|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 27, 19211-19219, July 2, 1999
From the Caspase-8 is the most proximal caspase in the
caspase cascade and possesses a prodomain consisting of two homologous
death effector domains (DEDs). We have discovered that caspase-8 and its homologs can physically interact with tumor necrosis factor receptor-associated factor family members and activate the c-Jun N-terminal kinase (JNK, or stress-activated protein kinase) pathway. This ability resides in the DED-containing prodomain of these proteins
and is independent of their role as cell death proteases. A point
mutant in the first DED of caspase-8 can block JNK activation induced
by several death domain receptors. Inhibition of JNK activation blocks
apoptosis mediated by caspase-10, Mach-related inducer of
toxicity/cFLIP, and Fas/CD95, thereby suggesting a cooperative role of
this pathway in the mediation of caspase-induced apoptosis.
Caspase-8 and its homologs play an essential role in the mediation
of apoptosis by several DD1
receptors such as tumor necrosis factor receptor 1 (TNFR1), Fas/Apo-1, death receptor 3 (DR3, or Wsl-1, APO-3, or TRAMP), death receptor 4 (DR4, or TNF-related apoptosis-inducing ligand receptor 1), and death
receptor 5 (DR5, or TNF-related apoptosis-inducing ligand receptor 1)
(1-4). Caspase-8 is recruited to these receptors via the interaction
of its prodomain with Fas-associated death domain (FADD, or MORT1),
which leads to formation of the death-inducing signaling complex (DISC)
(5-7). Upon its recruitment to the DISC, caspase-8 is activated by an
autoproteolytic mechanism involving the removal of the prodomain and
the release of its activated protease subunits into the cytosol (8).
Activated caspase-8 acts as the initiator caspase in the caspase
cascade, activation of which eventually results in cell death (9). The
DED-containing prodomains are also found in two additional cellular
proteins; caspase-10 (Mch4, FLICE2), a proteolytically active caspase-8 homolog (3, 10), and Mach-related inducer of toxicity (MRIT; also
called c-FLIP, Casper, I-FLICE, FLAME, CASH, and CLARP), a caspase-8
homolog devoid of protease activity (11-17).
In addition to its ability to activate the caspase cascade, TNFR1 is
also known to activate a kinase cascade involving activation of the
nuclear factor- The role of caspase cascade in JNK activation is also controversial.
Fas/Apo-1-induced JNK activation in a Fas-sensitive neuroblastoma cell
line was shown to be resistant to the caspase inhibitor z-VAD-fmk (23).
Similarly, Fas-induced JNK activation in 293 cells was resistant to
caspase inhibitors CrmA, z-VAD, and z-DEVD (19). On the contrary,
Fas-induced JNK activation was found to be sensitive to caspase
inhibitors in Jurkat (T cell leukemia) and SKW6.4 (B lymphoblastoid)
cells (31, 32). Finally, a recent study analyzed Fas-mediated JNK
activation in Jurkat cells and discovered that while the JNK activity
induced by low levels of Fas cross-linking could be blocked by z-VAD,
that activated at higher levels of Fas cross-linking was
z-VAD-resistant (30). Collectively, these studies suggest that,
depending on the cell type and the magnitude of the stimulus, Fas can
activate the JNK pathway by caspase-dependent and
-independent pathways.
Until recently, DED-containing proteins were mainly known for their
interaction with FADD and the resultant activation of the caspase
cascade. However, a recent study, using yeast two-hybrid interaction
and coimmunoprecipitation assays, demonstrated that MRIT/Casper can
also interact with various TRAF family members (12). This study,
however, did not test the functional significance of the above
interaction. In the present study, we explore the ability of MRIT and
other caspase family members to interact with the TRAFs and activate
the JNK pathway.
Cell Lines and Reagents--
293T and MCF7 cells were obtained
from Dr. David Han (University of Washington, Seattle). 293 EBNA cells
were obtained from Invitrogen. All cells were maintained in Dulbecco's
modified Eagle's medium with 10% fetal calf serum at 37 °C with
5% carbon dioxide. Rabbit polyclonal antibodies against FLAG,
hemagglutinin (HA), and Myc tags were obtained from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Murine antibody against human
FADD was obtained from Transduction Laboratories. Flag beads were
obtained from Kodak Scientific Imaging Systems. A pull-down kinase
assay kit for stress-activated protein kinase/JNK was obtained from New England Biolabs, and the constructs for Pathdetect luciferase reporter
assay were purchased from Stratagene.
Expression Constructs--
The expression constructs for the
death receptors, FADD, CrmA, MRIT isoforms, and the various caspases
have been described previously (11, 33). Constructs encoding caspase-1,
-2, -3, -6, -7, and -9 were gifts from Michael Wright (University of
Washington). Constructs encoding caspase-10 (Mch4 isoform) and its
C358A mutant (3) have been described previously and were obtained from
the indicated source. A construct encoding JBD-JIP1 was a gift from Dr.
Roger Davis, and dominant negative rMEKK1 was obtained from Dr. Richard
Gaynor. Constructs encoding mTRAF1, mTRAF2, TRAF3, mTRAF5, and I-TRAF
were prepared using the corresponding IMAGE consortium expressed
sequence tag clones (Genome Systems) as templates. The various point
mutants of caspase-8, -7, and -9 were generated using the Quickchange
site-directed mutagenesis kit (Stratagene). The various deletion and
fusion constructs were made by polymerase chain reaction using custom
primers. Epitope-tagged expression constructs for caspase-3, -8, and
-10; MRIT; TRAFs; and their deletion and point mutants were tagged at
the C termini. The sequence of all constructs was confirmed by
automated fluorescent dye-terminator sequencing on an ABI 373 sequencing machine. Expression of all constructs was confirmed by
Western analysis on total cell extracts.
c-Jun Transcriptional Activation Assay--
293 EBNA cells
(1.2 × 105) were transfected in duplicate with
various expression constructs (500 ng) along with a fusion
transactivator plasmid containing yeast GAL4 DNA-binding domain fused
to transcription factor c-Jun (pFA-cJun) (50 ng), a reporter plasmid
encoding the luciferase gene downstream of the GAL4 upstream activating
Sequence (pFR-luc) (500 ng), and a Rous sarcoma virus/LacZ
( Coimmunoprecipitation Assays--
For studying in
vivo interaction, 2 × 106 293T cells were plated
in a 100-mm plate and co-transfected 18-24 h later with 5 µg/plate
of each epitope-tagged construct along with 1 µg of a green
fluorescent protein (GFP)-encoding plasmid (pEGFP or HA-GFP) by calcium
phosphate co-precipitation. 18-36 h post-transfection, cells were
lysed in 1 ml of lysis buffer containing 0.1% Triton X-100, 20 mM sodium phosphate (pH 7.4), 150 mM NaCl, and
one EDTA free mini-protease inhibitor tablet per 10 ml (Roche Molecular Biochemicals). For immunoprecipitation, cells lysate (500 µl) was
incubated for 1 h at 4 °C with 10 µl of FLAG or control mouse Ig beads precoated with 2% bovine serum albumin. Beads were washed twice with lysis buffer, twice with a wash buffer containing 0.1% Triton X-100, 20 mM sodium phosphate (pH 7.4), 500 mM NaCl and again with lysis buffer. Bound proteins were
eluted by boiling, separated by SDS-polyacrylamide gel electrophoresis,
transferred to a nitrocellulose membrane, and analyzed by Western blot.
Activation of the JNK Pathway by Caspase-8 and Homologs--
As a
measure of JNK activation, we tested the ability of caspase-8 and its
homologs to induce phosphorylation of transcription factor c-Jun in a
"pull-down" kinase assay. As shown in Fig.
1A, expression of active site
mutants of caspase-8 and -10 and MRIT led to significant JNK activation
in 293 EBNA cells, which was comparable in magnitude with that observed
with CD40, a known activator of this pathway. All of the above
DED-containing proteins are missing the critical cysteine residue at
the putative caspase active site and are proteolytically inactive.
Therefore, JNK activation by them is not related to the activation of
the caspase cascade and the resultant activation of the stress-response
genes. Activation of the JNK pathway by the above DED-containing
proteins was confirmed using a reporter assay in which luciferase
expression was driven by JNK-mediated phosphorylation of the activation
domain of transcription factor c-Jun fused to the GAL4 DNA binding
domain (Fig. 1B). This assay further demonstrated that the
DED-containing prodomains of caspase-8 and MRIT were even more
effective than the full-length proteins in c-Jun transcriptional
activation (Fig. 1B). Additional experiments localized the
JNK-activating ability of caspase-8 to its prodomain, while no
significant JNK activation was observed with the construct expressing
its protease domain (Fig. 1C). Coexpression of the caspase-8
prodomain with either a GAL4-cFos fusion construct or a GAL4 DNA
binding domain construct failed to activate the luciferase reporter
construct, thereby demonstrating the specificity of the assay (Fig.
1C). The observed activation of the JNK pathway was not
limited to 293 EBNA cells, since the caspase-8 prodomain activated this
pathway in COS (African green monkey kidney) and 293 (human embryonic
kidney) cells as well (data not shown). However, we have failed to
observe significant activation of the JNK pathway by the above proteins
in the baby hamster kidney cells (data not shown), which indicates
tissue/species specificity of this response.
Failure of Non-DED-containing Caspases to Activate the JNK
Pathway--
We also tested the JNK activating ability of several
non-DED-containing caspases, such as caspase-1, -2, -3, -7, and -9 and active site mutants of caspase-7 and -9. However, none of these caspases were able to activate the JNK pathway in 293 EBNA cells (Fig.
2A and data not shown),
supporting the requirement of the DEDs for this process.
Effect of CrmA and p35 on the JNK Activation by Caspase-8 and Its
Homologs--
The ability of MRIT and the active site mutants of
caspase-8 and -10 to activate the JNK pathway suggests that this
ability is independent of their role in the activation of the caspase cascade and the resultant activation of the stress response genes. However, it is conceivable that the overexpression of these proteins leads to their oligomerization with the endogenous caspases and the
resultant activation of the caspase cascade. Similarly, the prodomain
of caspase-8 could activate the JNK pathway by oligomerizing and
activating the endogenously expressed caspase-8. To rule out this
possibility, we tested the ability of CrmA and p35 to block JNK
activation by the caspase-8 prodomain and full-length MRIT (MRIT- Virally Encoded DED-containing Proteins Fail to Activate the JNK
Pathway--
Several viruses were recently shown to encode proteins
that contain DEDs, and these proteins were found to block apoptosis mediated by death domain receptors by binding to FADD or caspase-8 (34-36). These proteins, also called vFLIPs (FLICE inhibitory
proteins), include MC159L from the molluscum contagiosum virus, E8 from
equine herpes virus 2, and ORF-K13 from Kaposi's sarcoma-associated
herpes virus. Based on the ability of cellular DED-containing proteins to activate the JNK pathway, we were interested in checking whether their viral homologs could do the same. However, as shown in Fig. 2C, none of the vFLIPs could activate the JNK pathway,
thereby indicating the specificity of this response. Since both
cellular and viral DED-containing proteins share the property of
binding to FADD, these negative results also argue against the
involvement of FADD in JNK activation by the cellular DED-containing proteins.
A Point Mutant of Caspase-8 DED Acts as Dominant Negative Inhibitor
of JNK Activation by Death Receptors--
To further define the role
of DEDs in caspase-8-mediated JNK activation, we mutated several
conserved residues in the DED1 of caspase-8 and tested their ability to
activate JNK activation in the 293 EBNA cells. One of these mutants,
containing an Asp to Ala substitution at amino acid residue 73 (caspase-8 D73A) was found to be incapable of JNK activation on its own
and was selected for further studies (Fig.
3A and data not shown). As shown in Fig. 3A, this mutant effectively blocked JNK
activation by caspase-8 prodomain and caspase-10 C358A in a dominant
negative fashion (Fig. 3A). This mutant was also highly
effective in blocking JNK activation induced by different DD receptors,
including TNFR1, Fas/CD95, DR3, and DR4, consistent with the
involvement of caspase-8 or its homologs in JNK activation by these
receptors (Fig. 3B). Finally, the caspase homology domain of
caspase-8 could partially block JNK activation induced by caspase-8 and
-10 as well (Fig. 3A), thus explaining the stronger JNK
activation seen with the construct encoding only the caspase-8
prodomain (Fig. 1B).
Inhibitors of Caspase-8-induced JNK Activation--
TRAF2 has been
shown to play an essential role in JNK activation by different members
of the TNFR family. To determine its role in caspase-8-induced
activation of the JNK pathway, we used a dominant negative mutant of
TRAF2 (amino acids 87-501) that has been previously shown to block JNK
activation by different TNFR family members. As shown in Fig.
3C, dominant negative TRAF2 could successfully block JNK
activation by the caspase-8 prodomain. TRAF2 is believed to activate
the JNK/stress-activated protein kinase pathway via mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) (37, 38). Therefore, we next tested the involvement of MEKK1 in
JNK activation by the caspase-8 prodomain. As shown in Fig.
3D, caspase-8 prodomain-induced JNK activation was
effectively blocked by a kinase-defective mutant of full-length rat
MEKK1.
JIP-1 is a recently identified cytoplasmic protein that binds to JNK
through its N-terminal JNK-binding domain (JBD) (27). JIP-1 and its JBD
cause cytoplasmic retention of JNK and are specific inhibitors of the
JNK signal transduction pathway in several cellular processes including
cell death (27). As shown in Fig. 3E, JBD of JIP-1 was
highly effective in blocking JNK activation mediated by the caspase-8 prodomain.
Interaction of Caspase-8 and Its Homologs with the TRAF
Proteins--
To delineate the mechanism of JNK activation by
caspase-8 and its homologs, we tested their ability to physically
interact with the TRAF family members using a
coexpression-coimmunoprecipitation assay in 293T cells. Caspase-8 could
coimmunoprecipitate TRAF1, -2, -3, and -5 but failed to
coimmunoprecipitate I-TRAF (Fig. 4,
A-C). This ability to immunoprecipitate TRAF proteins was
not limited to caspase-8, since caspase-10 could successfully
coimmunoprecipitate TRAF1 and TRAF2 as well (data not shown). We also
confirmed the previously demonstrated interaction between MRIT/Casper
and TRAFs (data not shown). However, caspase-3, a non-DED-containing
caspase, failed to coimmunoprecipitate TRAF1 in the above assay,
thereby demonstrating the specificity of the interaction (data not
shown). Caspase-8 also failed to coimmunoprecipitate GFP, which served as a negative control (Fig. 4, B, F, and
G). The ability of caspase-8 to interact with TRAF1 protein
was confirmed using bacterially expressed and purified proteins, which
demonstrated that this interaction does not require the presence of any
intermediate bridging protein(s) that could be present in 293T cells
(Fig. 4D).
We also tested the interaction between caspase-8 D73A and the TRAFs. As
shown in Fig. 4E, this mutant of caspase-8 successfully coimmunoprecipitated TRAF1 or TRAF2. These results indicate that activation of the JNK pathway involves interaction of specific residue(s) of caspase-8 prodomain with the TRAFs and not the simple act
of binding of the two proteins.
We next sought to determine which domains of caspase-8 interact with
the TRAF proteins. Both the prodomain (amino acids 1-180) and the
caspase homology domain (amino acids 217-479) of caspase-8 could
coimmunoprecipitate TRAF1 or TRAF2 (Fig. 4, F and
G). The ability of the prodomain of caspase-8 to interact
with TRAF proteins was unexpected, since a deletion construct encoding
the homologous prodomain of MRIT/Casper was recently shown not to
interact with the TRAF proteins (12). To resolve this discrepancy, we
tested the ability of the naturally occurring MRIT
The TRAF proteins are characterized by the presence of a TRAF domain at
their COOH termini, which, based on its functions and sequence
conservation among different family members, can be further subdivided
into TRAF-N and TRAF-C subdomains (39). For example, the TRAF-N domain
of various family members is required for self-association and
association with the cellular inhibitors of apoptosis proteins, whereas
the TRAF-C domain is required for interaction with receptors and
signaling molecules RIP and TNFR1-associated death domain (TRADD) (39).
We used deletion mutagenesis to map the domain(s) of TRAF1 that are
necessary for its interaction with caspase-8. As shown in Fig.
4H, a C-terminal deletion mutant of TRAF1 (encoding residues
1-262), which is missing the TRAF-C domain, could weakly interact with
caspase-8. However, an almost complete absence of interaction with
caspase-8 was observed in experiments that utilized a construct missing
the TRAF-N domain as well (encoding residues 1-190). These results are
in agreement with the previously demonstrated interactions between
MRIT/Casper and TRAF2 and suggest the importance of an intact TRAF
domain for the interaction with caspase-8 and its homologs.
Effect of FADD on Caspase-8-TRAF Interaction--
Caspase-8 is
recruited to the DISC through the interaction of its DED-containing
prodomain with the DED of the adaptor molecule FADD. Recruitment to
FADD leads to autoproteolytic activation of caspase-8, resulting in
activation of the caspase cascade and eventual cell death. We were
interested in determining whether FADD can influence the interaction
between caspase-8 and TRAF proteins. Coexpression of FADD had no
significant effect on the interaction between full-length caspase-8 and
either TRAF1 or TRAF2 protein (data not shown). We reasoned that these
results could be due to the inability of FADD to influence the
interaction between the protease domain of caspase-8 and the TRAF
proteins. Therefore, the above experiment was repeated with the
prodomain of caspase-8 and the TRAF proteins. As shown in Fig.
4I, FADD led to displacement of TRAF1 or TRAF2 proteins from
the caspase-8 prodomain. These results suggest that the multidomain
interactions of TRAFs with the DED-containing proteins probably
contribute to the stability of the interaction, thereby allowing the
formation of multiprotein signaling complex. Moreover, these results
argue against the possibility that the caspase-8-TRAF interactions are mediated via a FADD-TRADD bridge.
Role of JNK Activation in Caspase-induced Cell Death--
We next
sought to determine the role of JNK activation in caspase-mediated cell
death. We chose caspase-10 for these experiments, since it was found to
be a more efficient inducer of cell death than caspase-8 or MRIT. The
JBD of JIP-1 could partially block caspase-10-induced cell death in
293T and 293 cells (Fig. 5, A and B, and data not shown). The inhibitory role of the JBD
of JIP-1 was more pronounced when the amount of caspase-10 plasmid was
kept low and became less significant at higher levels of caspase-10 expression, at which point massive activation of the caspase cascade probably obviated any requirement of JNK activation for apoptosis (data
not shown). Consistent with the role of JNK activation in caspase-mediated cell death, JBD of JIP-1 could effectively block apoptosis mediated by Fas/CD95 (Fig. 5C). However, we did
not observe any significant inhibition of caspase-10-induced cell death
by the caspase-8 D73A mutant, which could reflect the simultaneous activation of the caspase cascade. Taken together, the above results suggest that while JNK activation may facilitate caspase-10-induced apoptosis in the cells tested, it may not be essential for this process.
Role of JNK Activation in MRIT-induced Cell Death--
Although
MRIT is proteolytically inactive, its overexpression has been shown to
lead to apoptosis (11, 12, 15, 16). Therefore, we were interested in
testing the role of the JNK activation in MRIT-induced apoptosis. We
began by performing a dose-response experiment to look for any
correlation between the JNK- and apoptosis-inducing abilities of MRIT.
As shown in Fig. 5D, transfection of MRIT- Death effector domain-containing caspases and caspase homologs
have been extensively studied in the activation of programmed cell
death. The proapoptotic ability of these caspases and caspase homologs
has been so far attributed to their ability to proteolytically cleave
and activate the downstream executioner caspases. In the present study,
we provide evidence that transient overexpression of the DED-containing
caspases and caspase homologs may lead to cell death by an indirect
route involving activation of the JNK pathway.
Based on the available data, it is likely that DED-containing proteins
activate the JNK pathway by virtue of their interaction with the TRAF
proteins and subsequent activation of MEKK1. This conclusion is
supported by the ability of dominant negative mutants of TRAF2 and
MEKK1 to block caspase-8 prodomain-induced JNK activation. In addition
to MEKK1, we have also tested the involvement of ASK1 in caspase-8
prodomain-induced JNK activation. However, a kinase-inactive mutant of
ASK1 (22) has been only partially effective in blocking caspase-8
prodomain-induced JNK activation in 293 EBNA cells, arguing against a
major role of ASK1 in caspase-8-induced JNK activation.2
The activation of the JNK pathway by the DED-containing proteins is
unlikely to be secondary to the activation of caspase cascade and the
resultant activation of stress-response genes based on the following
evidence. First, this activity was localized to the prodomain of these
proteins rather than the caspase homology domain. Second, active site
mutants of caspase-8 and -10, which are proteolytically inactive, could
efficiently activate the JNK pathway. Third, several non-DED-containing
caspases failed to activate this pathway. Finally, CrmA and p35, two
inhibitors of caspases and the apoptosis induced by them, could not
block JNK activation by the caspase-8 prodomain.
It is unlikely that the caspase-8 and its homologs activate the JNK
pathway due to the overexpression-induced reverse signaling via a
caspase-8/10/MRIT-FADD-TRADD-death receptors-TRADD-TRAF2 pathway due to
the following reasons. First, Shu et al. (12) have
previously shown that Casper/MRIT could interact with both TRAF1 and
TRAF2 in the yeast two-hybrid assay, which rules out the involvement of
any bridging protein(s) in this interaction. In fact, these researchers
successfully isolated a total of nine independent clones of the TRAFs
that interacted with Casper using the above assay, which points to the
strength of the interaction (12). Second, we have similarly observed an
interaction between bacterially expressed caspase-8 and TRAF1 in the
present study. Third, a reverse signaling complex containing
caspase-8/10/MRIT-FADD-TRADD-death domain receptors-TRADD-TRAF2 has
never been demonstrated. In fact, Shu et al. (12)
specifically looked for such a complex in the case of MRIT/Casper but
failed to recruit MRIT/Casper to TNFR1 complex by the addition of
TRADD, FADD, and TRAF2 either individually or in various combinations.
Similarly, they failed to generate a complex consisting of MRIT, TRADD,
and TRAF2 (12). We have similarly failed to detect the presence of
endogenous FADD in the coimmunoprecipitated complex of caspase-8 with
TRAFs (data not shown) and have found that cotransfection of FADD, in
fact, decreases the binding between caspase-8 prodomain and TRAF1 or -2. Fourth, virally encoded DED-containing proteins, which have been
previously shown to bind FADD (34-36), fail to activate the JNK
pathway. Similarly, we have observed that the caspase-8 D73A mutant,
which cannot activate the JNK pathway, continues to bind to FADD.
While both CrmA and p35 failed to block caspase-8-induced JNK
activation, they successfully blocked the activation of this pathway by
MRIT. This result is somewhat surprising, since CrmA does not directly
bind to MRIT (12). However, since CrmA is known to bind to caspase-8
(12), it is possible that it blocks MRIT-induced JNK activation by
blocking its interaction with caspase-8. Alternatively, MRIT may
require activation by caspases for activation of the JNK pathway, and
this step may be blocked by CrmA and p35.
An important discovery of the present study was the role of the JNK
pathway in MRIT-induced cell death. Overexpression of MRIT has been
shown to lead to cell death by several independent groups (11, 12, 15,
16). Based on the ability of CrmA to block MRIT-mediated cell death, we
and others had previously concluded that MRIT induces apoptosis by
activating the caspase cascade (11, 12, 15). However, overexpression of
MRIT/CLARP in 293T cells, under the conditions where it induces
significant apoptosis, failed to generate caspase activity (15). In the present study, we have discovered that CrmA is also capable of blocking
MRIT-induced JNK activation, which provides an alternative explanation
for the ability of CrmA to block MRIT-induced cell death.
A major limitation of the current study is that the activation of the
JNK pathway is observed under conditions of overexpression of caspase-8
(and its homologs), and it is not clear whether the same effect would
be observed under an endogenous level of expression of this protein. It
can be further argued that the observed effect is an artifact of
overexpression-induced aggregation of caspase-8. However, under
endogenous conditions, caspase-8 is present in the cells as a minimally
active zymogen and is even incapable of inducing cell death.
Recruitment to the DISC allows aggregation of caspase-8 zymogens and
generation of active caspase molecules via cross-proteolysis (8).
Therefore, overexpression-induced aggregation of caspase-8 may mimic a
physiological step in the activation of this protein and may not be
entirely artificial. While not the perfect approach, it currently
represents the only practical way for studying the signaling by
cytosolic proteins in their native form due to our inability to
cross-link them using antibodies. We have also attempted to generate
stable cell lines expressing the prodomain of caspase-8 with the hope
of subsequently testing them for JNK activation. However, we have so
far failed to generate such cell lines, which might reflect the
cytotoxic effect of constitutive JNK activation.
The physiological role of caspase-8 in the JNK activation by death
domain-containing receptors is also supported by two recent studies
(40, 41). The first study compared the activation of the JNK pathway in
embryonic fibroblast cell lines derived from caspase-8 +/+ and
caspase-8 We thank Drs. Vishva Dixit, Roger Davis,
Audrey Minden, Anning Lin, Emad Alnemri, Richard Gaynor, Melanie Cobb,
and Micheal Wright for various plasmids and Dr. David Han for helpful discussions.
*
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.
§
Supported by a Postdoctoral Fellowship from the Cancer Research
Fund of the Damon Runyon-Walter Winchell Foundation. To whom correspondence and reprint requests should be addressed: Hamon Center
for Therapeutic Oncology Research, UT Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas TX 75235-8593. Tel.: 214-648-1837; Fax:
206-648-4940; E-mail: chaudhary{at}simmons.swmed.edu.
2
P. M. Chaudhary, M. T. Eby, A. Jasmin,
and L. Hood, unpublished observation.
The abbreviations used are:
DD, death domain;
TNF, tumor necrosis factor;
TNFR, TNF receptor;
DR3, DR4, and DR5,
death receptor 3, 4, and 5, respectively;
FADD, Fas-associated death
domain;
TRADD, TNFR1-associated death domain;
DISC, death-inducing
signaling complex;
DED, death effector domain;
MRIT, Mach-related
inducer of toxicity;
TRAF, tumor necrosis factor-associated factor;
JNK, c-Jun N-terminal kinase;
NF-
Activation of the c-Jun N-terminal Kinase/Stress-activated
Protein Kinase Pathway by Overexpression of Caspase-8 and Its
Homologs*
§,,, and Hamon Center for Therapeutic Oncology
Research, University of Texas Southwestern Medical Center, Dallas,
Texas 75235-8593 and the ¶ Department of Molecular Biotechnology,
University of Washington, Seattle, Washington 98195
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B (NF-B) and the JNK pathways via recruitment of
tumor necrosis factor receptor-associated factor 2 (TRAF2) and
receptor-interacting protein (RIP) (18). On the other hand, Fas/Apo-1
was recently shown to activate the JNK pathway via the adaptor protein
Daxx (19). Activation of the JNK pathway leads to the
phosphorylation-induced activation of transcription factors c-Jun,
activating transcription factor-2, and Elk-1 (20, 21). While JNK
activation has been implicated in oncogenic transformation and immune
response in mammalian cells, its role in cell death is still
controversial. For example, overexpression of several kinases of this
pathway has been shown to induce cell death, whereas their
corresponding dominant negative mutants can block stress-induced and DD
receptor-induced apoptosis in several cell lines (22-25). JNK
activation has also been implicated in nerve growth factor withdrawal-induced apoptosis of PC12 cells, which can be blocked by
JNK-interacting protein-1 (JIP-1), a recently identified inhibitor of
the JNK pathway (26, 27). Daxx was recently shown to activate JNK via
apoptosis signal-regulating kinase 1 (ASK1) and act cooperatively with
the FADD-induced caspase cascade in the mediation of Fas-induced apoptosis (19, 28). Finally, gene knock-out of JNK3, a terminal kinase
of the JNK pathway leads to resistance against neuronal cell death
(29). On the contrary, JNK activation was found to be dispensable for
the TNF
- and/or Fas-induced apoptosis in HeLa and Jurkat cells,
suggesting that the requirement for the JNK pathway in apoptosis is
cell type- and stimulus-dependent (18, 30).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase) reporter construct (75 ng). Transfection was
performed using the calcium phosphate coprecipitation method. 32-40 h
later, cell extracts were prepared using the Luciferase cell culture
lysis reagent (Promega, Madison, WI), and luciferase assays were
performed using 20 µl of cell extract. The cell lysate was diluted
1:20 with phosphate-buffered saline (pH 7.4) and used for the
measurement of -galactosidase activity. Luciferase activity was
normalized relative to the -galactosidase activity to control for
the difference in the transfection efficiency. Western blot analysis on
cell extracts prepared from similarly transfected cells was used to confirm that the various expression constructs led to the production of
the desired proteins.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Activation of the JNK pathway by caspase-8,
caspase-10, and MRIT. A, 293 EBNA cells were
transfected with the indicated plasmids (5 µg) along with a plasmid
encoding GFP (1 µg) using a calcium phosphate co-precipitation
method. 36 h later, cells were examined under a fluorescent
microscope to ensure equal transfection efficiency. Cells were
subsequently lysed, and a "pull-down" kinase assay for the
activation of endogenously expressed stress-activated protein
kinase/JNK was performed using a stress-activated protein kinase/JNK
assay kit (New England Biolabs). GST-c-Jun coupled to agarose beads was
used for both pulling down the endogenously expressed stress-activated
protein kinase/JNK and as a substrate for activated JNK-induced
phosphorylation. B, activation of the JNK pathway as
measured by the PathDetect c-Jun Trans-Reporting system (Stratagene).
293 EBNA cells (1.2 × 105) were transfected in
duplicate with the indicated constructs (500 ng) along with a fusion
transactivator plasmid containing yeast GAL4 DNA-binding domain fused
to transcription factor c-Jun (pFA-cJun) (50 ng), a reporter plasmid
encoding the luciferase gene downstream of the GAL4 upstream activating
sequence (pFR-luc) (500 ng), and a Rous sarcoma virus/LacZ
( -galactosidase) reporter construct (75 ng). 40 h later, cell
extracts were prepared and used for the measurement of luciferase and
-galactosidase activities. Luciferase activity was normalized
relative to the -galactosidase activity to control for the
difference in transfection efficiency. Values shown are mean ± S.E. Control experiments demonstrated no activation of luciferase
reporter gene transcription in cells transfected with vector encoding
only the GAL4 DNA binding domain (pFC-DBD) instead of pFA-cJun plasmid.
Casp 8-Pro, caspase-8 prodomain (residues 1-180).
C, c-Jun transcriptional activation by the caspase-8
prodomain (Casp 8 PRO) and protease domain. The experiment was
performed as described for B and was repeated with a
GAL4-cFos fusion vector (c-Fos) or a vector encoding the
GAL4 DNA binding domain only (DBD). Values shown are
mean ± S.E. of a representative of two independent
experiments.
View larger version (15K):
[in a new window]
Fig. 2.
Further analysis of caspase-mediated JNK
activation. A, non-DEDs containing caspases fail to induce
c-Jun transcriptional activation. The active site mutants of caspase-7
and -9 were used for the above experiments as the wild-type proteins
induced massive cell death. Values shown are mean ± S.E. of a
representative of two independent experiments performed in duplicate.
Casp, caspase. B, CrmA and p35 failed to block
c-Jun transcriptional activation by the caspase-8 prodomain but
effectively blocked that induced by MRIT- 1. Values shown are
mean ± S.E. of a representative of two independent experiments
performed in duplicate. C, virally encoded DED-containing
proteins (vFLIPs) fail to induce c-Jun transcriptional activation.
Values shown are mean ± S.E. of a representative of two
independent experiments performed in duplicate.
1
isoform). CrmA is a cowpox virus-encoded inhibitor of the protease
activity of the proximal caspases, whereas p35 is a baculovirus-encoded
inhibitor of both proximal and distal caspases. As demonstrated in Fig.
2B, both CrmA and p35 failed to block JNK activation by the
caspase-8 prodomain, thereby confirming that it activates the JNK
pathway independent of the activation of the caspase cascade.
Surprisingly, both CrmA and p35 could block JNK activation by MRIT
(Fig. 2B), which suggests a difference in the mechanism of
JNK activation between MRIT and caspase-8.
View larger version (32K):
[in a new window]
Fig. 3.
Modulation of caspase-8-mediated JNK.
A, deletion and point mutants of caspase-8 block c-Jun
transcriptional activation in a dominant negative fashion. The
experiment was performed as described in the legend to Fig.
1B using 150 ng/well of empty vector, the caspase-8
prodomain, or caspase-10 C358A plasmid, and 750 ng/well of control
vector, caspase-8 D73A, or the caspase-8 protease domain plasmid.
Values shown are mean ± S.E. of a representative of two
independent experiments performed in duplicate. B, caspase-8
D73A mutant blocks JNK activation by various DD receptors. The
experiment was performed as described above using 150 ng/well of empty
vector or various receptors and 750 ng/well of empty vector or the D73A
plasmid. C, inhibition of caspase-8 prodomain-induced JNK
activation by a dominant-negative mutant of TRAF2 (DN-TRAF2). 293 EBNA
cells were transfected with an empty vector or an expression vector for
caspase-8 prodomain (4 µg each) with or without an expression vector
for DN-TRAF2 (4 µg), and the "pull-down" kinase assay performed
as described for Fig. 1A. The total amount of DNA was kept
constant by adding empty vector. D, inhibition of the
caspase-8 prodomain-induced JNK activation by dominant-negative
full-length rat MEKK1. 293 EBNA cells were transfected with the
indicated plasmids, and a "pull-down" kinase assay was performed as
described for Fig. 3C. Lack of JNK inhibition with wild-type
MEKK1 demonstrates the specificity of the assay. A representative of
two independent experiments is shown. E, inhibition of the
caspase-8 prodomain-induced JNK activation by JBD of JIP. The
experiment was performed as described for Fig. 3C. A
representative of two independent experiments.
View larger version (29K):
[in a new window]
Fig. 4.
Interaction of caspase-8 and its homologs
with TRAFs. A, interaction of caspase-8 with TRAF1, TRAF2,
and DN-TRAF2. 293T cells were transfected with expression vectors
encoding FLAG-tagged caspases along with HA-tagged murine TRAFs.
FLAG-tagged proteins were immunoprecipitated using beads containing an
antibody against the FLAG epitope or an irrelevant mouse antibody
(control). The coimmunoprecipitating HA-tagged TRAFs were detected by
Western blot analysis using rabbit polyclonal antibodies against the HA
tag. The expression of caspase-8 is shown by Western analysis on the
cell lysates. All constructs were tagged at the COOH termini.
B, interactions of caspase-8 with TRAF3. 293T cells were
transfected with expression vectors encoding FLAG-tagged caspases along
with HA-tagged TRAF3 and GFP-HA. The experiment was performed as
described in Fig. 4A. The lack of immunoprecipitation of GFP
with caspase-8 demonstrates the specificity of the interaction. The
expression of caspase-8 is shown by Western analysis on the cell
lysates. C, interaction of caspase-8 with TRAF5. 293T cells
were transfected with the indicated plasmids. Flag-tagged murine TRAF5
was immunoprecipitated with Flag beads or control beads, and the
coimmunoprecipitated Myc-caspase-8 was detected by Western blot with a
rabbit polyclonal antibody against the Myc tag. The expression of TRAF5
is shown by Western analysis on the cell lysates. D,
interaction of caspase-8 and TRAF1 in a cell-free system. Myc-caspase-8
and HA-mTRAF1 were cloned in pET28 bacterial expression vector, and the
proteins were expressed in the BL21(DE3)pLysS host cells following the
manufacturer's instructions (Novagen, Madison, WI). Cells were lysed
by three cycles of freeze-thaw and subsequently sonicated. The lysed
samples were centrifuged, and equal volumes of supernatant from
cultures expressing Myc-caspase-8 and mTRAF1-HA were incubated for
1 h at 4 °C. Coimmunoprecipitation was carried out with Myc
beads or control beads as described previously, and Western blot
analysis was performed using rabbit polyclonal antibodies against the
HA tag. E, interactions of the caspase-8 D73A mutant with
TRAF1 or TRAF2. The experiment was performed as described for Fig.
4A. F and G, TRAF1 and TRAF2 interact
with caspase-8 prodomain (residues 1-180), protease domain (residues
217-479), and MRIT 1 isoform. The experiment was performed as for
Fig. 4A. The lack of coimmunoprecipitation of GFP-HA
demonstrates the specificity of the interactions. The expression of
caspase-8 deletion constructs and MRIT1 is shown by Western analysis
on the cell lysates. H, the importance of an intact TRAF
domain for the interaction between caspase-8 and TRAF1. The experiment
was performed as in A. The expression of TRAF1 deletion
constructs is shown by Western analysis on the cell lysates.
I, FADD can displace TRAF1 and TRAF2 from caspase-8
prodomain. 293T cells were transfected with the indicated plasmids, and
the experiment was performed as described above for A.
Western blot analysis on total cellular lysates indicates equivalent
expression of TRAFs and FADD protein in various lanes
(middle and lower panels).
I.P., immunoprecipitate; L, lysate; C,
control antibody beads; F, Flag beads; mIg, mouse
immunoglobin.
1 isoform to
coimmunoprecipitate TRAF1. Like the prodomain construct used in the
previous study (12), the MRIT1 isoform contains both DEDs and has
additional 37 amino acids at the COOH terminus (11). MRIT1
successfully coimmunoprecipitated TRAF1 (Fig. 4F),
suggesting that the additional amino acids at the COOH terminus may
contribute to the stability of the MRIT1 isoform or alternatively to
its interaction with TRAF1. Taken together, the above results suggest
that the DED-containing proteins interact with TRAF family members
through multiple domains.
View larger version (36K):
[in a new window]
Fig. 5.
Role of the JNK pathway in caspase-10-induced
cell death. A, JBD blocks caspase-10-induced cell
death. 293T (1.2 × 105) cells were transfected with
50 ng/well of empty vector or caspase-10 along with 750 ng/well of
plasmids encoding empty vector, JBD of JIP-1 or CrmA, and a pRC/Rous
sarcoma virus-LacZ reporter plasmid in triplicate in each well of a
24-well plate. 24 h later, cells were fixed and stained with
5-bromo-4-chloro-3-indolyl- -D-galactopyranoside, and the
percentage of apoptotic cells was counted based on the criteria
described below for B. B, phase contrast
micrograph of 293T cells transfected with expression constructs
containing caspase-10 (50 ng/well) along with either a control vector
or JBD of JIP-1 (750 ng/well) as described above. 20 h later, the
cells were fixed and stained with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside, which
stains the transfected cells blue. Apoptotic cells have a dark rounded
appearance and membrane blebs and are getting detached from the plate.
C, inhibition of Fas-induced apoptosis by JBD of JIP-1. 293 EBNA (1.2 × 105) cells were transfected with the
indicated plasmids (250 ng each). The ratio of Fas to JBD-JIP1 (or
CrmA) plasmid was 1:1, and the total amount of transfected DNA was kept
constant by adding empty vector. 36 h later, cells were fixed and
stained with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside, and the
percentage of apoptotic cells was counted based on criteria described
for B. A representative of two independent experiments
performed in duplicate is shown. D, dose response of
MRIT-induced JNK activation and apoptosis. 293 EBNA (1.2 × 105) cells were transfected with an empty vector or the
indicated amounts of a MRIT-1 encoding plasmid in quadruplicate in
each well of a 24-well plate. The total amount of transfected DNA was
kept constant by adding empty vector. The activation of the JNK pathway
and the percentage of apoptotic cells were measured as described for
Figs. 2B and 5B, respectively. A representative
of two independent experiments performed in duplicate is shown.
E, inhibition of MRIT-induced apoptosis by JBD-JIP1. 293 EBNA cells were transfected with the indicated plasmids (250 ng each),
and the percentage of apoptotic cells was measured 36 h
post-transfection as described for A. The ratio of MRIT-1
to JBD-JIP1 plasmid was 1:1, and the total amount of transfected DNA
was kept constant by adding empty vector. A representative of two
independent experiments performed in duplicate is shown.
1 led to a
dose-dependent increase in both JNK activation and
apoptosis, and there was a close correlation between the two
activities, suggesting a causal link. To further test the involvement
of the JNK pathway in MRIT-induced cell death, we tested the effect of blocking the JNK activation on MRIT-induced apoptosis. As shown in Fig.
5E, coexpression of JBD-JIP1 led to effective inhibition of
MRIT-induced cell death in 293 EBNA cells. Taken together, the above
results suggest a role for the JNK pathway in MRIT-induced cell death.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/ embryos in response to signaling via TNFR1, Fas/Apo1,
and DR3 and concluded that the JNK pathway could be activated by these
receptors in both cell lines (40). However, a careful analysis of this
experiment has revealed a clear difference in the kinetics of JNK
activation between the two groups of cells. For example, stimulation
with TNF- resulted in almost equivalent peak JNK activation between
the two cell lines, which was observed after 10 min of treatment (40).
However, while this JNK activation was sustained in the caspase-8 +/+
cells for at least 30 min, there was a quick decline in the caspase-8 / cells and a return to the base-line level by the end of this time
period (40). Essentially similar results were obtained when the JNK
pathway was activated via Fas or DR3 signaling (40). caspase-8 /
cells also appeared to lag behind the wild-type cells in the initiation
of JNK activation response (40). Taken together, these results indicate
that there is a kinetic difference in JNK activation between the
caspase-8 +/+ and / embryonic fibroblast cells and suggest that
caspase-8 may play a contributory role in sustaining (and probably
initiating) this response in these cells. Future studies should confirm
this difference in additional tissues, since the contribution of
caspase-8 to JNK activation may be tissue-specific, as demonstrated by
its inability to activate the JNK pathway in the baby hamster kidney
cells. The latter conclusion is further supported by another recent
study that reported the isolation of a Jurkat T lymphocyte cell line deficient in caspase-8 that was resistant to Fas-induced cell death
(41). Detailed analysis revealed that this cell line was not only
incapable of activating caspases in response to Fas stimulation but,
unlike in the study by Varfolomeev et al. (40), also
completely failed to activate the JNK and p38 kinase pathways. This
study suggests that caspase-8 may be essential for Fas-induced JNK
activation in T cells (41) and underscores the importance of analyzing multiple tissues when testing the functional role of a gene/protein in
knock-out animals. Finally, caspase-8 and its homologs may play a
mutually redundant role in JNK activation by the death domain
receptors, and it may require the knock-out of all three cellular
DED-containing proteins to fully appreciate their contribution to this pathway.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
B, nuclear factor-B;
RIP, receptor-interacting protein;
JIP-1, JNK-interacting protein-1;
JBD, JNK binding domain;
GFP, green fluorescent protein;
MEKK1, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase kinase 1;
ASK1, apoptosis signal-regulating kinase 1.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Matta, L. Mazzacurati, S. Schamus, T. Yang, Q. Sun, and P. M. Chaudhary Kaposi's Sarcoma-associated Herpesvirus (KSHV) Oncoprotein K13 Bypasses TRAFs and Directly Interacts with the I{kappa}B Kinase Complex to Selectively Activate NF-{kappa}B without JNK Activation J. Biol. Chem., August 24, 2007; 282(34): 24858 - 24865. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Feeney and A. C. Clark Reassembly of Active Caspase-3 Is Facilitated by the Propeptide J. Biol. Chem., December 2, 2005; 280(48): 39772 - 39785. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Gumpricht, R. Dahl, M. W. Devereaux, and R. J. Sokol Licorice Compounds Glycyrrhizin and 18{beta}-Glycyrrhetinic Acid Are Potent Modulators of Bile Acid-induced Cytotoxicity in Rat Hepatocytes J. Biol. Chem., March 18, 2005; 280(11): 10556 - 10563. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hull, G. McLean, F. Wong, P. J. Duriez, and A. Karsan Lipopolysaccharide Signals an Endothelial Apoptosis Pathway Through TNF Receptor-Associated Factor 6-Mediated Activation of c-Jun NH2-Terminal Kinase J. Immunol., September 1, 2002; 169(5): 2611 - 2618. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Fernando, J. F. Kelly, K. Balazsi, R. S. Slack, and L. A. Megeney Caspase 3 activity is required for skeletal muscle differentiation PNAS, August 20, 2002; 99(17): 11025 - 11030. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ohtsuka and T. Zhou Bisindolylmaleimide VIII Enhances DR5-mediated Apoptosis through the MKK4/JNK/p38 Kinase and the Mitochondrial Pathways J. Biol. Chem., August 2, 2002; 277(32): 29294 - 29303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. H. Lee, S. E. Lee, K. Kwack, W. Yeo, T. H. Lee, S. S. Bae, P.-G. Suh, and H.-H. Kim Caspase-mediated cleavage of TRAF3 in FasL-stimulated Jurkat-T cells J. Leukoc. Biol., March 1, 2001; 69(3): 490 - 496. [Abstract] [Full Text]
|
|
|
|
 |
|
 N. ILAN, A. MOHSENIN, L. CHEUNG, and J. A. MADRI PECAM-1 shedding during apoptosis generates a membrane-anchored truncated molecule with unique signaling characteristics FASEB J, February 1, 2001; 15(2): 362 - 372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Villunger, D. C. S. Huang, N. Holler, J. Tschopp, and A. Strasser Fas Ligand-Induced c-Jun Kinase Activation in Lymphoid Cells Requires Extensive Receptor Aggregation But Is Independent of DAXX, and Fas-Mediated Cell Death Does Not Involve DAXX, RIP, or RAIDD J. Immunol., August 1, 2000; 165(3): 1337 - 1343. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. F. Law, G. M. O'Neill, S. J. Fashena, M. B. Einarson, and E. A. Golemis The Docking Protein HEF1 Is an Apoptotic Mediator at Focal Adhesion Sites Mol. Cell. Biol., July 15, 2000; 20(14): 5184 - 5195. [Abstract] [Full Text]
|
|
|
|
|
|
M. T. Eby, A. Jasmin, A. Kumar, K. Sharma, and P. M. Chaudhary TAJ, a Novel Member of the Tumor Necrosis Factor Receptor Family, Activates the c-Jun N-terminal Kinase Pathway and Mediates Caspase-independent Cell Death J. Biol. Chem., May 12, 2000; 275(20): 15336 - 15342. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A.C. Almeida, D. Ili, Q. Han, C. R. Hauck, F. Jin, H. Kawakatsu, D. D. Schlaepfer, and C. H. Damsky Matrix Survival Signaling: From Fibronectin via Focal Adhesion Kinase to c-Jun NH2-terminal Kinase J. Cell Biol., May 1, 2000; 149(3): 741 - 754. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zhuang, J. T. Demirs, and I. E. Kochevar p38 Mitogen-activated Protein Kinase Mediates Bid Cleavage, Mitochondrial Dysfunction, and Caspase-3 Activation during Apoptosis Induced by Singlet Oxygen but Not by Hydrogen Peroxide J. Biol. Chem., August 18, 2000; 275(34): 25939 - 25948. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kumar, M. T. Eby, S. Sinha, A. Jasmin, and P. M. Chaudhary The Ectodermal Dysplasia Receptor Activates the Nuclear Factor-kappa B, JNK, and Cell Death Pathways and Binds to Ectodysplasin A J. Biol. Chem., January 19, 2001; 276(4): 2668 - 2677. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Siegmund, D. Mauri, N. Peters, P. Juo, M. Thome, M. Reichwein, J. Blenis, P. Scheurich, J. Tschopp, and H. Wajant Fas-associated Death Domain Protein (FADD) and Caspase-8 Mediate Up-regulation of c-Fos by Fas Ligand and Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) via a FLICE Inhibitory Protein (FLIP)-regulated Pathway J. Biol. Chem., August 24, 2001; 276(35): 32585 - 32590. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |