From the Laboratory of Molecular Microbiology, NIAID,
National Institutes of Health, Bethesda, Maryland 20892-0460, the
¶ Department of Immunology, Juntendo University School of
Medicine, Tokyo 113, Japan, and the
Core Research for
Evolutional Science and Technology, Japan Science and Technology
Corporation, Tokyo 101-0062, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mechanisms by which the human T-cell leukemia
virus type I Tax oncoprotein activates NF- HTLV-I1 Tax is the
etiological oncoprotein associated with adult T-cell leukemia (1). Tax
is a 40-kDa nuclear phosphoprotein whose expression sufficiently
immortalizes T-lymphocytes (2, 3). Although its mechanisms for
immortalization are not fully understood, Tax has been shown to
dysregulate cell cycle progression (4-6) and to subvert host DNA
damage surveillance pathways (7-11). Tax is also a well characterized
transcriptional activator of the HTLV-I long terminal repeats as well
as many cellular promoters (12, 13) with abilities to activate
cAMP-responsive, NF- Through NF- Because of potential implications for cellular transformation, it is of
interest to understand how transforming viruses activate NF- Plasmids--
pHA is a derivative of pM
(CLONTECH) in which the Gal4 DNA-binding domain
(BD) has been replaced by an HA tag (YPYDVPDYA). pHA-derived plasmids
pHA GST Pull-down Assay--
Expression and purification of GST,
GST-IKK- Protein Analysis--
Immunoprecipitations, Western blotting,
yeast two-hybrid assay, electrophoretic mobility shift assay (EMSA),
and CAT assay were performed as described (10, 17, 38, 39). Luciferase assay was according to Promega.
IKK Assay--
Cells were lysed in buffer (20 mM
Hepes, pH 7.3, 2.5 mM MgCl2, 10 mM
EGTA, 40 mM Tax Interacts Directly with IKK-
To address this issue, we co-expressed Tax (pIEX) and HA-tagged human
IKK-
IKK-
To confirm the association between Tax and IKK- Tax and IKK-
Previously, we have constructed and characterized 47 Tax point mutants
(16). The phenotypes of two mutants, Tax S258A and Tax L320G,
distinguish clearly between activity through the cellular CREB/ATF
versus the NF-
HeLa cells were separately co-transfected with pHA Evidence Supports Direct Contact of Tax with IKK-
To define better the details of protein-protein contact, we performed
modified protein hybrid assays in yeast (Fig. 3C). First we
sought to determine whether there was a direct contact of Tax with
either IKK-
That IKK-
The model deduced from results in yeast can be further tested in
mammalian cells. Indeed, if IKK- IKK-
Two salient points emerge from the current work. The first is the
unexpected finding of IKK-
The second instructive point from this work is that IKK-
An obvious question raised by our results is why should
oncoproteins interact via an adapter rather than directly with the IKK-B remain incompletely
understood. Although others have described an interaction between Tax
and a holo-I
B kinase (IKK) complex, the exact details of
protein-protein contact are not fully defined. Here we show that Tax
binds to neither IKK-
nor IKK-
but instead complexes directly
with IKK-
, a newly characterized component of the IKK complex. This
direct interaction with IKK-
correlates with Tax-induced I
B-
phosphorylation and NF-
B activation. Thus, our findings establish
IKK-
as a key molecule for adapting an oncoprotein-specific
signaling to IKK-
and IKK-
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
B-responsive, and serum-responsive promoters
(14-16). Despite its pleiotropic effects, specificity of Tax action
has been shown to occur through direct contacts with cellular proteins
(e.g. Refs. 10 and 17-19).
B, Tax modulates the expression of several cytokines and
proto-oncogenes. NF-
B is a key regulator of inflammatory responses
as well as cell death (20, 21). Ambiently, NF-
B exists in the
cytoplasm tightly associated with inhibitory proteins, I
Bs. Activity
of NF-
B is stimulated widely by cytokines, oxidative stress, phorbol
ester, and virus infection. One mechanism of NF-
B activation
involves site-specific phosphorylation at serines 32 and 36 of
I
B-
, followed by its ubiquitination and degradation (20, 21). To
date, several I
B kinases, including IKK-
and IKK-
, have been
cloned and characterized (22-27). IKK-
and IKK-
have been
suggested to be components of a larger multiprotein complex, which
includes NF-
B/RelA, I
B-
, MEKK-1, and NF-
B-inducing kinase
(NIK) (26, 28-30). Recently, two new proteins, IKK complex-associated protein (IKAP) and IKK-
/NEMO, with functions yet to be completely defined, have been identified in this holo complex (31-33).
B.
HTLV-I represents an attractive model; its oncoprotein, Tax, has been
suggested to target both IKK-
(34-36) and IKK-
(34-37),
presumably through direct protein-protein contacts. Mechanistically, how IKK-
and IKK-
are impinged upon by Tax has not been defined. Here we show that Tax binds to neither IKK-
nor IKK-
but instead contacts directly IKK-
. We propose that IKK-
functionally adapts oncoprotein signaling to IKK-
/IKK-
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and pHA
C contain full-length and C-terminally truncated
(amino acids 1-306) human IKK-
, respectively. pGST-IKK-
contains
IKK-
cDNA inserted into pGEX 4T-1 (Amersham Pharmacia Biotech).
pMBP-Tax, pIE-Tax, pIE-Tax mutants (pIEX, pIEX S258A, and pIEX L320G),
and pBD-Tax have been described previously (16, 17, 38). Plasmids
expressing IKK-
fused to Gal4 activation domain (AD-IKK-
),
AD-IKK-
, and AD-IKK-
in yeast are derived from pGAD424
(CLONTECH). Plasmid expressing IKK-
in yeast is based on histidine-marked pGHnf (17). Mouse IKK-
/
cDNAs (29), IKK-
/
mutants (29), and NF-
B-dependent reporter
(p
B-CAT) and control plasmids (10, 38, 39) have been described elsewhere.
, and MBP-Tax from Escherichia coli were
performed using protocols from Amersham Pharmacia Biotech and New
England BioLabs. Protein affinity chromatography was performed as
described previously (10).
-glycerophosphate, 1% Nonidet P-40, 1 mM dithiothreitol, 2 mM orthovanadate, and
protease inhibitor mixture). Clarified lysates were precipitated with
anti-Tax. Precipitates were washed with kinase buffer (20 mM Hepes, pH 7.3, 10 mM MgCl2, 2 mM MnCl2, 0.5 mM EGTA, 0.5 mM NaF, 0.5 mM vanadate, and 12.5 mM
-glycerophosphate). Kinase assay was performed for 30 min at 30 °C in kinase buffer supplemented with 3.3 µM
dithiothreitol, 20 µM ATP, 5 µCi of
[
-32P]ATP, and 3 µg of GST-I
B-
(Santa Cruz).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
--
Recently, full-length
cDNA clones for both mouse and human IKK-
(also termed NEMO)
have been isolated (31, 33, 40). Genetically, IKK-
is linked to Tax
activation of NF-
B (31). In assessing this, we considered two other
observations. First, IKK-
contains a striking coiled-coil domain in
its 51-353 residues (33, 40). Second, several other cellular
Tax-binding proteins share a characteristically structured coiled-coil
domain. Examples include GPS2 (17), ATF4/CREB2 (18, 19), TXBP181/HsMAD1
(10), TXBP151 (19), and TXBP121/KIAA0445 (GenBankTM
accession number AB007914). Thus, circumstantially, a particular coiled-coil structure likely represents a recognition motif for Tax.
Hence, we wondered whether the coiled-coil IKK-
protein could be a
direct Tax adapter that would help to explain NF-
B activation by
this oncoprotein.
(pHA
) in HeLa cells and performed reciprocal co-immunoprecipitations (Fig.
1A). HeLa cells transfected
singularly with pHA
abundantly expressed HA-IKK-
, which was
detected easily by direct immunoblotting with a mouse anti-HA antibody
(Fig. 1A, lane 1,
-HA (m)) and by
immunoprecipitation with a rabbit anti-HA antiserum (Fig.
1A, lane 2,
-HA (r)) followed by
immunoblotting with mouse anti-HA antibody. In HeLa cells
co-transfected with pHA
and pIEX (Fig. 1A, lanes
5 and 8), HA-IKK-
was observed in the mouse anti-Tax
precipitate (lane 5), and Tax was found in the rabbit
-HA
antiserum precipitate (lane 8). These reciprocal findings
are consistent with an intracellular Tax-IKK-
complex.
View larger version (34K):
[in a new window]
Fig. 1.
Tax binds IKK-
directly. A, co-immunoprecipitation of Tax and
IKK-
from cells. HeLa cells were transfected with pHA
alone
(lanes 1 and 2) or the indicated plasmids
(lanes 3-8). The crude cell extract was analyzed by Western
blotting with mouse anti-HA (
-HA (m), lane 1).
Cell lysates were also immunoprecipitated (i.p.) with either
rabbit
-HA (
-HA (r); lanes 2 and 6-8) or
rabbit anti-Tax (
-Tax (r), lanes 3-5). The
precipitates were analyzed by Western blotting with mouse
-HA
(
-HA (m), lanes 3-5) or mouse anti-Tax
(
-Tax (m), lanes 6-8). Relative migrations of
molecular mass markers are as shown. B, in vitro
GST pull-down assay. GST (lanes 1 and 2) and
GST-IKK-
(lanes 3 and 4) proteins were bound
to Sepharose beads. Beads were incubated with MBP-Tax, and bound
proteins were then eluted. Flow-through (FT, lanes
1 and 3) and eluates (lanes 2 and
4) were analyzed by Western blotting with rabbit anti-Tax.
Anti-Tax-reactive proteins are marked by an arrow and an
asterisk. The band marked by an
asterisk likely represents a degradation product.
C, yeast two-hybrid assay. Yeast SFY526 was transformed with
plasmids expressing the indicated proteins. Stable transformants were
selected and assayed for relative
-galactosidase activity in
chlorophenol red-
-D-galactopyranoside (CPRG)
units (38). Results are representative of three independent
experiments.
is one component of a larger holo-IKK complex (31, 33). In view
of this, the observed co-immunoprecipitation of IKK-
and Tax does
not exclude the possibility that IKK-
is simply a passenger protein
recovered as a consequence of Tax interaction with another member of
the IKK complex. To challenge this possibility, we performed in
vitro pull-down assays with GST-IKK-
and MBP-Tax purified from
E. coli. In agreement with a direct contact between Tax and
IKK-
, Fig. 1B verified that MBP-Tax bound to GST-IKK-
(lane 4) but not to GST alone (lane 2).
within a eukaryotic
cell, we checked for interactions in yeast (Fig. 1C). When
BD-Tax was co-expressed in yeast with AD-IKK-
, we observed that this
pair (Fig. 1C, column 4) conferred >20-fold
stimulation over the background
-galactosidase activity induced by
each plasmid singularly (Fig. 1C, columns 1 and
2). The interaction measured for Tax and IKK-
(Fig.
1C, column 4) is comparable with the previously characterized binding between Tax and HsMAD1 (Fig. 1C,
column 6, and Ref. 10). Because no IKK homologs have been
identified in the complete sequence of the yeast genome, we are
reassured that the observed results are unlikely to be consequences of
bridging fortuitously supplied by yeast IKKs. Hence, the GST pull-down assays and the yeast two-hybrid results collectively support a direct
interaction between Tax and IKK-
.
Interaction Correlates with I
B-
Phosphorylation and NF-
B Activation--
Tax activates NF-
B (14,
34-36). Although the involvement of MEKK-1, IKK-
, IKK-
, and NIK
have been suggested (34-37), the exact mechanisms for this activation
are not fully understood. Based on the above observation, we next asked
whether binding of IKK-
to Tax correlates functionally with Tax
activation of NF-
B.
B pathways. Thus, Tax S258A activates CREB/ATF but not NF-
B; whereas Tax L320G activates NF-
B but not
CREB/ATF (3, 16). We used these two mutants to clarify the significance
of Tax-IKK-
interaction for NF-
B activation.
and pIE vector
(Fig. 2, lane 1), pHA
and
pIEX (Tax wild type; lane 2), pHA
and pIEX S258A
(lane 3), or pHA
and pIEX L320G (lane 4). The
equivalent expression of Tax and Tax mutants was verified by Western
blotting (Fig. 2A). In parallel, nuclear NF-
B DNA binding
activity and cytoplasmic IKK activity associated with Tax were assessed
by EMSA (Fig. 2B) and in vitro kinase assay (IKK
act.; Fig. 2C), respectively. Consistent with previous
findings (16), Tax L320G activated NF-
B and IKK in a manner similar to wild type Tax (Fig. 2, B and C, compare
lanes 4 with lanes 2). By contrast, Tax S258A was
defective for this activation (Fig. 2, B and C,
compare lanes 3 to lanes 1 and 2).
Concordantly, anti-Tax serum (
-Tax; Fig. 2D)
co-immunoprecipitated IKK-
(as detected by immunoblotting with
-IKK-
serum) from cells that expressed Tax or Tax L320G (Fig.
2D, lanes 2 and 4), but not from cells that expressed Tax S258A (Fig. 2D, lane 3). In
the same immunoprecipitations, we also found that Tax or Tax L320G but
not Tax S258A associated with HA-IKK-
(Fig. 2E).
Considered together, these findings correlated Tax/Tax mutant binding
to IKK-
(Fig. 2E) with nuclear NF-
B activity (Fig.
2B), I
B-
phosphorylation (Fig. 2C), and
co-immunoprecipitation with IKK-
(Fig. 2D).
View larger version (27K):
[in a new window]
Fig. 2.
Tax interaction with IKK-
correlates with NF-
B activation.
HeLa cells were transfected with pHA
and pIE (lanes 1),
pHA
and pIEX (lanes 2), pHA
and pIEX S258A
(lanes 3), or pHA
and pIEX L320G (lanes 4).
A, expression of wild type and mutant Tax. HeLa extracts
with 12 µg of protein were analyzed by Western blotting with mouse
anti-Tax antibody. B, NF-
B DNA binding activity by EMSA.
HeLa nuclear extracts (1.5 µg of protein) were used for each lane.
Positions of the NF-
B shifted band and the free probe
(FP) are indicated. C, in vitro IKK
assay. Extracts from 5 × 106 HeLa cells were
incubated with 3 µg of mouse anti-Tax antibody. Immunoprecipitates
were assayed for IKK activity using GST-I
B-
as substrate.
Phosphorylated I
B-
was separated by 10% SDS-polyacrylamide gel
electrophoresis, and the autoradiograph was visualized with a Fuji
FLA-2000 phosphorimager. D, Tax association with IKK-
.
Monoclonal anti-Tax (
-Tax) immunoprecipitates
(i.p.) were probed with rabbit anti-IKK-
in Western
blotting. E, Tax association with IKK-
. Anti-Tax
immunoprecipitates were probed with rabbit anti-HA in Western
blotting.
but Not with
IKK-
nor IKK-
--
Data presented here and elsewhere are
compatible with the following: (a) direct binding of Tax to
IKK-
(Fig. 1); (b) direct/indirect association of Tax
with IKK-
and/or IKK-
(Fig. 2D and Ref. 34); and
(c) a Tax-induced increase in IKK activity (Fig.
2C and Refs. 34-36). Others have shown that IKK-
can
bind IKK-
directly (31) and associates with IKK-
in
vivo (33). Based on these observations, one cannot formally
distinguish between a model in which Tax contacts all three
IKK-proteins (IKK-
, IKK-
, and IKK-
; Fig.
3A) equally versus
another model in which Tax contacts only IKK-
, which then intermediates a signal to IKK-
and IKK-
(Fig. 3B).
View larger version (24K):
[in a new window]
Fig. 3.
Tax association with
IKK- /
is mediated by
IKK-
. A, schematic
representation of direct contacts between Tax and IKK-
/
and
IKK-
. Binding of IKK-
to IKK-
is also depicted. B,
alternative representation of direct interaction of Tax with only
IKK-
. C, IKK-
bridges Tax to IKK-
/
as revealed
by modified yeast protein hybrid assay. Yeast SFY526 was transformed
with plasmids carrying different markers and expressing the indicated
proteins. D, IKK-
activates NF-
B through IKK-
/
.
HeLa cells were transfected with p
BCAT (column 1),
p
BCAT plus pHA
(column 2), or p
BCAT plus pHA
plus increasing amounts of plasmids expressing the indicated DN mutants
(columns 3-6). These mutants have little effect on the
basal CAT activity (data not shown). Results are representative of
three independent experiments. CPRG, chlorophenol red
-D-galactopyranoside.
or IKK-
. We co-expressed either BD-Tax and AD-IKK-
(Fig. 3C, column 2) or BD-Tax and AD-IKK-
(Fig. 3C, column 3) in yeast. Neither pair showed
any increased reporter activity over the background level observed with
BD-Tax alone (Fig. 3C, column 1). By contrast,
yeast transformants that co-expressed BD-Tax and AD-IKK-
showed an
activity >10-fold over background (Fig. 3C, compare
column 4 with column 1). These results are most simply interpreted by a direct Tax interaction with IKK-
but not
with IKK-
nor IKK-
(Fig. 3B).
adapts Tax to either IKK-
or IKK-
is supported by
further functional evidence. Thus, if one co-expresses three proteins
simultaneously in yeast, either a nonfused IKK-
with BD-Tax and
AD-IKK-
(Fig. 3C, column 5) or a nonfused
IKK-
with BD-Tax and AD-IKK-
(Fig. 3C, column
6), then >5-fold IKK-
-dependent increases in
reporter activity are measured (Fig. 3C, compare columns 5 and 6 with columns 1-3).
This role for unfused IKK-
in reconstructing BD-Tax and
AD-IKK-
/AD-IKK-
activity is fully consistent with a model in
which IKK-
contacts IKK-
, IKK-
, and Tax independently while
bridging a functional interaction between Tax and either IKK-
or
IKK-
(Fig. 3B). Thus, for Tax oncoprotein signaling,
IKK-
is positioned upstream of IKK-
/IKK-
activation and
I
B-
phosphorylation.
represents an upstream adapter
whereas IKK-
/IKK-
represent downstream effectors, then one
prediction is that a dominant negative (DN) form of the latter would
repress activity from the former. When overexpressed in HeLa cells,
IKK-
conferred a 4-fold activation of an
NF-
B-dependent reporter (Fig. 3D, compare
column 2 with column 1). Notably, this activation
was abrogated by co-expression of DN mutants of either IKK-
or
IKK-
(Fig. 3D, columns 3-6). We observed that
compared with DN IKK-
(Fig. 3C, columns 5 and
6), a higher dosage of DN IKK-
is required for the
inhibition of IKK-
-mediated activation (columns 3 and
4). This might be explained by the intrinsic differences in
specific kinase-activity for IKK-
and IKK-
(27).
Is a Mediator for Tax Activation of
NF-
B--
Previously, a C-terminal truncated form of IKK-
(IKK-
C) has been described as a DN inhibitor of IKK-
function
(33). We used a similarly constructed IKK-
C mutant to explore the role of IKK-
in Tax activation of NF-
B. In HeLa and Jurkat cells, expression of wild type IKK-
enhanced Tax activation of an
NF-
B-dependent reporter (Fig.
4, compare group 3 with
group 2). In contrast, activation by Tax was progressively
diminished with increased expression of IKK-
C (Fig. 4,
groups 4-6, compared with group 2), consistent
with a DN inhibition of function. This finding agrees with the
demonstration that a genetic defect in IKK-
led to loss of
responsiveness to several NF-
B-activating stimuli including Tax
(31).
View larger version (21K):
[in a new window]
Fig. 4.
A dominant negative IKK-
protein abrogates Tax activation of
NF-
B. Left y axis and
open bars, CAT assay in HeLa cells. The reporter plasmid is
p
BCAT. Right y axis and filled bars:
luciferase (luc.) assay in Jurkat cells. The reporter
plasmid is pNFkB-Luc (Stratagene). Results are representative of three
independent repetitions. The IKK-
C mutant has minimal effect on
the basal CAT and luciferase activity (data not shown).
as a direct Tax-binding factor. Previously, several studies have shown an association of Tax with IKK
complex (34-37). Although contact with IKK-
and IKK-
was suggested (34), the exact details were not defined. We now show that
the functional ability of Tax to activate IKK-
/IKK-
is unlikely
to stem from immediate contact but occurs indirectly through binding
between Tax and IKK-
.
, previously
shown to be essential for IKK-
/IKK-
activation of NF-
B (31,
33), is located functionally and physically upstream of its kinase
counterparts. Although our work does not directly address how Tax
activates IKK-
/IKK-
, the finding that Tax binds IKK-
but not
IKK-
/IKK-
ascribes physical recruitment of Tax into the holo-IKK
complex to the functional adapter function suggested for IKK-
.
IKK-
-tethered Tax protein is further expected to bring Tax-associated kinase(s) (e.g. MEKK-1 and/or NIK (35-37))
to the local proximity of IKK-
/IKK-
for phosphorylation-mediated activation.
/IKK-
kinases? Although this is not completely understood, one suggestive explanation is provoked by our recent finding that the
human gene for IKK-
localizes on the X chromosome (40). If IKK-
is indeed essential (as has been shown in Refs. 31 and 33) for a
multitude of signals that activate NF-
B, then some hints of
gender-linked NF-
B-based diseases might have been expected. In the
absence of evidence for such, a reasonable deduction is that many yet
to be described IKK-associated proteins (another example is IKAP; Ref.
32) might also provide adapter function. The predicted existence of
many adapters could explain specificity and redundancy of signaling
that cannot otherwise be easily reconciled if each of the numerously
different NF-
B-activating signals all contacted directly
IKK-
/IKK-
. Future studies are needed to clarify the identities of
other adapters and how they function in dictating specificity of
IKK activation.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Ko Okumura.
![]() |
FOOTNOTES |
---|
* 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.
§ Present address: Inst. of Molecular Biology, The University of Hong Kong, Pokfulam, Hong Kong.
** To whom correspondence should be addressed: LMM/NIAID/NIH, Bldg. 4, Rm. 306, 9000 Rockville Pike, Bethesda, MD 20892-0460. Tel.: 301-496-6680; Fax: 301-480-3686; E-mail: kjeang{at}niaid.nih.gov.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
HTLV-I, human T-cell
leukemia virus type I;
IKK, IB kinase;
MEKK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase kinase;
NIK, NF-
B-inducing kinase;
IKAP, IKK complex-associated protein;
NEMO, NF-
B essential modulator;
HA, hemagglutinin;
BD, Gal4
DNA-binding domain;
AD, Gal4 activation domain;
GST, glutathione
S-transferase;
MBP, maltose-binding protein;
EMSA, electrophoretic mobility shift assay;
CAT, chloramphenicol
acetyltransferase;
CREB, cAMP-responsive element-binding protein;
ATF, activating transcription factor;
DN, dominant negative.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|