Y,
Kanamaru A,
and
Akira S.
IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IB kinases.
Int Immunol
11:
1357-1362,
1999
56.
Su, F,
and
Schneider RJ.
Hepatitis B virus HBx protein activates transcription factor NF-B by acting on multiple cytoplasmic inhibitors of rel-related proteins.
J Virol
70:
4558-4566,
1997[Abstract].
57.
Tanaka, M,
Fuentes ME,
Yamaguchi K,
Durnin MH,
Dalrymple SA,
Hardy KL,
and
Goeddel DV.
Embryonic lethality, liver degeneration, and impaired NF-B activation in IKK-
-deficient mice.
Immunity
10:
421-429,
1999[ISI][Medline].
58.
Thanos, D,
and
Maniatis T.
NF-B: a lesson in family values.
Cell
80:
529-532,
1995[ISI][Medline].
59.
Traenckner, EB,
Wilk S,
and
Baeuerle PA.
Phosphorylation of human IB-
on serines 32 and 36 controls I
B-
proteolysis and NF-
B activation in response to diverse stimuli.
EMBO J
13:
5433-5441,
1994[Abstract].
60.
Truant, R,
Antunovic J,
Greenblatt J,
Prives C,
and
Gromlish JA.
Direct interaction of the hepatitis B virus HBx protein with p53 leads to inhibition by HBx of p53 response element-directed transactivation.
J Virol
69:
1851-1859,
1995[Abstract].
61.
Ueda, H,
Ullrich SJ,
Gangemi JD,
Kappel CA,
Ngo L,
Feitelson MA,
and
Jay G.
Functional inactivation but not structural mutation of p53 causes liver cancer.
Nat Genet
9:
41-47,
1995[ISI][Medline].
62.
Uhlik, M,
Good L,
Xiao G,
Harhaj EW,
Zandi E,
Karin M,
and
Sun SC.
NF-B-inducing kinase and I
B kinase participate in human T-cell leukemia virus I Tax-mediated NF-
B activation.
J Biol Chem
273:
21132-21136,
1998
63.
Verma, IM,
Stevenson JK,
Schwarz EM,
Van Antwerp D,
and
Miyamoto S.
Rel/NF-B/I
B family: intimate tales of association and dissociation.
Genes Dev
9:
2723-2735,
1995[ISI][Medline].
64.
Wang, H-D,
Trivedi A,
and
Johnson DL.
Regulation of RNA polymerase I-dependent promoters by the hepatitis B virus X protein via activated Ras and TATA-binding protein.
Mol Cell Biol
18:
7086-7094,
1998
65.
Wei, Y,
Fourel G,
Ponzetto A,
Silvestro M,
Tiollais P,
and
Buendia MA.
Hepadnavirus integration: mechanisms of activation of the N-myc2 retrotransposon in woodchuck liver tumors.
J Virol
66:
5465-5276,
1992.
66.
Weil, R,
Sirma H,
Giannini C,
Kremsdorf D,
Bessia C,
Dargemont C,
Brechot C,
and
Israel A.
Direct association and nuclear import of the hepatitis B virus X protein with the NF-B inhibitor I
B
.
Mol Cell Biol
19:
6345-6354,
1999
67.
Whiteside, T,
Ernst KM,
Lebail O,
Laurent-Winter C,
Rice N,
and
Israel A.
N- and C-terminal sequences control degradation of MAD3/IB
in response to inducers of NF-
B activity.
Mol Cell Biol
15:
5339-5345,
1995[Abstract].
68.
Woronicz, JD,
Gao X,
Cao Z,
Rothe M,
and
Goeddel DV.
IB kinase-
: NF-
B activation and complex formation with I
B kinase-
and NIK.
Science
278:
866-869,
1997
69.
Yamaoka, S,
Courtois G,
Bessia C,
Whiteside ST,
Weil R,
Agou F,
Kirk HE,
Kay HE,
and
Israel A.
Complementation cloning of NEMO, a component of the IB kinase complex essential for NF-
B activation.
Cell
93:
1231-1240,
1998[ISI][Medline].
70.
Yin, M-J,
Christerson LB,
Yamamoto Y,
Kwak Y-T,
Xu S,
Mercurio F,
Barbosa M,
Cobb MH,
and
Gaynor RB.
HTLV-I Tax protein binds to MEKK1 to stimulate IB kinase activity and NF-
B activation.
Cell
93:
875-884,
1998[ISI][Medline].
71.
Zandi, E
Rothwarf DM, Delhase M, Hayakawa M, and Karin M. The IB kinase complex (IKK) contains two kinase subunits, IKK
and IKK
, necessary for I
B phosphorylation and NF-
B activation.
Cell
91:
243-252,
1997[ISI][Medline].
72.
Zhao, Q,
and
Lee FS.
Mitogen-activated protein kinase/ERK kinase kinases 2 and 3 activate nuclear factor-B through I
B kinase-
and I
B kinase-
.
J Biol Chem
274:
8355-8358,
1999
73.
Zheng, C,
Xiang J,
Hunter T,
and
Lin A.
The JNKK2-JNK1 fusion protein acts as a constitutively active c-Jun kinase that stimulates c-Jun transcription activity.
J Biol Chem
274:
28966-28971,
1999
74.
Zoulim, F,
Saputelli J,
and
Seeger C.
Woodchuck hepatitis virus X protein is required for viral infection in vivo.
J Virol
68:
2026-2030,
1994[Abstract].