From the Center for Apoptosis Research and the Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Received for publication, November 8, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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vCLAP, the E10 gene product of equine
herpesvirus-2, is a caspase-recruitment domain (CARD)-containing
protein that has been shown to induce both apoptosis and NF- Equine herpesvirus-2 (EHV-2)1 is a gammaherpesvirus
related to other lymphotropic
herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. EHV-2
contains 79 reading frames that encode 77 distinct molecules, several
of which show significant similarity to cellular genes. One of these
molecules, vCLAP (also called vCIPER/E10/vCARMEN) (1-4), a
CARD-containing apoptotic protein, was recently found to induce both
apoptosis and activation of the transcription factor NF- In most resting cells, NF- The kinase activity responsible for phosphorylation of I Cell Culture, Transfection, and Treatment--
Cells were
cultured either in Dulbecco's modified Eagle's medium (DMEM) (HeLa,
Rat-1, or 5R cells) or DMEM/F12 (293T cells; Life Technologies, Inc.),
supplemented with 10% fetal bovine serum, 200 µg/ml penicillin, and
100 µg/ml streptomycin sulfate. Transfections were carried out using
LipofectAMINE (Life Technologies, Inc.). Cells were stimulated with
either 20 ng/ml recombinant human TNF- Expression Vectors and Antibodies--
Constructs encoding
full-length IKK Biochemical Analysis--
Immunoprecipitations were performed as
described previously (20), and the precipitated proteins were analyzed
by SDS polyacrylamide gel electrophoresis followed by immunoblotting.
GST pull-down assays, luciferase reporter gene assays, and IKK kinase
assays were performed as described (20, 22).
Confocal Microscopy--
293T cells were grown on coverslips and
then transfected with the GFP-tagged IKK vCLAP Interacts with the IKK Complex and Persistently Activates the
IKK Kinases--
Whereas in resting cells the IKK kinases are
inactive, potent activators, such as TNF- vCLAP Interacts Directly with and Requires IKK
To rule out the possibility that other proteins were necessary for the
vCLAP-IKK
To address the physiological relevance of this finding, we transiently
expressed vCLAP in wild type or IKK
In contrast to vCLAP, Bcl10 was unable to interact with IKK Mapping of the Interaction Domains of vCLAP and IKK
To extend the characterization of the vCLAP-IKK
To confirm that vCLAP associates intercellularly with IKK vCLAP Mediates Activation of the IKKs through Oligomerization of
IKK
We then tested whether enforced oligomerization of the CTD of vCLAP
could induce NF- Although previous studies have shown that the EHV-2-encoded vCLAP
protein activates NF- Activation of NF- Recently, several independent groups have demonstrated that the
cellular homologue of vCLAP, Bcl10 (also called
cCLAP/CIPER/hE10/CARMEN), was also able to activate NF-B
activation in mammalian cells. vCLAP has a cellular counterpart,
Bcl10/cCLAP, which is also an activator of apoptosis and
NF-
B. Recent studies demonstrated that vCLAP activates NF-
B
through an I
B kinase (IKK)-dependent pathway, but
the underlying mechanism remains unknown. In this report, we
demonstrate that vCLAP associates stably with the IKK complex through
direct binding to the C-terminal region of IKK
. Consistent with this
finding, IKK
was found to be essential for vCLAP-induced NF-
B
activation, and the association between vCLAP and the IKK complex
induced persistent activation of the IKKs. Moreover, enforced
oligomerization of the isolated C-terminal region of vCLAP, which
interacts with IKK
, can trigger NF-
B activation. Finally,
substitution of the C-terminal region of IKK
, which interacts with
vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that
was able to activate NF-
B when ectopically expressed in
IKK
-deficient cells. These data suggest that vCLAP-induced oligomerization of IKK
, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of
NF-
B.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B in
mammalian cells. vCLAP, like its cellular counterpart Bcl10, contains
two domains, an N-terminal CARD that can oligomerize via homotypic
interactions and a C-terminal domain that probably functions as the
NF-
B activation domain. Because NF-
B activation is considered to
be a survival signal, virally encoded proteins, such as vCLAP, may be
utilized by viruses as a strategic tool to initiate self-replication or
to suppress apoptosis in infected cells (5).
B is sequestered in the cytoplasm through
interaction with the I
B inhibitory proteins. I
Bs mask the NF-
B
nuclear localization signal, thereby preventing its nuclear uptake.
Exposure of cells to a wide variety of stimuli, such as viral or
bacterial infection, inflammatory cytokines, or UV irradiation leads to
the rapid phosphorylation, ubiquitination, and ultimately proteolitic
degradation of the I
Bs (6-9). This allows the activated NF-
B to
translocate to the nucleus and activate the transcription of several
NF-
B target genes.
Bs is
present in a large (700-900 kDa) cytoplasmic complex composed of two
catalytic subunits, IKK
and IKK
(10-14), and a noncatalytic subunit termed IKK
(also called NEMO, IKKAP1, or FIP-3) (15-18). We
and others have recently demonstrated that activation of the IKK
complex could be achieved through IKK
-mediated oligomerization of
the IKK kinases, indicating that IKK
functions as an adaptor to link
the IKKs with the upstream regulators of NF-
B (19, 20). Here we show
that vCLAP associates directly and specifically with IKK
through its
C-terminal glycine-rich domain and may regulate the activity of the IKK
complex through CARD-mediated oligomerization of IKK
.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(Sigma) or 0.1 µg/ml AP1510
(Arriad) for the indicated times. NEMO/IKK
-deficient Rat-1 cells
(5R) are a gift from S. Yamaoka.
, IKK
, IKK
, or vCLAP or truncated mutants have
been described previously (1, 20, 21). The FKBP12 fusion of vCLAP
C-terminal domain (CTD) was constructed in a modified pcDNA3-T7
vector, which contains a T7 tag sequence, by fusing three tandem
repeats of FKBP12 cDNA in frame with the cDNA of vCLAP-CTD
(residues 108-311) as described previously (1). The plasmids
expressing the green fluorescent protein (GFP) (pEGFP-C1) and the red
fluorescent protein (RFP) (pDsRed1-N1) were from
CLONTECH. FLAG-M5 antibody was from Sigma. T7-horseradish peroxidase conjugate antibody was from Novagen. IKK
and IKK
polyclonal antibodies were from Santa Cruz.
or RFP-tagged vCLAP
separately or together, with the indicated vectors. 24 h after
transfection, cells were fixed with 4% paraformaldehyde in
phosphate-buffered saline for 30 min. The coverslips were mounted on a
glass slide, and the fluorescence was detected by confocal microscopy
using an excitation wavelength of 488 nm and a detection wavelength of
522 nm (GFP) or an excitation wavelength of 568 nm and a detection
wavelength of 585 nm (RFP). Images were Kalman-averaged with a Kalman
filter to increase the signal/noise ratio.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, interleukin-1, or
lipopolysaccharide, induce a very rapid IKK activation, detectable
within minutes. However, numerous studies have shown that this
activation is only transient and after ~30 min decreases to about
25% of its peak value (12, 14, 23). Using a luciferase reporter assay,
we and others demonstrated that expression of vCLAP results in a robust
activation of NF-
B (1-4). Because of the very high level of this
activation, we asked whether vCLAP could persistently activate the
IKKs, resulting in a sustained rather than transient activation of
NF-
B. To answer this question, endogenous IKK
was isolated by
immunoprecipitation from extracts prepared from vCLAP-transfected or
TNF-
-treated HeLa cells and assayed for IKK catalytic activity using
a GST-I
B
fusion protein as a substrate. Consistent with previous
observations, TNF-
stimulation of HeLa cells induced high but
transient IKK
kinase activity (Fig.
1A); the activity, which was
maximum after 10 min of stimulation, declined sharply with time and was
barely detectable after 90 min of stimulation. However, compared with
TNF-
stimulation, overexpression of vCLAP in HeLa cells induced a
robust and sustained IKK
kinase activity in the absence of any
external stimulation (Fig. 1B). IKK
protein expression
was comparable in vCLAP-transfected and nontransfected cells,
indicating that vCLAP activates endogenous IKK
by a
post-translational mechanism. To determine whether vCLAP associates
stably with the IKK complex, immunoprecipitates obtained using
anti-IKK
or anti-FLAG antibodies were assayed for the presence of
vCLAP or the IKK components, respectively. As shown in Fig.
1B, vCLAP was readily detected after precipitation of
endogenous IKK
. Moreover, IKK
and IKK
were also detected in
immunocomplexes obtained after precipitation of vCLAP (Fig.
1C). The vCLAP immunoprecipitates also possessed IKK kinase
activity (Fig. 1C). These results provide direct biochemical evidence that vCLAP associates stably with the IKK complex and is able
to persistently activate the IKK kinases.
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Fig. 1.
vCLAP associates stably with the IKK complex
and persistently activates the IKK kinases. A, HeLa
cells were either left untreated or were incubated with TNF- for the
indicated times and then lysed. The lysates were immunoprecipitated
with anti-IKK
antibody, and the IKK activity associated with IKK
was determined by immune complex kinase assay (KA).
Expression of endogenous IKK
was determined by immunoblotting
(IB). B and C, HeLa cells were
transfected with expression construct for FLAG-vCLAP. At the indicated
times after transfection, cells were lysed, and equal amount of
proteins were immunoprecipitated with anti-IKK
(B) or
anti-FLAG (C) antibody. The immunoprecipitates were assayed
for IKK activity by immune complex kinase assay and analyzed by
SDS-PAGE and immunoblotted with anti-FLAG antibody (B) or
anti-IKK
and anti-IKK
antibodies (C). The cellular
extracts were also immunoblotted with anti-IKK
and anti-FLAG
(B) or anti-FLAG (C) antibodies.
for Activation of
NF-
B--
To determine which component of the IKK complex interacts
with vCLAP, 293T cells were transfected with expression vectors for
FLAG-tagged vCLAP and T7-IKK
with or without T7-IKK
. As shown in
Fig. 2A, a small amount of
IKK
was coimmunoprecipitated with vCLAP in the absence of ectopic
IKK
. However, a remarkably higher amount of IKK
was
coimmunoprecipitated with vCLAP in the presence of coexpressed IKK
(Fig. 2A). The ectopic T7-IKK
was also detected in these
complexes (Fig. 2A). No IKK
or IKK
were precipitated
with the FLAG antibody in the absence of FLAG-vCLAP (Fig.
2A). This result shows that IKK
mediates the interaction of vCLAP with the IKK complex.
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Fig. 2.
IKK mediates the
assembly of the vCLAP-IKK complexes and is required for vCLAP-induced
NF-
B activation. A, 293T cells
were transfected with expression constructs for T7-IKK
, T7-IKK
,
and FLAG-vCLAP as indicated. 24 h after transfection, cells were
lysed, and the lysates immunoprecipitated with anti-FLAG antibody. The
immunoprecipitates were immunoblotted (IB) with anti-T7
antibody. Expression of T7-IKK
, T7-IKK
, or FLAG-vCLAP was
determined by immunoblotting with anti-T7 or anti-FLAG antibodies,
respectively. B, in vitro interaction of vCLAP
with GST-IKK
. 35S-Labeled vCLAP (lane 1,
10% input) was incubated with an equal amount of GST
(lane 2) or GST-IKK
(lane 3) proteins bound to
glutathione-Sepharose beads. Bound proteins were then eluted and
analyzed by SDS-PAGE and autoradiography. C, Rat-1 cells or
the IKK
-deficient Rat-1 derivative (5R) cells were transfected with
5×
B-luciferase reporter together with either empty vector or vCLAP
or Bcl10 expression construct. 24 h after transfection, cells were
either left untreated or incubated with TNF-
for 5 h. Cells
were then collected and lysed, and the luciferase activity in the cell
lysates was determined. pRSC-LacZ was included in all transfection
reactions to normalize the transfection efficiency. Mean values ± S.E. are shown from three independent experiments performed in
duplicate.
interaction, we analyzed the ability of a GST-IKK
fusion protein to associate with an in vitro-translated 35S-labeled vCLAP. In agreement with a direct interaction
between vCLAP and IKK
, 35S-labeled vCLAP bound to the
GST-IKK
fusion protein but not the GST control (Fig. 2B).
-deficient Rat-1 cells (15). In
contrast to wild type Rat-1 cells, no NF-
B activation was elicited
in the IKK
-deficient 5R cells after transfection with the vCLAP
construct or treatment with TNF-
(Fig. 2C). This result
provides genetic proof for the requirement of IKK
in vCLAP-induced activation of NF-
B, confirming its role as a molecular adaptor in
the assembly of the vCLAP-IKK complexes. The inability of vCLAP to
induce NF-
B activation in 5R cells cannot be attributed to defects
in the NF-
B pathway downstream of IKK
, because transfection of
these cells with IKK
can restore NF-
B activation by Tax, which is
expressed stably in this cell line (Ref. 15 and data not shown).
in
vitro (data not shown). However, like vCLAP, Bcl10 was able to
induce NF-
B activation in Rat-1, but not in 5R cells (Fig. 2C), suggesting that Bcl10 could relay its signal to IKK
indirectly.
--
We
next mapped the regions of vCLAP and IKK
that are required for their
interaction. FLAG-tagged IKK
was expressed in 293T cells with
T7-tagged full-length domain, CARD (residues 1-107), or CTD (residues
108-311) of vCLAP. Extracts prepared from the transfected cells
were immunoprecipitated with an anti-FLAG antibody, and the resulting
immune complexes were analyzed by Western blotting with an anti-T7
antibody that recognizes the T7-vCLAP variants. Both the full-length
domain and the CTD of vCLAP were able to bind to IKK
(Fig.
3A). In contrast, the CARD did
not interact with IKK
(Fig. 3A). The same results were
obtained using a GST-IKK
pull-down assay, which showed that the
recombinant GST-IKK
fusion protein is able to bind the in
vitro-translated 35S-labeled full-length domain or the
CTD of vCLAP, but not the CARD of vCLAP (not shown). Combined, these
results show that the CTD of vCLAP mediates its interaction with
IKK
.
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Fig. 3.
Interaction domains of IKK
and vCLAP. The CTD of vCLAP mediates its interaction
with IKK
. vCLAP, IKK
, and their deletion mutants are represented
on the top. A, 293T cells were transfected with
FLAG-IKK
together with either empty vector or T7-tagged full-length
(FL) vCLAP, vCLAP-CARD, or vCLAP-CTD expression constructs.
24 h after transfection, cells were lysed, and the lysates were
immunoprecipitated (IP) with anti-FLAG antibody. The
immunoprecipitates were immunoblotted (IB) with anti-T7
antibody. The expression of FLAG-IKK
and the different T7-CLAP
chimeras was determined by immunoblotting with anti-FLAG or anti-T7
antibodies, respectively. B, IKK
interacts with vCLAP via
its C terminus. 293T cells were transfected with T7-vCLAP and different
FLAG-tagged full-length (FL) or C-terminally truncated
IKK
expression constructs. 24 h after transfection, cells were
lysed, and the lysates were immunoprecipitated (IP) with
anti-FLAG antibody. The immunoprecipitates were immunoblotted
(IB) with anti-T7 antibody. The expression of T7-vCLAP and
the different FLAG-IKK
truncated mutants was determined by
immunoblotting with anti-T7 or anti-FLAG antibodies, respectively.
LZ, leucine zipper. C, recruitment of
IKK
into vCLAP filaments. RFP-tagged vCLAP and GFP-tagged IKK
were expressed either alone or together in 293T cells. 24 h after
transfection, cells were then examined and photographed under a
fluorescent microscope.
interaction, we
expressed T7-tagged vCLAP in 293T cells together with several FLAG-tagged full-length or truncated IKK
. vCLAP was found to specifically associate with full-length IKK
but not with the C-terminally truncated IKK
() or IKK
() (Fig.
3B). Removal of the last 119 amino acids of IKK
strongly
reduced its interaction with the vCLAP (Fig. 3B). Taken
together, these data indicate that the interaction between vCLAP and
IKK
involves sequences in the C-terminal region of IKK
.
when the
two proteins are coexpressed in the same cell, we cotransfected 293T
cells with constructs encoding GFP-IKK
and RFP-vCLAP fusion proteins and then monitored the subcellular localization of these proteins by confocal microscopy. As shown in Fig. 3C, the
two proteins exhibited different patterns of cellular localization when
expressed alone. Whereas vCLAP exhibited a clear pattern of discrete
and interconnecting cytoplasmic filaments, IKK
displayed a somewhat
punctuate cytoplasmic or whole-cell distribution. However, coexpression
of the two proteins resulted in redistribution of IKK
to the vCLAP
filaments. This observation is consistent with a direct interaction
between vCLAP and IKK
.
--
Our data demonstrate that IKK
mediates the association
of vCLAP with the IKK kinases and is required for vCLAP-induced
activation of NF-
B. This suggest that IKK
is an adaptor molecule
with a C-terminal half that binds to NF-
B activators like vCLAP (see above), RIP (18-20, 24), or Tax (25-27) and an N-terminal half that
is required for interaction with the IKK kinases (20, 28). vCLAP has a
bipartite structure consisting of an N-terminal CARD and a C-terminal
domain that interacts with IKK
(see above). Interestingly, we and
others have shown that the CARDs of cellular and viral CLAP proteins
are important for homo- and heterotypic interactions (1-4). It is
therefore likely that CARD-mediated self-association of vCLAP could
induce oligomerization of IKK
resulting in activation of the IKK
complex. To test this hypothesis, we generated a fusion protein
(CARD-IKK
-
C) composed of the CARD of vCLAP linked to the
N-terminal part (residues 1-200) of IKK
(IKK
-
C) and
determined its ability to induce NF-
B activation. To rule out the
possibility that the CARD-IKK
-
C chimera functions through
interaction with the endogenous IKK
protein, we examined its ability
to activate NF-
B in the IKK
-deficient 5R cells. As shown in Fig.
4A, transient transfection of
the CARD-IKK
-
C chimera resulted in a large increase of NF-
B
activity in a dose-dependent manner. In contrast, neither
the separate CARD of vCLAP nor IKK
-
C were able to activate the
NF-
B when transfected at either low or high doses (Fig.
4A). Moreover, a single point mutation of a conserved
residue in the CARD of vCLAP that abrogates homodimerization (L49R) (1)
prevented NF-
B activation by the chimeric protein (Fig.
4A). Similar results were obtained when the CARD of Bcl10 was used instead of vCLAP-CARD in the above experiments (data not
shown).
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Fig. 4.
Activation of NF- B
through vCLAP-induced oligomerization of
IKK
. A, IKK
-deficient (5R)
cells were transfected with 5×
B-luciferase reporter together with
different amounts (0.1 and 0.3 µg) of the indicated expression
constructs, with or without kinase-inactive mutant of IKK
(DN
IKK
). 24 h after transfection, cells were lysed, and the
luciferase activity was determined as in the legend of Fig. 2. The
CARD-IKK
-
C chimera is represented by a bar diagram.
B, enforced oligomerization of the CTD of vCLAP induces
activation of NF-
B. Rat-1 cells or the IKK
-deficient Rat-1
derivative (5R) cells were transfected with 5×
B-luciferase
reporter together with either empty vector or the indicated expression
constructs. 24 h after transfection, cells were either left
untreated or incubated with AP1510 for 6 h. The luciferase
activity in the transfected cell lysates was assayed and normalized as
in the legend of Fig. 2.
B activation. For this purpose, the CTD of vCLAP was
fused to a 3-fold repeat of the FKBP12 polypeptide, which oligomerizes
when it binds to the cell-permeable synthetic organic ligand AP1510
(29). As shown in Fig. 4B, transient tranfection of this
construct into Rat-1 cells, but not in the IKK
-deficient 5R cells,
induced a large NF-
B activation in a ligand-dependent manner. No NF-
B activation was detected when the FKBP12-CTD
construct was cotransfected with kinase-inactive IKK
(not shown) or
after treatment of empty vector-transfected 293T cells with AP1510
(Fig. 4B). Taken together, these results demonstrate that
vCLAP-induced oligomerization of IKK
is the triggering event leading
to activation of the IKK complex.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B in mammalian cells (1, 4), the mechanism by
which vCLAP interfaces with the cell's NF-
B-activating machinery
remains unclear. In this paper, we report several observations that,
when combined, provide a potential mechanism for vCLAP-induced NF-
B
activation. First, we show that the IKK kinases are persistently activated in vCLAP-expressing cells, which might explain the robust NF-
B activity observed in vCLAP-transfected cells (1, 4). Second, we
demonstrate that vCLAP, via its C-terminal domain, interacts physically
with the IKK complex through direct binding to the C-terminal part of
IKK
. Consistent with this observation, IKK
was found to be
essential for vCLAP-induced activation of NF-
B. Third, we
demonstrate that vCLAP activates the IKK complex through
oligomerization of IKK
. Indeed, CARD-dependent
clustering of the N-terminal part of IKK
, which we have previously
shown to interact with the IKK kinases (20), was able to activate NF-
B. Moreover, enforced oligomerization of the CTD of vCLAP was
able to induce a large increase in NF-
B activity in Rat-1 cells but
not in the IKK
-deficient 5R cells. This mechanism of NF-
B
activation by vCLAP, namely oligomerization-induced activation of the
IKK kinases via IKK
, is reminiscent of the model we proposed for
RIP-induced activation of NF-
B after ligation of TNF-R1 (20). Based
on this model, TNF-
stimulation induces binding of RIP to, and
concomitant oligomerization of IKK
, which in turn passes the
oligomerization signal to the effector kinases, resulting in their
activation through autophosphorylation of their T-loop serines (20).
However, in contrast to vCLAP, which binds stably to the IKK complex,
RIP releases the activated IKK complex after its oligomerization,
resulting in transient rather than persistent activation. Therefore,
our results illustrate the ubiquitous role of oligomerization in IKK activation.
B by expression of a single viral protein has been
described in several studies, indicating that infection with an intact
virus is not always required for NF-
B activation. One well
documented example of this is the human T-cell leukemia virus-Tax
protein (30-33). Tax has been shown to interact with the IKK complex
through direct interaction with IKK
(25-27). Moreover, Tax mutants
defective in IKK
binding failed to activate NF-
B (34).
Interestingly, Tax has been shown to function as a dimerizer that
stabilizes dimer formation in other proteins (35). It is therefore
possible that Tax-induced oligomerization of IKK
is, as for vCLAP,
the triggering event in the activation of the IKK kinases by Tax.
B when
expressed in cells, although at a lesser degree than vCLAP. Bcl10
requires IKK
for activation of NF-
B (Fig. 2C). Our
preliminary results suggest that cCLAP interacts with the IKK complex,
as immunoprecipitation of the endogenous cCLAP results in isolation of
the IKK components. However, in a GST-IKK pull-down assay, in
vitro-translated 35S-labeled cCLAP was not able to
bind to recombinant GST-IKK
or GST-IKK
(data not shown),
indicating that the cCLAP-IKK complex interaction could be indirect or
regulated by a post-translational modification of cCLAP, such as
phosphorylation. Future studies will reveal the precise physiological
function of cCLAP and how it activates the IKK complex.
![]() |
ACKNOWLEDGEMENT |
---|
We thank W. C. Green for the IKK kinase
inactive construct and S. Yamaoka and A. Israel for the
NEMO/IKK
-deficient Rat-1.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant CA85421 (to E. S. A.).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.
Contributed equally to this work.
¶ Special fellow of the Leukemia and Lymphoma Society.
§ To whom correspondence should be addressed: Thomas Jefferson University, Kimmel Cancer Inst., Bluemle Life Sciences Bldg., Rm. 904, 233 S. 10th St., Philadelphia, PA 19107. Tel.: 215-503-4632; Fax: 215-923-1098; E-mail: E_Alnemri@lac.jci.tju.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.C000792200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EHV, equine
herpesvirus;
TNF, tumor necrosis factor;
CARD, caspase-recruitment
domain;
IKK, IB kinase;
RIP, receptor-interacting protein;
GST, glutathione S-transferase;
GFP, green fluorescent protein;
RFP, red fluorescent protein;
CTD, C-terminal domain;
PAGE, polyacrylamide gel electrophoresis;
DMEM, Dulbecco's modified Eagle's
medium.
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