From the a Institute and Department of Microbiology and Immunology, National Yang-Ming University, Taipei 11221, Taiwan, the c Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan, the i Immunology Research Center, National Yang-Ming University, Taipei 11221, Taiwan, the e Department of Microbiology, Immunology, and Molecular Genetics, Jonsson Comprehensive Cancer Center and Molecular Biology Institute, UCLA, Los Angeles, California 90095, the f Department of Immunology, Juntendo University School of Medicine, Tokyo 113, Japan, and the g Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
Received for publication, August 23, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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LIGHT (homologous to
lymphotoxins, shows inducible expression, and
competes with herpes simplex virus glycoprotein D for
herpesvirus entry mediator, a receptor expressed by
T lymphocytes) is a member of the tumor necrosis factor
superfamily that can interact with lymphotoxin- Lymphotoxin- The cytoplasmic domains of TNFR families function as docking sites for
downstream signaling molecules. Signaling occurs mostly through two
classes of cytoplasmic adaptor proteins: death domain-containing molecules and TNFR-associated factors (TRAFs). The death
domain-containing molecules or TRAFs are recruited to the cytoplasmic
domain of members of TNFR after engagement with ligands. The
cytoplasmic domain of LT Apoptosis signal-regulating kinase 1 (ASK1), also called
mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 5 (MEKK5), can be activated in response to various stress signals, including genotoxic stress (21), oxidative stress, reactive
oxygen species (ROS) (22), and laminar flow (23). Furthermore, the
kinase-inactive mutant of ASK1 inhibits cell death induced by tumor
necrosis factor, Fas ligation, anti-cancer drugs, or withdrawal of
neurotrophic factors (21, 24-27). ASK1 functions as an upstream
component of the kinase cascades and interacts with a variety of
molecules involved in stress-induced signaling pathways (21, 24). ASK1
phosphorylates and activates MKK4/7, which then activates the c-Jun
NH2-terminal protein kinases (JNKs), also known as the
stress-activated protein kinases. JNK activation requires
phosphorylation at a specific motif (TPY). Moreover, ASK1
phosphorylates and activates MKK3 and MKK6, leading to activation of
the p38 mitogen-activated protein kinases (24, 28, 29). It has been
reported that JNK and p38 activations are abolished in
ASK1 Signaling mediated by death domain-containing receptors, such as TNFRI
and Fas, could be inhibited efficiently by caspase inhibitors. However,
caspase inhibitor has only a partial effect to prevent
LIGHT/IFN- Cell Culture--
The human hepatoma cells (Hep3BT2), human
cervical carcinoma cells (HeLa), human embryonic kidney (HEK293) cells,
and traf knockout mouse embryonic fibroblasts (MEFs) were
maintained in Dulbecco's modified Eagle's medium (Invitrogen),
supplemented with 10% (v/v) heat-inactivated fetal bovine serum
(Invitrogen) at 37 °C in 5% (v/v) CO2.
Plasmids and Transfection--
Plasmids containing the hLT Generation of Anti-LT Generation of LIGHT Mutein--
The cDNA of extracellular
region of LIGHT was cloned into pIZ/V5-His-FLAG (Invitrogen).
Substitution of Arg228 by glutamic acid was performed by
overlap extension using polymerase chain reaction (33). The primers
used for polymerase chain reaction were designed to introduce an
XhoI site as described in the followings: 5'-GAGGATGGTACCCGGTCTTACTTC-3' (sense) and 5'-GAGTCGAACCAGGCGTTCATC-3' (antisense). The PCR products were ligated at the XhoI
site of pIZ/V5-His-FLAG-LIGHT to create pIZ/V5-His-FLAG-LIGHT(R228E). The construct was autosequenced (MB Mission Biotech) for verification of the mutation. The pIZ/V5-His-FLAG-LIGHT(R228E) construct was transfected into Sf21 cells by LipofectinTM
(Invitrogen). Stable transfectants were selected with 500 µg/ml Zeocin (Invitrogen). Protein was purified by agarose beads conjugated with anti-FLAG antibody (M2) and followed by dialysis in
phosphate-buffered saline as described (30).
Generation of ASK1-KE Stable Transfectants--
ASK1-KE DNA
construct (a gift from Dr. Wen-Chen Yeh) was transfected into Hep3BT2
using LipofectAMINETM (Invitrogen) as suggested by the
vendor. Stable transfectants were selected with G418 (800 µg/ml
Geneticin; Sigma), followed by immunoblot analysis to confirm the
expression of ASK1-KE.
Antibodies and Other Reagents--
The expression of ASK1-HA and
TAK1-HA was detected by using anti-HA mAb (clone 3F10; Roche Molecular
Biochemicals) or anti-human ASK1 antibodies (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA). The expression of c-Myc-tagged TRAF2-DN,
TRAF3-DN, and TRAF5-DN was detected by using anti-c-Myc tag polyclonal
antibody (Upstate Biotechnology, Inc.). Rabbit polyclonal antibody
against TRAF6 was obtained from Santa Cruz Biotechnology. Recombinant
human IFN- Immunoblot Analysis--
Cell lysates were prepared by the
addition of lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml
aprotonin). Equal amounts of protein were subjected to electrophoresis,
transferred onto nitrocellulose membrane (Hybond-C extra, Amersham
Biosciences), and reacted with appropriate antibodies in
phosphate-buffered saline containing 5% nonfat dry milk, 0.02% Tween
20. Blots were then incubated with horseradish peroxidase-conjugated
secondary antibodies and reacted with enhanced chemiluminescence
reagents subsequently (Amersham Biosciences).
Surface Plasmon Resonance--
Association and
dissociation rates of the interaction of LIGHT or LIGHT-R228E with
human LT Immunocomplex Kinase Assay--
To measure the activity of ASK1
in cell extracts, the immune complex was incubated at 30 °C for 30 min with 2 µg of substrates (such as myelin basic protein (MBP)) in
30 µl of solution containing 20 mM Tris-HCl (pH 7.5)/10
mM MgCl2/0.5 µCi of
[ Determination of Cell Death--
Cell death induced by
overexpression of LT Measurement of Caspase Activity--
Cytosolic extracts were
prepared by freezing and thawing of cells in extraction buffer (50 mM PIPES-NaOH, pH 7.0, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin A) as described (34). Cell lysates (50 µg) were diluted with 500 µl of ICE standard buffer (100 mM HEPES-KOH buffer, pH 7.5, 10% sucrose,
0.1% CHAPS, 10 mM dithiothreitol, 0.1 mg/ml ovalbumin) and
incubated at 30 °C for 60 min with 20 µM fluorescent
substrates. Fluorescence intensity was measured using a fluorescence
spectrophotometer (Hitachi F-4500) at an excitation wavelength of 325 nm and emission wavelength of 392 nm.
Characterization of Agonistic Anti-LT
To further confirm this argument, we designed a recombinant LIGHT
mutein to bind LT
The association and dissociation rates of wild type LIGHT and
LIGHT-R228E to LT Activation of ASK1 by 31G4D8 mAb and LIGHT-R228E--
Oxidative
stress was reported to disrupt the ASK1-thioredoxin complex and thereby
to activate ASK1 (39). It has been shown that ROS play essential roles
in LIGHT/IFN- Inhibition of ASK1 Activation by TRAF Mutants--
ASK1 has been
implicated in transmitting TRAF-dependent signaling (32,
40). In order to investigate the roles of TRAFs on ASK1 activation
induced by LT
To further confirm this observation, we investigated the
endogenous ASK1 activation induced by LIGHT in
traf2 Involvement of ASK1 in LT
We further examined the relationship between ASK1 activation and cell
death induced by LIGHT/IFN- ASK1 Is Inhibited by ROS Scavenger but Not Caspase
Inhibitor--
It has been shown that ROS can induce dimerization of
ASK1 and cause its activation in TNF
It has been reported that ASK1-mediated cell death is via either a
caspase-dependent or caspase-independent pathway (41, 42);
thus, we ask whether caspase-3 activation is dependent on ASK1
activation induced by IFN- LT In a previous study, we have demonstrated that LIGHT/IFN- Moreover, we further demonstrate that both LIGHT-R228E and agonistic
antibody against LT The ROS has been demonstrated to play a crucial role in
stress-activated mitogen-activated protein kinase kinase kinase
signaling pathway (22, 39), and the activation of ASK1 by LT Although TRAF2 is essential for TNF-induced ASK1 activation (47),
LIGHT-induced ASK1 activation is apparently independent of TRAF2. It
has been shown that overexpression of TRAF2 or TRAF5, but not TRAF3, is
able to activate ASK1 directly (40). However, we found that ASK1
activation is impaired not only in traf5 In a previous study, we demonstrated that activation of LT Unlike ROS inhibitor, ASK1 only provides a partial effect on
LT receptor (LT
R),
herpes virus entry mediator, and decoy receptor (DcR3). In our
previous study, we showed that LIGHT is able to induce cell death via
the non-death domain containing receptor LT
R to activate both
caspase-dependent and caspase-independent pathway. In this
study, a LIGHT mutein, LIGHT-R228E, was shown to exhibit similar
binding specificity as wild type LIGHT to LT
R, but lose the ability
to interact with herpes virus entry mediator. By using both LIGHT-R228E
and agonistic anti-LT
R monoclonal antibody, we found that signaling
triggered by LT
R alone is sufficient to activate both
caspase-dependent and caspase-independent pathways. Cross-linking of LT
R is able to recruit TRAF3 and TRAF5 to activate ASK1, whereas its activity is inhibited by free radical scavenger carboxyfullerenes. The activation of ASK1 is independent of
caspase-3 activation, and kinase-inactive ASK1-KE mutant can
inhibit LT
R-mediated cell death. This suggests that ASK1 is one of
the factors involved in the caspase-independent pathway of
LT
R-induced cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor
(LT
R)1 is a member of the
tumor necrosis factor receptor (TNFR) superfamily and is
ubiquitously expressed on the surface of most cell types, except T and
B lymphocytes (1, 2). It has been reported that LT
R interacts
specifically with two ligands: lymphotoxin LT
1/
2 (3, 4) and LIGHT
(5, 6). There is ample evidences to demonstrate that LT
R plays an
essential role in the development of lymphoid organs. Lymphoid nodes
are deficient in LT
gene-deleted (LT
/
) mice (7),
and the impairment of lymph node development as well as the loss of
splenic architecture was also observed in LT
knockout mice (8).
Furthermore, LT
R-deficient mice are shown to lack Peyer's patches,
colon-associated lymphoid tissues, and all lymph nodes (9).
Interestingly, the administration of agonistic antibody to LT
R can
induce lymph node development in LT
/
mice (10). In addition to its role in lymphoid organ formation, LT
R
is also involved in host immune responses to foreign antigens. Blockade
of LT
R with LT
R-Fc not only prevents germinal center formation in
spleen but also results in impaired IgG antibody responses to sheep red
blood cells (11). Moreover, administration of LT
R-Fc is shown
to enhance host survival after virus challenge (12) and is effective in
preventing the onset of Th2 cell-mediated colitis (13).
R does not contain consensus sequences
characteristic of death domain; thus, LT
R-transduced signaling is
mainly mediated by TRAFs. TRAF molecules consist of amino-terminal RING
finger domain, central zinc finger loop, and carboxyl-terminal TRAF
domain. The TRAF domain mediates interactions between TRAF proteins and both their upstream and downstream effectors, whereas the RING finger
domain is reported to be necessary for TRAF effector activation (14).
Among the six TRAF proteins, TRAF2, TRAF3, and TRAF5 are found to
associate with LT
R (15-17). Further study has indicated that TRAF3
plays an important role in mediating LT
R-induced cell death (15, 16,
18, 19), whereas TRAF2 and TRAF5 have been shown to be involved in the
activation of NF-
B (17). Moreover, two serine/threonine protein
kinases (p50 and p80) are reported to be associated with cytoplasmic
region of LT
R (20), but their roles in LT
R-mediated signaling
have not been elucidated yet.
/
embryonic fibroblasts (28).
-induced cell death (30). In contrast, free radical
scavenger carboxyfullerenes (C60) can completely inhibit
LIGHT/IFN-
-induced cell death (30), indicating the important roles
of ROS in LIGHT/IFN-
-induced cell death (30). Since ROS are key
mediators to activate ASK1, which contributes to
progression of cell death (22, 31), we investigated the role of ASK1 in
LIGHT-LT
R-induced cell death. Here we report that activation of
LT
R alone, without the necessity to trigger HVEM activation, by
either agonistic anti-LT
R mAb or a LIGHT mutein (LIGHT-R228E)
incapable of HEVM binding, could induce the production of free radicals
and the activation of ASK1. Blockade of ASK1 activation by free radical
scavenger C60 could inhibit LT
R-mediated cell death.
Thus, in addition to caspase activation, the activation of ASK1 also
contributes to LT
R-mediated apoptotic pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R
and hLT
R-CD proteins have been described (19). The hemagglutinin
(HA)-tagged expression constructs of ASK1, catalytically inactive
ASK1-KE-HA, were kindly provided by Dr. Wen-Chen Yeh (32). The dominant
negative TRAF mutants were provided by Dr. Wen-Chen Yeh (TRAF2 mutant)
and Dr. Bharat B. Aggarwal (TRAF3, -5, and -6 mutants) vector. All of
the TRAF mutants contained the c-Myc tag except TRAF6 mutant. For DNA
transfection, cells were plated and grown for 16 h and transfected
with expression vectors by the calcium phosphate method or by using
LipofectAMINETM (Invitrogen).
R Monoclonal Antibody--
Monoclonal
antibodies were prepared by immunizing Balb/c mice with recombinant
human lymphotoxin
receptor-Fc (hLT
R-Fc) protein (6). Spleen
cells were fused with NS-1 cells, and hybridomas were screened by
enzyme-linked immunosorbent assay. Anti-hLT
R monoclonal antibodies
were selected by their specific binding to hLT
R but not to the Fc
portion of human IgG1.
was purchased from Roche Molecular Biochemicals.
R-Fc or HVEM-Fc were determined by surface plasmon resonance
using a BIAcore® 2000 biomolecular interaction analysis system
(BIA-core Inc., Piscataway, NJ). The Fc fusion proteins (50 µg/ml)
were coupled to a CM5 sensor chip by amine coupling at pH 7.0. The sensor surface was equilibrated with phosphate-buffered
saline, and sensorgrams were collected at 25 °C and a flow rate at
30 µl/min. A 120-µl injection of LIGHT or LIGHT-R228E was passed
over the sensor surface. After the association phase, 600 s of
dissociation data were collected. The sensor surface was regenerated
after each cycle with a 15-µl pulse of 10 mM glycine (pH
2.0) twice with a 30-s interval. Sets of eight analyte concentrations, 100-800 nM, were collected and analyzed.
-32P]ATP. Reactions were stopped by the addition of
Laemmli sample buffer. Samples were then fractionated by SDS-PAGE, and
proteins were visualized by Coomassie Blue staining. Phosphorylated
proteins were identified by autoradiography and quantified by a
densitometer (Amersham Biosciences).
R was determined by
-galactosidase-based cell
morphology assay, and the killing effect of LIGHT/IFN-
treatment was
detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For the
-galactosidase-based cell morphology assay, HeLa cells were co-transfected with lacZ
expression vector, pBKCMV-lacZ. After 24 h of transfection, cells
were fixed and then were stained with
5-bromo-4-chloro-3-indolyl-
-D-galatopyranoside (X-gal)
to determine the percentage of apoptotic cells as described previously
(19). The survival rate of Hep3BT2 cells was determined by MTT assay.
Briefly, cells were seeded in 96-well flat bottom plates at a density
of 5 × 103 cells/well. After treatment, 10 µl of 5 mg/ml MTT per well was added and incubated at 37 °C for 4 h.
Cells were then lysed by the addition of 50 µl of 10% SDS in 0.4 N HCl per well and incubated at 37 °C for another
16 h. The optical density of each sample was determined by
measuring the absorbance at 570 versus 650 nm using an
enzyme-linked immunosorbent assay reader (TECAN; RainBow) (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R-mAb (31G4D8) and
LIGHT-R228E Mutein--
Cross-linking of cell surface receptor
by ligand or by agonistic antibodies can trigger signal
transduction, and members of the TNFR superfamily are reported
to be activated by agonistic antibodies, such as anti-human
Fas antibody (CH11) and anti-mouse Fas antibody (Jo2). To study the
signaling transduced by LT
R, monoclonal antibodies against human
LT
R were raised. One of the selected clones, 31G4D8, is found to
bind to LT
R specifically. Anti-LT
R mAb 31G4D8 does not have any
cytotoxic effect to Hep3BT2 or HT29, which are sensitive to
LIGHT/IFN-
-mediated cell death. However, in conjunction with
IFN-
, 31G4D8 mAb is able to induce cell death with similar extent as
that induced by wild type LIGHT (Fig. 1).
This observation is in agreement with the previous observation that
overexpression of LT
R is able to induce cell death (19), and LIGHT
mutein incapable of binding to LT
R loses its ability to induce
cell death (35).
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Fig. 1.
Cytotoxic effects of
anti-LT R monoclonal antibody 31G4D8. HT29
cells (A) or Hep3BT2 cells (B) were seeded in
culture plates precoated with a series of diluted 31G4D8 mAb in
conjunction with 50 units/ml IFN-
(for HT29 cells) or 100 units/ml
IFN-
(for Hep3BT2 cells) and were incubated for 72 h. Cell
viability was determined by MTT assays, whereas the percentage of cell
survival was determined by measurement of
A280 for cells treated with cytokines
versus cells cultured in medium alone.
R but not HVEM, using a strategy of molecular modeling. A three-dimensional model for the interaction of LIGHT and
its receptors (LT
R, HVEM, and DcR3) was generated by homology modeling (Molecular Simulation Inc., San Diego, CA) based on the crystallographic complex structure of LT
and TNFRI (Protein Data Bank code 1TNR) (36-38). Residues of the receptor-binding sites of
this system, conventionally denoted as the A-R interaction domain and
the A-S interaction domain, were identified. A few charge or polar
residues were chosen for site-specific mutagenesis with the prediction
that their mutations would, depending on the type of receptor, either
enhance or interrupt receptor binding through altered electrostatic
interactions. One of the LIGHT muteins that we have substantially
characterized, the mutation at amino acid 228 from arginine to glutamic
acid (LIGHT-R228E) at the A-R interaction domain (see the model in
Table I), met the modeling objective of
the present study.
Kinetics of ligand binding to receptors determined by surface
plasmon resonance
R (two
units, one in orange the other in green), showing both the A-R
interface (blue-orange) and the A-S interface (blue-green). The two
amino acids whose mutants exhibited selective binding of HVEM and not
LT
R (glycine 119) (35) and vice versa (arginine 228) (this work) are
labeled (pink).
R and HVEM were determined by surface plasmon resonance. As shown in Fig. 2 and Table
I, the binding affinity of wild type LIGHT to both HVEM
(KD = 8.81 ± 3.2 nM) and LT
R
(KD = 8.72 ± 3.21 nM) is similar,
whereas the binding affinity of LIGHT-R228E to HVEM is almost
undetectable, and its binding affinity to LT
R (KD = 77.8 ± 41 nM) is reduced from that of the wild type
but is clearly evident (Fig. 2B). The reduction in affinity
of R228E for LT
R-Fc was due to a decrease in association rate and an
increase in dissociation rate (Table I). The binding of
LIGHT-R228E to LT
R and the lack of it to HVEM were
further confirmed by a competition analysis using LT
R-Fc or HVEM-Fc
to inhibit wild type LIGHT and LIGHT-R228E-mediated cell death (Fig.
2C). Namely, wild type LIGHT/IFN-
-induced cell death
could be blocked by either LT
R-Fc or HVEM-Fc in a
dose-dependent manner (Fig. 2C, upper
panel), whereas LIGHT-R228E/IFN-
-induced cell death was only
blocked by LT
R-Fc and not by HVEM-Fc (Fig. 2C, lower
panel). These observations provided direct evidence that the amino
acid arginine 228 is essential for the interaction between LIGHT and
HVEM, and LT
R alone is sufficient for LIGHT-mediated cell death.
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Fig. 2.
Binding specificity of LIGHT-R228E
mutant. A and B, kinetic analysis for the
interaction of LIGHT and LT R-Fc and HVEM-Fc by surface plasmon
resonance. Human IgG1 or OPG-Fc was first mobilized on channel one of a
CM5 chip as the blank to determine the bulk effect of injection itself,
whereas HVEM-Fc and LT
R-Fc were immobilized on channel two and
three, respectively, for analysis of its kinetic interaction with wild
type LIGHT or LIGHT-R228E mutant. LIGHT or LIGHT-R228E, as the analyte,
was injected from 100 to 800 nM, respectively. The
interaction between LIGHT or LIGHT-R228E with LT
R-Fc (A)
or HVEM-Fc (B) was determined by surface plasmon resonance
using a BIAcore 2000. C, Hep3BT2 cells were incubated with
50 ng/ml wild type LIGHT (upper panel) or
LIGHT-R228E (lower panel) in conjunction with
IFN-
(100 units/ml). The LT
R-Fc, HVEM-Fc, DcR3-Fc, and human IgG1
(ranging from 10
6 to 10 µg/ml) were added to culture
medium, respectively, and incubated for 72 h to determine their
inhibitory effect on LIGHT and LIGHT-R228E-mediated cell
death.
-induced cell death (30); thus, we ask whether
signaling through LT
R alone is enough to activate ASK1 activation to
induce cell death. To address this question, HeLa cells were
transfected with HA-tagged ASK1, followed by incubation with agonistic
31G4D8 mAb (Fig. 3A) or
LIGHT-R228E (Fig. 3B) to test their ability to activate
HA-tagged ASK1 by in vitro kinase assay. As shown in Fig.
3A, a rapid increase of ASK1 activity was observed at 5 min
after 31G4D8 treatment and observed to last for at least 60 min (Fig.
3A). LIGHT-R228E had a similar effect as 31G4D8 mAb in ASK1
activation but with distinct kinetics. ASK1 activity increased at 15 min, peaked at 60 min, and returned to basal level at 90 min when
stimulated with LIGHT-R228E. The kinetics of endogenous ASK1 activation
in Hep3BT2 was similar to that of transfected HA-tagged ASK1 after
31G4D8 mAb stimulation (Fig. 3C). This demonstrated that
ASK1 could be activated by LT
R-transduced signaling.
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Fig. 3.
Activation of ASK1 induced by cross-linking
of LT R. HeLa cells were transfected with
HA-tagged ASK1, followed by incubation with 10 µg/ml anti-LT
R
antibody 31G4D8 (A) or 100 ng/ml LIGHT-R228E (B)
for various time intervals. ASK1 was immunoprecipitated by anti-HA
antibody, whereas the ASK1 kinase activity contained in the
immunocomplex was determined by incubation with MBP as a substrate by
in vitro kinase assay. C, the endogenous ASK1 of
HeLa cells was precipitated by polyclonal anti-ASK1 antibody after
treatment with 10 µg/ml 31G4D8 mAb, whereas its activity was
determined by incubation with MBP as a substrate by in vitro
kinase assay.
R, we examined the effects of TRAF dominant negative
(TRAF-DN) mutants in ASK1 activation. To address this question, HeLa
cells were transfected with ASK1-HA in conjunction with TRAF-DN
mutants. It was obvious that TRAF3-DN and TRAF5-DN, but not TRAF2-DN
and TRAF6-DN mutants, effectively inhibited transfected ASK1 activation
in the in vitro kinase assay (Fig.
4A). The endogenous ASK1
activity was also inhibited by TRAF3-DN and TRAF5-DN, but not by
TRAF2-DN, to the same extent as catalytic inactive ASK1-KE mutant (Fig.
4B).
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Fig. 4.
Inhibition of ASK1 activity by dominant
negative mutant of TRAF (TRAF-DN) proteins and impairment of ASK1
activation in traf-deficient MEFs. HeLa cells
were transfected with HA-tagged ASK1 in conjunction with TRAF-DNs and
ASK-KE for 24 h, followed by incubation with 10 µg/ml 31G4D8 for
30 min. To determine the ASK1 activity, cells were incubated with
anti-HA antibody (A) or polyclonal anti-ASK1 antibody
(B) to precipitate HA-tagged ASK1 (A) or
endogenous ASK1 (B), followed by incubation with MBP to
determine their activities by in vitro kinase assay.
Expression of TRAFs in the transfectants was detected by Western blot
analysis using anti-Myc and anti-TRAF6 antibodies. Data shown are
representative of three independent experiments. C,
traf-deficient and wild type MEF cells were stimulated with
TNF (200 ng/ml) for 15 min or with LIGHT (200 ng/ml) for 45 min, and
cell lysates were collected and incubated with polyclonal anti-ASK1
antibody to precipitate endogenous ASK1 for an in vitro
kinase assay.
/
,
traf3
/
, and
traf5
/
MEFs. The ASK1 activation induced by
TNF
is impaired in traf2
/
MEFs,
which is consistent with previous report that TNF
-induced ASK1
activation is TRAF2-dependent (32). In contrast, the
activation of ASK1 by LIGHT is not affected in
traf2
/
MEFs (Fig. 4C,
upper panel). However, the activation of endogenous ASK1 is
inhibited in either traf3
/
or
traf5
/
MEFs (Fig. 4C, lower
panel), suggesting that LT
R-mediated ASK1 activation is via
TRAF3 and TRAF5 but not TRAF2.
R-induced Cell Death--
We further
asked whether activation of ASK1 is involved in LT
R-induced cell
death. It has been shown that overexpression of LT
R could induce
HeLa cell death (19); thus, we co-transfected ASK1-KE, LT
R, and
-galactosidase to test its effect in LT
R-mediated cell death. At
24 h after transfection, the percentage of cell death in cells
overexpressing full-length LT
R or cytoplasmic LT
R was ~56.6 and
53.7%, respectively, whereas the co-expression of ASK1-KE reduced the
percentage of apoptotic cells to 19% (Fig. 5A). This suggests that ASK1
is involved in LT
R-mediated cell death. ASK1-KE and TRAF3-DN are not
toxic to HeLa cells under the same condition.
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Fig. 5.
Effects of ASK1 dominant negative mutant
(ASK1-DN) on LT- R-mediated cell death.
A, HeLa cells were co-transfected with DNA constructs
expressing full-length LT
R (pFLAG-LT
R) or its cytoplasmic domain
(FLAG-LT
R-CD), in conjunction with dominant mutants of ASK1, TRAF3,
and pCMV-lacZ in a ratio of 7:1:7. Cells were stained with X-gal at
24 h after transfection, followed by examination under a
phase-contrast microscope. The percentage of apoptotic cells was
calculated as the number of blue cells with apoptotic morphology
divided by the total number of blue cells. At least 1000 blue cells
were counted for each sample. The data shown here are the averages ± S.D. of triplicate experiments. B and C,
Hep3BT2 cells overexpressing ASK1-KE (clones 3, 12, 13, and 52) and
control vector were treated with LIGHT (50 ng/ml)/IFN-
(100 units/ml) (B) or LIGHT-R228E (50 ng/ml)/IFN-
(100 units/ml) (C) for 72 h. Cell viability was determined
by MTT assays, whereas the percentage of survival rate was determined
by measurement of A280 for cells treated with
cytokines compared with cells cultured in medium alone.
. Hep3BT2 cells stably expressing ASK1-KE
were incubated with IFN-
in conjunction with LIGHT (Fig. 5B) or LIGHT-R228E (Fig. 5C) to test its
resistance to cell death. Compared with cells transfected with control
plasmid pcDNA3 (survival rate 57%), Hep3BT2 cells stably
expressing ASK1-KE are relatively resistant to LIGHT/IFN-
killing
(survival rate 67-78%) or LIGHT-R228E/IFN-
-mediated apoptosis
(survival rate 73-81%). Thus, ASK1 is clearly involved in
LT
R-mediated cell death.
signaling (22), and
inhibition of ROS production by C60 can inhibit
LIGHT/IFN-
-mediated cell death (30); thus, we are interested to know
whether C60 can inhibit LT
R-mediated ASK1 activation. As
shown in Fig. 6A (upper panel), pretreatment of C60 completely inhibits ASK1
activation in Hep3BT2 cells treated with LIGHT-R228E. This indicates
that LT
R-mediated ASK1 activation is regulated by ROS. Moreover, the production of ROS induced by LT
R activation is not affected by ASK1-KE mutant; this further suggests that production of ROS is upstream to ASK1 activation in LT
R-mediated signaling (Fig.
6B).
View larger version (34K):
[in a new window]
Fig. 6.
ASK1 activation triggered by
LT R cross-linking is regulated by ROS.
A, Hep3BT2 cells pretreated with 50 µM
C3 isoform of carboxyfullerene were incubated with 100 ng/ml LIGHT-R228E for 30 min, and the endogenous ASK1 activity was
determined by immunoprecipitation using polyclonal anti-ASK1 antibody,
followed by incubation with MBP as a substrate by an in
vitro kinase assay. B, generation of ROS in wild
type Hep3BT2 cells (upper panel) or Hep3BT2 cells
overexpressing ASK1-KE (lower panel). After
incubation with 100 ng/ml LIGHT, 100 ng/ml LIGHT-R228E, or 10 µg/ml
31G4D8 in conjunction with 100 units/ml IFN-
for 6 h, Hep3BT2
cells or ASK1-KE/Hep3BT2 cells were stained with 5 µM
2',7'-dihydrodichlorofluorescein diacetate at 37 °C for 15 min,
followed by flow cytometry analysis to determine their fluorescence
intensity. Line, medium; shadow,
LIGHT/IFN-
or 31G4D8 mAb; mean fluorescence intensity is indicated.
C, Hep3BT2 cells or ASK1-KE/Hep3BT2 were incubated with 100 ng/ml LIGHT, 100 ng/ml LIGHT-R228E, or 10 µg/ml 31G4D8 in conjunction
with 100 units/ml IFN-
, and caspase activities were determined by
incubating the cell lysates with fluorescence substrate
MCA-DEVD.APK
(7-methoxycoumarin-4-yl)acetyl-Asp-Glu-Val-Asp- Ala-Pro-Lys(2,4-dinitrophenyl)-OH).
D, Hep3BT2 cells pretreated with 100 µM
z-VAD-FMK were stimulated with 100 ng/ml LIGHT-R228E for 30 min,
followed by immunoprecipitation using polyclonal anti-ASK1 antibody to
determine endogenous ASK1 activity by an in vitro
kinase assay. E, failure of caspase inhibitors to protect
ASK1-KE/Hep3BT2 cells form LIGHT-R228E/IFN-
-mediated cell
death. Hep3BT2 and ASK1-KE/Hep3BT2 cells were pretreated with 100 µM z-VAD-FMK or 20 µM C60 (C3
form) for 1 h, followed by incubation in medium supplemented with
100 units/ml IFN-
and 50 ng/ml LIGHT-R228E for 72 h. Cell
viability was determined by MTT assay.
/LIGHT, IFN-
/LIGHT-R228E, or
IFN-
/31G4D8. In Hep3BT2 cells stably expressing ASK1-KE, activation of caspase-3 by IFN-
/LIGHT, IFN-
/LIGHT-R228E, or IFN-
/31G4D8 is partially inhibited (50%) (Fig. 6C), but ASK-KE does not
have any effect on caspase activation induced by transforming growth factor-
1 (data not shown). This demonstrated the important role of
ASK1 for caspase-3 activation in LT
R-mediated signaling pathway. Moreover, caspase-3 inhibitor does not have any effect on ASK1 activation in Hep3BT2 cells when treated with LIGHT-R228E, suggesting that ASK1 is upstream to caspase-3 activation (Fig. 6D). To
further determine the role of ASK1 in LT
R-mediated cell death, wild
type Hep3BT2 and Hep3BT2/ASK-KE cells were incubated with LIGHT-R228E in the presence or absence of caspase inhibitor z-VAD-FMK.
Compared with wild type Hep3BT2 cells, cells overexpressing ASK-KE
(Hep3BT2/ASK-KE) are more resistant to LIGHT-R228E-mediated cell death
(Fig. 6E). Moreover, the addition of z-VAD-FMK provides
partial protective effect in both wild type Hep3BT2 and Hep3BT2/ASK-KE
cells. The protective effect of caspase inhibitor z-VAD-FMK is less
than the ASK1-KE dominant negative mutant, indicating that ASK1 plays a
more important role than caspase-3 activation in LT
R-mediated cell
death. In contrast, C60 could fully protect both wild type Hep3BT2 and Hep3BT2/ASK-KE cells from LT
R-mediated cell death. Since
the activation of ASK1 is regulated by free radicals, we conclude that
ASK1 is one of the factors activated by free radicals contributing
to LT
R-induced cell death.
R-induced ROS Release Is Not Affected in Caspase-3-deficient
Cells or by z-VAD-FMK--
After confirming the role of ROS in ASK1
activation, we further ask whether caspase activation lies upstream or
downstream to ROS production. To address this question, Hep3BT2 cells
were pretreated with general caspase inhibitor z-VAD-FMK, followed by
incubation with IFN-
/LIGHT-R228E to determine its effect on LT
R-induced ROS release by flow cytometry using
2',7'-dihydrodichlorofluorescein diacetate as probe. As shown in Fig.
7A, the addition of z-VAD-FMK did not suppress mean fluorescence intensity, suggesting that the
release of ROS is not affected, indicating that ROS release is not
suppressed by general caspase inhibitor. In caspase-3-deficient MCF-7
cells, the mean fluorescence intensity is still increased after
IFN-
/LIGHT-R228E treatment. This suggests that ROS release is not
dependent on the activation of caspase-3 and other caspases (such as
caspase-1, -3, -5, -6, -7, -8, and -9), which are sensitive to
z-VAD-FMK (43).
View larger version (25K):
[in a new window]
Fig. 7.
ROS release induced by
LT R is not regulated by caspases. Hep3BT2
cells (A) or MCF-7 (B) pretreated with 100 µM z-VAD-FMK were incubated with 100 ng/ml LIGHT-R228E in
conjunction with 100 units/ml IFN-
for 8 h; Hep3BT2 cells or
MCF-7 cells were stained with 5 µM
2',7'-dihydrodichlorofluorescein diacetate at 37 °C for 15 min,
followed by flow cytometry analysis to determine their fluorescence
intensity. Line, medium; shadow,
IFN-
/LIGHT-R228E or z-VAD-FMK/IFN-
/LIGHT-R228E; mean fluorescence
intensity is indicated. C, putative model of LT
R-mediated
apoptotic pathway. Activation of LT
R by agonistic mAb 31G4D8 or
LIGHT-R228E induces the production of ROS, which is enhanced by
IFN-
. ROS initiate both caspase-3-dependent and
caspase-independent pathways to induce cell death. One of the
caspase-3-independent pathways is the activation of ASK1, via the
recruitment of TRAF3 and TRAF5, to LT
R. *, caspases insensitive to
z-VAD-FMK cannot be ruled out.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
can
induce the production of free radicals, which in turn induce cell death
via both caspase-dependent and -independent pathways (30).
Moreover, signaling triggered by LT
R overexpression or agonistic
anti-LT
R mAb is shown to be sufficient for LIGHT/IFN-
-mediated cell death (19, 35). However, it is unclear whether signaling triggered
by LT
R is still able to activate both caspase-dependent and caspase-independent pathways to induce cell death. Previously we
have demonstrated that the caspase-dependent pathway plays a minor role in LIGHT/IFN-
-mediated cell death, since caspase inhibitor z-VAD-FMK only provides partial protective effect to LIGHT/IFN-
-induced cell death. In this study, we further ask whether
signaling triggered by LT
R alone is enough to activate a
caspase-dependent pathway and/or caspase-independent
pathway to induce cell death. To clarify this issue, LIGHT muteins and agonistic antibody against LT
R were generated to test the questions raised above. Among the LIGHT-muteins generated, we find that the amino
acid arginine 228 is crucial for LIGHT-HVEM interaction, since mutation
of arginine 228 to glutamic acid 228 abolished the
interaction between LIGHT and HVEM (Fig. 2). It has been shown that
amino acid glycine 119 is critical for LIGHT-LT
R interaction (35);
in complementation, we showed here that amino acid arginine 228 is
essential for LIGHT-HVEM interaction. According to the homology model
(shown in Table I), both glycine 119 and arginine 228 interact with the
receptor in the A-R interaction domain, but from different regions of
LIGHT; whereas glycine 119 is located in the N-terminal A-A' loop of
LIGHT, arginine 228 is located in the G-H loop of the C-terminal. It
will be of interest, and also of considerable use, for further studies
to identify amino acid residues that are essential for LIGHT-DcR3 interactions.
R still have the ability, like wild type
LIGHT, to induce the production of free radicals and activate both
caspase-dependent and -independent pathways to induce cell death. We find that LT
R-transduced signaling is able to activate ASK1 via the induction of free radicals (Fig. 6A), and
activation of ASK1 also contributes to LT
R-mediated cell death (Fig.
6E); this observation thus reveals one of the mechanisms of
LT
R-mediated caspase-independent pathway to induce cell death.
Although ASK1 activity is not required in the caspase-independent cell
death in the ASK1 overexpression system (42), the kinase activity of
ASK1 is essential for LT
R-mediated cell death, since the
kinase-inactive ASK1-KE can inhibit the cell death triggered by LT
R
activation (Fig. 6E). Previous study has shown that
kinase-inactive mutant of ASK1 is capable of inhibiting cell death
induced by genotoxic stress, Fas, and tumor necrosis factor
overexpression (21, 24, 25); this implies that catalytic active ASK1
may contribute to a kinase-dependent, but
caspase-independent, mechanism to cell death triggered by various cell
death-inducing signals. In our recent study, we also demonstrate that
signaling transduced by LT
R induces the secretion of IL-8 in HEK 293 via the activation of ASK1-MKK4/MKK7-JNK1/2-AP1 and NIK-IKK-NF-
B
signaling cascades (44). Since activation of JNK/stress-activated
protein kinase also contributed to cell death (28, 45, 46), the
ASK-1-dependent cell death in our model system might be
mediated by a JNK/stress-activated protein kinase signaling cascade.
R
activation further provides an example of how free radical-regulated
mitogen-activated protein kinase kinase kinase can mediate cell death.
Recently, thioredoxin, a redox-sensing protein, has been shown to
associate with ASK1 in its reduced form. Tumor necrosis factor can
stimulate the production of ROS to activate ASK1 via the dissociation
of ASK1 from thioredoxin, followed by binding to TRAF2 to form a TNFR-TRAF2-ASK1 complex (47). In our study, we find that the LT
R-mediated ASK1 activation is dependent on TRAF3 and TRAF5 but not
on TRAF2 and TRAF6 (Fig. 4). This is consistent with the previous
finding that the LT
R-mediated signaling cascade is transduced by
TRAF3 and TRAF5 (17, 18) and that the dominant negative mutant
of TRAF3 provides partial protection to LT
R-mediated cell death
(19).
/
MEF cells but also in traf3
/
MEF cells (Fig.
4C). This suggests that even TRAF3 could interact with ASK1
directly (40), but TRAF3 alone is not enough to activate ASK1.
Therefore, TRAF3-dependent ASK1 activation after LT
R
activation might be via its interaction with TRAF5 to recruit ASK1, and
further investigation is needed to clarify this question.
R can
trigger both a caspase-3-dependent and -independent pathway to induce cell death (30). Moreover, free radical scavenger C60 can completely inhibit LT
R-mediated cell death,
whereas general caspase inhibitor z-VAD-FMK has only a partial
protective effect, suggesting the important role of ROS in
LT
R-mediated cell death (30). Here we provide further evidence that
LT
R-induced ROS release is apparently independent from caspase-3 and
other caspases that are sensitive to z-VAD-FMK, such as caspase-1, -3, -5, -6, -7, -8, and -9 (43). Whether caspase-2, -4, and -10 or other newly identified caspases affect LT
R-induced ROS needs to be tested
in the future.
R-mediated cell death, although ASK1-KE is more potent than caspase inhibitor z-VAD-FMK. This indicates that a caspase-independent or z-VAD-sensitive caspase-independent pathway distinct from ASK1 activation is also responsible for LT
R activation. Fig.
7C summarizes our current understanding to LT
R-mediated
cell death; HVEM apparently is dispensable for LIGHT-mediated free
radical production as well as the activation of ASK1 and caspase-3. The
recruitment of TRAF3 and TRAF5 to LT
R induces the production of free
radicals to activate both caspase-3-dependent and
z-VAD-sensitive caspase-independent pathways. Since IFN-
enhances
LT
R-mediated cell death, an IFN-
-regulated pathway distinct from
ASK1 activation might be one of the major pathways responsible for
LT
R-mediated cell death. Identification of an IFN-
-regulated
pathway distinct from ASK1 activation might be very helpful to
elucidate the caspase-independent pathway transduced by LT
R and
other members of the TNFR superfamily.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Wen-Chen Yeh (Amgen, Inc.) and Dr. Bharat B. Aggarwal for providing traf2 knockout mouse embryonic fibroblasts and TRAF-dominant negative constructs, respectively. We also thank Dr. Chi-Ying F. Huang (NHRI) for BIAcore technical support. We also thank Dr. Nien-Jung Chen for fluorescence-activated cell sorting analysis.
![]() |
FOOTNOTES |
---|
* This work was mainly supported by National Science Council, Taiwan, Grants NSC 91-2320-B-010-053, NSC 91-2320-B010-092. Additional support came from the National Health Research Institute, Taiwan (NHRI-CN-BP-8902S) and the Ministry of Education (89-B-FA22-2-4) under the Program for Promoting Academic Excellence of Universities. This work was also supported by Chi-Mei Foundational Hospital, Tainan, Grant CMYM 8902.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.
b Present address: Dept. of Microbiology and Immunology, Taipei Medical University, Taipei 110, Taiwan.
d Present address: United Biomedical, Inc., New York, NY 11788.
h Present address: Anawrahta Biotechnology, Taipei 251, Taiwan.
j To whom all correspondence should be addressed: Institute of Microbiology and Immunology, National Yang-Ming University, Shih-Pai, Taipei 11221, Taiwan. Tel.: 886-2-28267161; Fax: 886-2-28212880; E-mail: slhsieh@ym.edu.tw.
k Supported by Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, and a grant from Human Frontier Science Program.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M208661200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
LTR, lymphotoxin-
receptor;
ASK1, apoptosis signal-regulating kinase
1;
IFN-
, interferon-
;
HVEM, herpes virus entry mediator;
TRAF, tumor necrosis factor receptor-associated factor;
ROS, reactive
oxygen species;
C60, carboxyfullerenes;
HA, influenza
hemagglutinin;
TNFR, tumor necrosis factor receptor;
LT
, lymphotoxin-
;
MEKK5, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase 5;
JNK, c-Jun N-terminal kinase;
mAb, monoclonal antibody;
MEF, mouse embryonic fibroblast;
MBP, myelin
basic protein;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galatopyranoside;
PIPES, 1,4-piperazinediethanesulfonic acid;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic
acid;
z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone;
hLT
R, human
LT
R.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Force, W. R., Walter, B. N., Hession, C., Tizard, R., Kozak, C. A., Browning, J. L., and Ware, C. F. (1995) J. Immunol. 155, 5280-5288[Abstract] |
2. | Ware, C. F., VanArsdale, T. L., Crowe, P. D., and Browning, J. L. (1995) Curr. Top. Microbiol. Immunol. 198, 175-218[Medline] [Order article via Infotrieve] |
3. |
Browning, J. L.,
Dougas, I.,
Ngam-ek, A.,
Bourdon, P. R.,
Ehrenfels, B. N.,
Miatkowski, K.,
Zafari, M.,
Yampaglia, A. M.,
Lawton, P.,
and Meier, W.
(1995)
J. Immunol.
154,
33-46 |
4. | Crowe, P. D., VanArsdale, T. L., Walter, B. N., Ware, C. F., Hession, C., Ehrenfels, B., Browning, J. L., Din, W. S., Goodwin, R. G., and Smith, C. A. (1994) Science 264, 707-710[Medline] [Order article via Infotrieve] |
5. | Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu, G. L., Ruben, S., Murphy, M., Eisenberg, R. J., Cohen, G. H., Spear, P. G., and Ware, C. F. (1998) Immunity 8, 21-30[Medline] [Order article via Infotrieve] |
6. |
Zhai, Y.,
Guo, R.,
Hsu, T. L., Yu, G. L.,
Ni, J.,
Kwon, B. S.,
Jiang, G. W.,
Lu, J.,
Tan, J.,
Ugustus, M.,
Carter, K.,
Rojas, L.,
Zhu, F.,
Lincoln, C.,
Endress, G.,
Xing, L.,
Wang, S.,
Oh, K. O.,
Gentz, R.,
Ruben, S.,
Lippman, M. E.,
Hsieh, S. L.,
and Yang, D.
(1998)
J. Clin. Invest.
102,
1142-1151 |
7. | De Togni, P., Goellner, J., Ruddle, N. H., Streeter, P. R., Fick, A., Mariathasan, S., Smith, S. C., Carlson, R., Shornick, L. P., and Strauss-Schoenberger, J. (1994) Science 264, 703-707[Medline] [Order article via Infotrieve] |
8. |
Koni, P. A.,
and Flavell, R. A.
(1998)
J. Exp. Med.
187,
1977-1983 |
9. | Futterer, A., Mink, K., Luz, A., Kosco-Vilbois, M. H., and Pfeffer, K. (1998) Immunity 9, 59-70[Medline] [Order article via Infotrieve] |
10. | Rennert, P. D., Browning, J. L., and Hochman, P. S. (1997) Int. Immunol. 9, 1627-1639[Abstract] |
11. | Mackay, F., Majeau, G. R., Lawton, P., Hochman, P. S., and Browning, J. L. (1997) Eur. J. Immunol. 27, 2033-2042[Medline] [Order article via Infotrieve] |
12. | Puglielli, M. T., Browning, J. L., Brewer, A. W., Schreiber, R. D., Shieh, W. J., Altman, J. D., Oldstone, M. B., Zaki, S. R., and Ahmed, R. (1999) Nat. Med. 5, 1370-1374[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Dohi, T.,
Rennert, P. D.,
Fujihashi, K.,
Kiyono, H.,
Shirai, Y.,
Kawamura, Y. I.,
Browning, J. L.,
and McGhee, J. R.
(2001)
J. Immunol.
167,
2781-2790 |
14. | Wajant, H., Henkler, F., and Scheurich, P. (2001) Cell. Signal. 13, 389-400[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Force, W. R.,
Cheung, T. C.,
and Ware, C. F.
(1997)
J. Biol. Chem.
272,
30835-30840 |
16. |
Force, W. R.,
Glass, A. A.,
Benedict, C. A.,
Cheung, T. C.,
Lama, J.,
and Ware, C. F.
(2000)
J. Biol. Chem.
275,
11121-11129 |
17. |
Nakano, H.,
Oshima, H.,
Chung, W.,
Williams-Abbott, L.,
Ware, C. F.,
Yagita, H.,
and Okumura, K.
(1996)
J. Biol. Chem.
271,
14661-14664 |
18. |
VanArsdale, T. L.,
VanArsdale, S. L.,
Force, W. R.,
Walter, B. N.,
Mosialos, G.,
Kieff, E.,
Reed, J. C.,
and Ware, C. F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
2460-2465 |
19. |
Wu, M. Y.,
Wang, P. Y.,
Han, S. H.,
and Hsieh, S. L.
(1999)
J. Biol. Chem.
274,
11868-11873 |
20. |
Wu, M. Y.,
Hsu, T. L.,
Lin, W. W.,
Campbell, R. D.,
and Hsieh, S. L.
(1997)
J. Biol. Chem.
272,
17154-17159 |
21. | Chen, Z., Seimiya, H., Naito, M., Mashima, T., Kizaki, A., Dan, S., Imaizumi, M., Ichijo, H., Miyazono, K., and Tsuruo, T. (1999) Oncogene 18, 173-180[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Gotoh, Y.,
and Cooper, J. A.
(1998)
J. Biol. Chem.
273,
17477-17482 |
23. |
Liu, Y.,
Yin, G.,
Surapisitchat, J.,
Berk, B. C.,
and Min, W.
(2001)
J. Clin. Invest.
107,
917-923 |
24. |
Ichijo, H.,
Nishida, E.,
Irie, K.,
ten Dijke, P.,
Saitoh, M.,
Moriguchi, T.,
Takagi, M.,
Matsumoto, K.,
Miyazono, K.,
and Gotoh, Y.
(1997)
Science
275,
90-94 |
25. |
Chang, H. Y.,
Nishitoh, H.,
Yang, X.,
Ichijo, H.,
and Baltimore, D.
(1998)
Science
281,
1860-1863 |
26. |
Wang, T. H.,
Popp, D. M.,
Wang, H. S.,
Saitoh, M.,
Mural, J. G.,
Henley, D. C.,
Ichijo, H.,
and Wimalasena, J.
(1999)
J. Biol. Chem.
274,
8208-8216 |
27. |
Kanamoto, T.,
Mota, M.,
Takeda, K.,
Rubin, L. L.,
Miyazono, K.,
Ichijo, H.,
and Bazenet, C. E.
(2000)
Mol. Cell. Biol.
20,
196-204 |
28. |
Tobiume, K.,
Matsuzawa, A.,
Takahashi, T.,
Nishitoh, H.,
Morita, K.,
Takeda, K.,
Minowa, O.,
Miyazono, K.,
Noda, T.,
and Ichijo, H.
(2001)
EMBO Rep.
2,
222-228 |
29. |
Wang, X. S.,
Diener, K.,
Jannuzzi, D.,
Trollinger, D.,
Tan, T. H.,
Lichenstein, H.,
Zukowski, M.,
and Yao, Z.
(1996)
J. Biol. Chem.
271,
31607-31611 |
30. |
Chen, M. C.,
Hsu, T. L.,
Luh, T. Y.,
and Hsieh, S. L.
(2000)
J. Biol. Chem.
275,
38794-38801 |
31. | Adler, V., Yin, Z., Tew, K. D., and Ronai, Z. (1999) Oncogene 18, 6104-6111[CrossRef][Medline] [Order article via Infotrieve] |
32. | Hoeflich, K. P., Yeh, W. C., Yao, Z., Mak, T. W., and Woodgett, J. R. (1999) Oncogene 18, 5814-5820[CrossRef][Medline] [Order article via Infotrieve] |
33. | Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985) Science 230, 1350-1354[Medline] [Order article via Infotrieve] |
34. | Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996) Nature 380, 723-726[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Rooney, I. A.,
Butrovich, K. D.,
Glass, A. A.,
Borboroglu, S.,
Benedict, C. A.,
Whitbeck, J. C.,
Cohen, G. H.,
Eisenberg, R. J.,
and Ware, C. F.
(2000)
J. Biol. Chem.
275,
14307-14315 |
36. | Banner, D. W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H. J., Broger, C., Loetscher, H., and Lesslauer, W. (1993) Cell 73, 431-445[Medline] [Order article via Infotrieve] |
37. |
Eck, M. J.,
and Sprang, S. R.
(1989)
J. Biol. Chem.
264,
17595-17605 |
38. |
Eck, M. J.,
Ultsch, M.,
Rinderknecht, E.,
de Vos, A. M.,
and Sprang, S. R.
(1992)
J. Biol. Chem.
267,
2119-2122 |
39. |
Saitoh, M.,
Nishitoh, H.,
Fujii, M.,
Takeda, K.,
Tobiume, K.,
Sawada, Y.,
Kawabata, M.,
Miyazono, K.,
and Ichijo, H.
(1998)
EMBO J.
17,
2596-2606 |
40. | Nishitoh, H., Saitoh, M., Mochida, Y., Takeda, K., Nakano, H., Rothe, M., Miyazono, K., and Ichijo, H. (1998) Mol. Cell 2, 389-395[Medline] [Order article via Infotrieve] |
41. |
Hatai, T.,
Matsuzawa, A.,
Inoshita, S.,
Mochida, Y.,
Kuroda, T.,
Sakamaki, K.,
Kuida, K.,
Yonehara, S.,
Ichijo, H.,
and Takeda, K.
(2000)
J. Biol. Chem.
275,
26576-26581 |
42. |
Charette, S. J.,
Lambert, H.,
and Landry, J.
(2001)
J. Biol. Chem.
276,
36071-36074 |
43. |
Garcia-Calvo, M.,
Peterson, E. P.,
Leiting, B.,
Ruel, R.,
Nicholson, D. W.,
and Thornberry, N. A.
(1998)
J. Biol. Chem.
273,
32608-32613 |
44. | Chang, Y., Hsieh, S., Chen, M., and Lin, W. (2002) Exp. Cell Res. 278, 166-174[CrossRef][Medline] [Order article via Infotrieve] |
45. | Hochedlinger, K., Wagner, E. F., and Sabapathy, K. (2002) Oncogene 21, 2441-2445[CrossRef][Medline] [Order article via Infotrieve] |
46. | Herr, I., Wilhelm, D., Meyer, E., Jeremias, I., Angel, P., and Debatin, K. M. (1999) Cell Death Differ. 6, 130-135[CrossRef][Medline] [Order article via Infotrieve] |
47. |
Liu, H.,
Nishitoh, H.,
Ichijo, H.,
and Kyriakis, J. M.
(2000)
Mol. Cell. Biol.
20,
2198-2208 |