From the Burnham Institute, La Jolla,
California 92037 and the § Department of
Hematology-Oncology, University of Heidelberg,
Heidelberg 69115, Germany
Received for publication, February 2, 2000, and in revised form, October 17, 2000
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ABSTRACT |
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TRAF family proteins are signal-transducing
adapter proteins that interact with the cytosolic domains of tumor
necrosis factor (TNF) family receptors. Here we show that TRAF1 (but
not TRAF2-6) is cleaved by certain caspases in vitro and
during TNF- TRAFs represent a group of structurally similar adapter proteins
sharing a conserved C-terminal ~180-amino acid-long region known as
the TRAF domain that binds the cytosolic tail of several TNF1 family cytokine
receptors, linking them to downstream signaling pathways (reviewed in
Ref. 1). Thus far, six TRAF family proteins have been described in
mammals, TRAF1-6 (2-9). Several of these TRAF family proteins can
homodimerize/oligomerize with themselves. Some also selectively form
heterocomplexes (2, 10, 11), the significance of which is largely unknown.
A variety of signal-transducing functions have been described for
several TRAFs. For example, TRAF2, -5, and -6 are known to activate
protein kinases (NIK (12), RIP2 (13), and MEKK (14)) that
are involved in inducing the transcription factor NF- In contrast to TRAF2, -5, and -6, the TRAF1 protein lacks a conserved
N-terminal RING domain found in TRAF family proteins and fails to
induce NF- Several of the TRAFs play roles in regulating apoptosis (41, 42). The
best established mechanism for apoptosis regulation by TRAFs is through
NF- Interestingly, although TRAF1 may influence apoptosis indirectly
through modulation of NF- In this report, we provide evidence that TRAF1 is a substrate of
caspase family proteases that are specifically activated by TNF family
death receptors, but not by other types of stimuli that primarily
activate an alternative mitochondrion-dependent pathway for
caspase activation. Interestingly, one of the TRAF1 cleavage products
can function as a suppressor of NF- Plasmids and Reagents--
Plasmids containing cDNAs
encompassing the complete open reading frames of human TRAF1
(pSG5-TRAF1), human TRAF2
(pcDNA3-HA-TRAF2), human TRAF3b
(pcDNAHA-TRAF3b), human TRAF6
(pcDNA3-Myc-TRAF6), human TNFR-I
(CD120a), and human Fas/CD95
(pCMV-Fas) have been described previously (11). The cleavage
fragments of TRAF1 (Fragments I and II) were generated by standard
two-step polymerase chain reaction methods and subcloned into
EcoRI/XhoI sites of pcDNA3-Myc. Non-cleavable
TRAF1 was prepared by mutating Asp163 to alanine by
QuickChangeTM site-directed polymerase chain reaction-based
mutagenesis as described previously (57). The promoter-containing
reporter gene plasmid pUC13-4xNF- In Vitro Caspase Cleavage Assays and Caspase Activity
Assays--
TRAF cDNAs in pcDNA3 were
in vitro transcribed/translated with
L-[35S]methionine (Amersham Pharmacia
Biotech) using reticulocyte lysates (Promega), and then 2 µl of
lysate were incubated at 37 °C for 1 h in 100 µl of caspase
buffer (50 mM HEPES (pH 7.4), 100 mM NaCl, 10%
sucrose, 1 mM EDTA, 0.1% CHAPS, and 10 mM
dithiothreitol) containing recombinant active caspases at 1 µM. Proteins were eluted in 20 µl of Laemmli buffer for
SDS-PAGE/autoradiography analysis. Recombinant caspases were prepared
as described previously (59), and their active concentration was
determined by titration against the unmethylated
benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone derivative purchased
from Bachem.
Caspase activities were assayed as previously described (59, 60).
Briefly, caspase activity was assayed at 37 °C in 100 µl of
caspase buffer containing the indicated fluorogenic peptide at 100 µM. Activity was measured continuously over the indicated times by the release of fluorogenic AFC from the peptide substitute acetyl-DEVD-AFC using a Molecular Devices fluorometer in the kinetic mode and a 405-510-nm filter pair. To assess caspase activity generated in cells, 10 µl of packed cells were lysed by addition of
20 µl of buffer A (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol) containing
0.25% Triton X-100. Suspensions were vortexed briefly and normalized
for protein concentration, and 2 µl of the normalized lysates were
assayed for caspase activity in 100 µl of caspase buffer containing
100 µM DEVD-AFC at 37 °C.
Cell Culture and Transfections--
MCF-7-Fas and BJAB cells
were a kind gift of Vishva Dixit. HEK293T and HT1080 cells were
obtained from American Type Culture Collection (Manassas, VA).
Wild-type and caspase-8-deficient Jurkat cells were generously provided
by J. Blenis (61). MCF-7-Fas, HEK293T, and HT1080 cells were
cultured in high glucose Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (Hyclone
Laboratories), 1 mM L-glutamine, and
antibiotics. Jurkat and BJAB cells were similarly cultured, except that
RPMI 1640 medium was used. Cisplatin, adriamycin (doxorubicin), etoposide, and staurosporine were added to cultures as indicated.
HT1080 and MCF-7-Fas cells were transfected with Superfect (QIAGEN
Inc.) in six-well plates and treated with various concentrations of anti-Fas antibody (usually 100 ng/ml). Lysates were prepared using
radioimmune precipitation assay buffer (10 mM Tris (pH
7.4), 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 0.5%
sodium deoxycholate, and 1 mM EDTA) containing a mixture of
protease inhibitors (Complete, Roche Molecular Biochemicals).
Reporter Gene Assays--
For NF- Immunoblotting--
For immunoblotting, cell lysates were
normalized for total protein content (50 µg), separated by SDS-PAGE
(12% gels), and transferred to 0.2-µm nitrocellulose membranes
(Bio-Rad) by transverse electrophoresis. Membranes were pre-blocked in
Tris-buffered saline (20 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20) containing 5% (v/v) skim milk
and 1% (v/v) goat serum (Sigma) for 1 h and incubated overnight
with primary antibody (at 1:1000, v/v) in the same solution. After
washing three times with Tris-buffered saline, blots were incubated for
1 h at room temperature in the same solution containing
horseradish peroxidase-conjugated secondary antibody (1:3000, v/v;
Bio-Rad). Membranes were washed again three times with Tris-buffered
saline, and proteins were detected by enhanced chemiluminescence (ECL,
Amersham Pharmacia Biotech). Anti-TRAF1 (SC-1831), anti-TRAF2 (C-20),
and anti-Myc antibodies were purchased from Santa Cruz Biotechnology
and used at 1:1000 (v/v) dilution.
TRAF1 Is Cleaved by Caspase-3, -6, -8, and -10, but Not Caspase-7
and -9, in Vitro at Aspartic Acid 163--
In vitro
translated, 35S-labeled TRAF1 was incubated with
recombinant active caspase-3 and -6-10, and in vitro
cleavage was monitored by SDS-PAGE. Caspase-3, -6, -8, and -10, but not
caspase-7 and -9, cleaved TRAF1 under these conditions, generating two
distinct fragments (Fig. 1A)
of ~28 and 22 kDa. The cleavage pattern revealed identical sizes for
the fragments, independent of the particular caspase employed,
suggesting the presence of a single cleavage site. By taking into
account the size of the generated fragments, a
160LEVD163 motif within TRAF1 appeared to be
the most likely target for cleavage. Mutation of aspartic acid 163 to
alanine confirmed this hypothesis and led to a non-cleavable form of
TRAF1 (Fig. 1B). In contrast, a control mutation (L160A) did
not prevent caspase-mediated cleavage of TRAF1 (data not shown).
TRAF1 Is Cleaved by Caspases at D163A in Cells--
To investigate
whether TRAF1 cleavage occurs in cells, the fibrosarcoma cell line
HT1080 was transiently transfected with a Myc-tagged TRAF1 expression
plasmid. After inducing caspase activation by adding anti-Fas antibody
(CH-11), cells were lysed at various times, and cell lysates were
prepared for immunoblot analysis. Within 3 h after Fas
stimulation, a faint band representing the N-terminal fragment of TRAF1
became detectable. Between 6 and 12 h, the cleaved TRAF1 product
increased in relative amount, whereas the full-length ~49-kDa TRAF1
protein was depleted. The cleavage of TRAF1 in Fas-stimulated HT1080
cells was completely prevented by preincubation (~30 min) with the
broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp
fluoromethyl ketone, indicating that cleavage of TRAF1 in mammalian
cells is caspase-dependent. In contrast to wild-type TRAF1,
the TRAF1(D163A) protein was not cleaved in Fas-stimulated HT1080
cells, even during late stages of apoptosis when most of the HT1080
cells had become rounded, shrunken, and detached from the culture
dishes (12-18 h). To investigate whether other TRAF molecules undergo
cleavage by caspases in Fas-stimulated HT1080 cells, TRAF2-6 were
transfected into the cells in similar experiments. No visible cleavage
or degradation of the full-length TRAF molecules was observed (Fig.
2 and data not shown).
Similar observations were made in MCF-7 breast cancer cells, a
caspase-3- and -10-deficient cell line (62-64) that had been stably
transfected with Fas. Interestingly, cleavage of TRAF1 was evident in
these cells even before addition of anti-Fas antibody (CH-11) (Fig.
2B), presumably reflecting constitutive caspase activation
in these cells as a result of overexpression of Fas. Addition of
anti-Fas antibody led to rapid cleavage of the remaining wild-type
TRAF1. The non-cleavable TRAF1(D163A) mutant as well as wild-type TRAF2
were not cleaved in Fas-stimulated MCF-7 cells (Fig. 2B).
Similar results were obtained when HT1080 and MCF-7 cells were
stimulated with TNF plus cycloheximide (data not shown), suggesting
that TRAF1 cleavage occurs in response to activation of this death
receptor, in addition to Fas (CD95).
Endogenous TRAF1 Is Up-regulated in Activated B- and T-cells and
Cleaved during TNF- and Fas-mediated Apoptosis--
To determine
whether the same effect can be seen for endogenous TRAF1, we stimulated
the human T-cell line Jurkat with phorbol 12-myristate 13-acetate and
the human B-cell line BJAB with CD40. These stimuli result in NF- Overexpression of a C-terminal TRAF1 Fragment Enhances TNF-mediated
Apoptosis--
To investigate the possible functional significance of
the cleavage of TRAF1, we coexpressed TRAF1(D163A), TRAF1 Fragment I
(N-terminal), and TRAF1 Fragment II (C-terminal) together with TNFR1
(CD120a) or Fas expression plasmids. The levels of TRAF1 and TRAF1
mutants produced by transient transfection were comparable to those
expressed endogenously in lymphoid cell lines (data not shown), arguing
that the levels of proteins are within physiologically relevant ranges.
Whereas TRAF1 and the non-cleavable TRAF1(D163A) had no remarkable
influence on TNFR1 or Fas cell death, TRAF1 Fragment II led to
significant enhancement of TNF-mediated apoptosis (Fig.
4A). This increase in
apoptosis was accompanied by elevations in caspase activity, as
measured in lysates of these cells (Fig. 4B and data not
shown). None of the TRAF constructs when expressed alone had any
significant influence on cell viability in the absence of TNFR1 or Fas.
Coexpression of TRAF1 Fragment II with Fas also resulted in enhanced
apoptosis (Fig. 4C), although the effect of TRAF1 Fragment
II was less potent for Fas than for TNFR1. Similar results were
obtained in experiments using HT1080 cells (data not shown).
Influence of TRAF1 and Its Fragments on TNF- Cleavage of TRAF1 and Enhancement of Apoptosis Are Limited to
Receptor-mediated Apoptosis--
To further localize the significance
of the cleavage of TRAF1 during apoptosis, HT1080 cells transfected
with Myc-tagged wild-type TRAF1 were exposed to various apoptotic
stimuli. Cisplatin, staurosporine (a broad-spectrum kinase inhibitor),
and UV irradiation resulted in little or no cleavage of TRAF1 compared
with TNF and Fas (Fig. 6). In contrast,
adriamycin (doxorubicin) treatment resulted in accumulation of the
cleaved TRAF1. Overexpression of TRAF1 Fragment II, however, did not
enhance cell death induced by these stimuli (Fig. 6). Thus, enhancement
of apoptosis by TRAF1 Fragment II appears to be specific for TNF family
death receptors.
TRAF1 Cleavage Is Impaired in TNF-stimulated Cells with Reduced
Levels of Caspase-8--
The preceding data demonstrated that TRAF1
cleavage occurs in cells following stimulation with activators of TNF
family death receptors (TNF- In this report, we demonstrate that TRAF1 is a target of caspases
that become activated during apoptosis inducted by TNF family death
receptors. The observation that Fas and TNFR1 induce cleavage of TRAF1,
whereas a variety of other apoptosis inducers do not, suggests that
TRAF1 cleavage results from an upstream caspase that is uniquely
activated by these TNF family cytokine receptors. In this regard, at
least two major pathways for caspase activation and apoptosis have been
delineated, including an "extrinsic" pathway triggered by TNF
family death receptors and an "intrinsic" pathway activated by
damage to mitochondria (reviewed in Refs. 69 and 70). The intrinsic and
extrinsic cell death pathways both induce activation of similar
downstream effector caspases such as caspase-3, -6, and -7, but differ
in their activation of upstream initiator caspases, with TNF family
death receptors typically activating procaspase-8 (and perhaps
procaspase-10) and mitochondria triggering activation of procaspase-9
via release of cytochrome c.
Although caspase-3, -6, -8, and -10 are capable of cleaving TRAF1
in vitro, the specific caspase chiefly responsible for
cleavage of TRAF1 in vivo appears to be caspase-8, based on
experiments performed with a mutant of the Jurkat cell line in which
levels of procaspase-8 are greatly reduced (61). Further evidence
consistent with cleavage of TRAF1 by an upstream initiator-type caspase
rather than a downstream effector-type caspase comes from inspection of
the TRAF1 cleavage site sequence: LEVD. The optimal tetrapeptide substrate sequence for cleavage by caspase-8 has been determined to be
LETD/LEVD, based on screening of combinatorial peptide libraries (71).
Also, caspase-10 is known to cleave substrates preferably at
(I/L/V)EXD sites (72). In contrast, the optimal tetrapeptide cleavage sequence for the downstream effector protease, caspase-3, is
DEVD/DEID. Thus, the presence of a hydrophobic residue at P4' in the
TRAF1 cleavage site argues in favor of an upstream initiator-type caspase.
The observation that TRAF1 undergoes cleavage in MCF-7 breast cancer
cells, which are entirely deficient in caspase-3 and -10 (62-64),
indicates that these caspases are not essential for proteolysis of
TRAF1, further supporting the contention that caspase-8 is primarily
responsible for this phenomenon. Moreover, although caspase-6 can
cleave TRAF1 in vitro, it has been shown that the apical
caspase in the TNF/Fas pathway, caspase-8, fails to induce processing
of procaspase-6 in cell extracts if caspase-3 is absent (73),
suggesting that the TNFR/Fas-induced cleavage of TRAF1 seen in
caspase-3-deficient MCF-7 cells is also unlikely to involve caspase-6.
Given that both initiator- and effector-type caspases are capable of
cleaving TRAF1 in vitro, it seems probable that factors other than preferred tetrapeptide cleavage sequences may play a role in
dictating which specific caspases cleave TRAF1 in cells. One potential
explanation is that interactions of TRAF1 with other proteins may place
it into the proximity of certain activated caspases, particularly
caspase-8. For example, TRAF1 reportedly interacts directly or
indirectly with a variety of proteins that are recruited to TNF family
death receptor complexes, including TRADD, TRAF2, RIP2, FLIP, cIAP1,
and cIAP2 (2, 13, 35, 37, 52). Thus, these protein interactions could
place TRAF1 into close proximity with active caspase-8 during
triggering of TNFR1 or related receptors.
The observation that adriamycin (doxorubicin) stimulated TRAF1 cleavage
in cultured tumor cell lines suggests differences in the caspases
activated by this anticancer drug compared with cisplatin,
staurosporine, and UV irradiation, which induced little or no
TRAF1 cleavage. In this regard, doxorubicin has been reported to induce
apoptosis through a Fas-dependent pathway involving autocrine up-regulation of Fas and Fas ligand expression in some tumor
and leukemia cell lines (74-76). The TRAF1 cleavage seen in HEK293
cells following treatment with doxorubicin may reflect activation of a
death receptor-dependent pathway for apoptosis, thus
explaining the differences in TRAF1 cleavage in tumor cells treated
with various anticancer drugs. However, overexpression of TRAF1
Fragment II failed to enhance apoptosis induced by doxorubicin (although it did augment apoptosis induced by TNF and by Fas), suggesting that this anticancer drug may simultaneously activate both
death receptor-dependent (TRAF1-sensitive) and -independent (TRAF1-insensitive) pathways (77).
As shown here, one of the cleavage products generated by
caspase-mediated proteolysis of TRAF1 is capable of suppressing NF- In addition to NF- Expression of TRAF1 is highly restricted in vivo, with
mRNA found at readily detectable levels only in spleen, lung, and
testis (2), unlike other TRAFs, which are generally more ubiquitously expressed. Moreover, levels of TRAF1 mRNA and protein
are dynamically regulated, becoming induced by activation of various
cytokine receptors, anti-CD3 antibody, anti-IgM antibody, and stimuli
that induce the transcription factor NF-- and Fas-induced apoptosis in vivo.
160LEVD163 was identified as the caspase
cleavage site within TRAF1, generating two distinct fragments.
Significant enhancement of TNF receptor-1 (CD120a)- and, to a
lesser extent, Fas (CD95)-mediated apoptosis was observed when
overexpressing the C-terminal TRAF1 fragment in HEK293T and HT1080
cells. The same fragment was capable of potently suppressing TNF
receptor-1- and TRAF2-mediated nuclear factor-
B activation in
reporter gene assays, providing a potential mechanism for the
enhancement of TNF-mediated apoptosis. Cell death induced by other
death receptor-independent stimuli such as cisplatin, staurosporine,
and UV irradiation did not result in cleavage of TRAF1, and
overexpression of the C-terminal TRAF1 fragment did not enhance cell
death in these cases. TRAF1 cleavage was markedly reduced in cells that
contain little procaspase-8 protein, suggesting that this apical
protease in the TNF/Fas death receptor pathway is largely responsible.
These data identify TRAF1 as a specific target of caspases activated
during TNF- and Fas-induced apoptosis and illustrate differences in the
repertoire of protease substrates cleaved during activation of
different apoptotic pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and to
activate MAP3K and MAP4K kinases (apoptosis signal kinase 1 (15), germinal center kinase (16), MEKK1 (14), and TAK1 (17)) involved
in activation of the transcription factor c-Jun. TRAF6 is additionally
involved in the activation of the pp60c-Src protein-tyrosine
kinase (18) and the ERKs (19).
B or to activate stress kinases when overexpressed in
cells. TRAF1 was initially cloned as a protein indirectly recruited to
TNF type II receptors (TNFR2; CD120b) through associations with
TRAF2 and was found to be expressed only in a limited number of tissues
under physiological conditions (2). Although interacting indirectly
with TNFR2 (CD120b), TRAF1 directly interacts with the cytoplasmic tail
of several TNFR family members, including CD30 (20, 21) (at a region
that mediates cell death by CD30 in a T-cell hybridoma (20)), 4-1BB
(22), herpesvirus entry mediator (23), TRANCE/RANK (24-26), perhaps
CD40 (10, 27), AITR (28), and the viral transforming protein LMP-1
(29). TRAF1 binds also to a number of intracellular proteins, including several associated with the TNF family receptor signaling complexes and/or regulation of NF-
B activation: TANK/I-TRAF (30, 31), TRIP
(32), A20 (33), the NF-
B-inducing STP oncoproteins of herpesvirus
(34), RIP (35), CARDIAK (RIP2) (36), and the caspase-8-binding protein
FLIP (CASPER/CASH/I-Flice/Usurpin/Flame) (37). Despite interactions
with multiple NF-
B-regulating proteins, the role of TRAF1 in NF-
B
activation remains unclear. A recent report in which a N-terminal
truncated TRAF1 protein was overexpressed demonstrated suppression of
TNF-mediated NF-
B and JNK activation (38), suggesting that
trans-dominant inhibitory mutants of TRAF1 interfere with
TNF signaling. However, overexpression of full-length TRAF1 also
reportedly can suppress NF-
B induction by TNF, interleukin-1, TRAF2,
and TRAF6 (39). Conversely, TRAF1 interactions with LMP-1 have been
implicated in induction rather than suppression of NF-
B (40).
B, although contributions by JNK-activating kinases and Akt may
also play a role in some circumstances (18, 43). NF-
B induces
expression of several anti-apoptotic genes, including the
Bcl-2 family member Bfl-1 (44), IAP
(inhibitor of apoptosis proteins)
family genes (45, 46), A20 (47), and IEX-1L (48).
Interestingly, some TNF family cytokine receptors such as TNFR1
simultaneously stimulate pathways for apoptosis induction via
receptor-associated proteins that activate caspase family cell death
proteases (particularly caspase-8) and pathways for suppression via
NF-
B (reviewed in Refs. 41, 49, and 50). Thus, the balance of these
two signaling pathways dictates whether cells ultimately live or die
when stimulated through TNFR1.
B-inducible gene expression,
TRAF1 was also identified together with TRAF2,
cIAP1, and cIAP2 as NF-
B-inducible genes that
may form a protein complex capable of directly blocking caspase-8
activation induced by TNFR1 (51). In this regard, TRAF1 binds the BIR
domain region of IAPs cIAP1 and cIAP2 (52-54), which directly inhibit
certain caspase family cell death proteases (55). Finally, experiments
with TRAF1 transgenic mice have revealed a role for
TRAF1 in inhibition of antigen-induced programmed cell death of
CD8-positive T-cells (56).
B activation induced by TNFR1 and
TRAF2 and thus could potentially play a role in committing
TNF-stimulated cells to an apoptotic demise. The findings demonstrate
differences in the repertoire of substrates cleaved by caspases that
become activated by alternative apoptosis pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B-Luc, which contains four tandem
human immunodeficiency virus NF-
B response elements and the minimal c-fos promoter, has been described (58). The caspase
inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone was
purchased from Alexis, TNF-
from Genzyme Corp., and anti-Fas
antibody CH-11 from Medical Biological Laboratories Co., LTD
(Nagoya, Japan). Cycloheximide, phorbol 12-myristate 13-acetate, and
staurosporine were obtained from Sigma.
B reporter gene assays, 293N
cells at 60-80% confluency in 12-well plates were transfected using a
cationic lipid reagent (Superfect) with various amounts of plasmid DNA,
including 0.1 µg of pUC13-4xNF-
B-Luc and 0.1 µg of pCMV-
gal.
After 36 h, cells were suspended in 0.1 ml of Promega lysis
buffer. Lysates were measured for luciferase activity using a
luminometer (EG&G Berthold) and Promega luciferase kits, normalizing
relative to
-galactosidase activity (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TRAF1 is cleaved by caspase-3, -6, -8, and
-10 in vitro at Asp163. A,
in vitro translated, Myc-tagged, 35S-labeled
TRAF1 was incubated in caspase buffer with recombinant active
caspase-3, -6, -7, -8, -9, or -10 (at 1 µM each) for
1 h at 37 °C. Samples were then solubilized in Laemmli buffer
and subjected to SDS-PAGE/autoradiography. Arrowheads
indicate full-length TRAF1 and resulting fragments. Data represent one
of at least two assays. B, in vitro translated,
Myc-tagged, 35S-labeled TRAF1(D163A) was incubated under
the same conditions as described for A. The
arrowhead indicates non-cleavable TRAF1. Mutation of another
amino acid in the LEVD motif (L160A) did not prevent in
vitro cleavage by caspase-3, -6, -8, or -10 (data not shown).
C, shown is a schematic representation of TRAF1 cleavage,
accounting for the formation of Fragments I and II.
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Fig. 2.
Overexpressed TRAF1 is cleaved by caspases at
D163A in vivo in HT1080 and MCF-7-Fas cells.
A, empty pcDNA3-Myc vector, pcDNA3-Myc-TRAF1,
pcDNA3-Myc-TRAF1(D163A), or pcDNA3-HA-TRAF2 was transfected
into HT1080 cells. Anti-Fas antibody was added 24 h later (100 ng/ml), and cells were lysed after 0.5, 1.0, 3.0, 6.0, and 12.0 h.
Lysates were then subjected to SDS-PAGE and immunoblotting using
anti-Myc antibody. Addition of the caspase inhibitor
benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (zVAD-Fmk;
100 µM) at 0.5 h prevented cleavage of TRAF1.
Note that Myc-tagged TRAF1(D163A) was not cleaved. Caspase-8 activation
(marked by the disappearance of the pro form) was seen as early as +30
min in these cells, whereas the active fragments of effector caspase-3
were not detected until +3 h (data not shown). B,
experiments were performed in MCF-7-Fas cells as described for
A. MCF-7-Fas cells contain constitutively processed
caspase-8 (data not shown), but lack caspase-3 and -10 (62-64).
B
activation and increased expression of endogenous TRAF1, a
NF-
B-inducible gene (38, 39, 65). Inducing apoptosis with either
anti-Fas antibody or TNF-
(+cycloheximide) led to the complete
disappearance of endogenous full-length TRAF1 within 24 h.
Cleavage of TRAF1 was evident within 6 h in these experiments
(Fig. 3, A and B).
In contrast, apoptosis induction by a death receptor-independent
stimulus, staurosporine (10 µM) (66), did not result in
TRAF1 cleavage (data not shown).
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Fig. 3.
Endogenous TRAF1 is up-regulated in activated
B- and T-cells and cleaved during TNF- and Fas-mediated apoptosis.
Jurkat (A) and BJAB (B) cell lines were
stimulated with phorbol 12-myristate 13-acetate (PMA; 0.1 µM) and CD40 ligand (CD40L; 250 ng/µl),
respectively, for 2 days, leading to increased production of endogenous
TRAF1 (open arrowheads). Subsequently, cells were stimulated
with either 100 ng/ml anti-Fas (CH-11) antibody or 100 ng/ml TNF-
(in combination with 10 µg/ml cycloheximide (CHX)).
Lysates were prepared at various times thereafter and analyzed by
SDS-PAGE/immunoblotting using anti-TRAF1 antibody directed against the
N-terminal region of TRAF1, revealing cleaved TRAF1 (closed
arrowheads).
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Fig. 4.
Overexpression of Fragment II of TRAF1
enhances TNFR1-mediated apoptosis in 293T and HT1080 cells.
A, 293T cells were transiently cotransfected with
0.1 µg of pEGFP together with 1.25 µg of pcDNA3-Myc-tagged
TRAF1, TRAF1(D163A), TRAF1 Fragment (Frg) I, or TRAF1
Fragment II, with or without 0.25 µg of a TNFR1 (p55) expression
plasmid, in six-well plates at ~40% cell confluence. Plates were
photographed 1 day later using fluorescence microscopy, and the
morphology of the apoptotic (rounded, shrunken) versus
non-apoptotic (flat, adherent) green fluorescent protein-expressing
successfully transfected cells was noted. B, caspase
activity in lysates prepared from cells transfected as described for
A was measured, based on cleavage of the substrate
acetyl-DEVD-AFC. Data are expressed as relative caspase activities to
TNF and are representative of one of two independent experiments.
C, 293T and HT1080 cells were cotransfected as described for
A with various TRAF expression plasmids and plasmids
encoding either TNFR1 (p55) or Fas. Viability of the green fluorescent
protein-positive cells was determined after 36 h by trypan blue
staining (mean ± S.E., n = 3).
- and TRAF2-mediated
NF-
B Activation--
Since TRAF1 has been implicated in regulation
of TNF-mediated NF-
B activation and is known to bind TRAF2, the
effect of the TRAF1 fragments on NF-
B induction was investigated. No
significant NF-
B induction was observed in cells overexpressing
TRAF1, TRAF1(D163A), or the TRAF1 fragments. Cotransfecting TRAF1 or
TRAF1 Fragment II with TNFR1 in 293 cells resulted in
dose-dependent suppression of TNFR1-mediated NF-
B
activation, with TRAF1 Fragment II exhibiting a more potent inhibitory
effect (Fig. 5A). In contrast,
the non-cleavable protein and TRAF1 Fragment I had no significant
effect on TNFR1-mediated NF-
B induction. Similar results were
obtained when TRAF2 rather than TNFR1 was employed as the stimulus for
inducing NF-
B (Fig. 5B). In contrast to TRAF1 Fragment
II, transfection of cells with plasmids encoding similar C-terminal
fragments of TRAF3 or TRAF4 did not interfere with NF-
B induction by
TNF (data not shown), providing further evidence of the specificity of
these results.
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Fig. 5.
Effect of TRAF1 and its fragments on
TNF- - and TRAF2-mediated
NF-
B activation. 293 cells were
transiently transfected in 12-well plates with 2.5 µg of total DNA
including TNFR1 (0.75 µg) (A) or TRAF2 (0.75 µg)
(B) and pcDNA-Myc vector (vector; 0.75 µg)
as a control, together with 0.125 µg of pCMV-
gal and 0.125 µg of
pUC13-4xNF-
B-Luc. pcDNA3-Myc plasmids encoding TRAF1 or its
fragments (Fragments I (Frg I) and II (Frg II))
were cotransfected in various amounts (0.5, 1.0, and 1.5 µg),
normalizing total DNA by addition of empty pcDNA3-Myc vector. After
36 h, cells lysates were prepared and analyzed for luciferase and
-galactosidase activities to determine relative luciferase units
(RLU) (mean ± S.D., n = 3).
View larger version (22K):
[in a new window]
Fig. 6.
Cleavage of TRAF1 occurs preferably during
death receptor-mediated apoptosis. A, HT1080 cells were
UV-irradiated (240 mJ) or cultured with cisplatin (100 µM), doxorubicin (Dox; 2 µM), or
staurosporine (Stauro; 100 ng/ml) for 6-36 h (time was
adjusted for individual stimuli so that ~50% cell death was achieved
at the time of assay). Floating and adherent cells were recovered from
cultures, and lysates were prepared, normalized for total protein
content (25 µg), and analyzed by SDS-PAGE/immunoblotting using
anti-TRAF1 antibody. B, 293T cells were transfected with
control vector or TRAF1 Fragment II (TRAF1-FrgII) in
combination with TNF, Fas, or control vector and, in the latter case,
exposed to various drugs (300 µM cisplatin, 4 µM doxorubicin, or 100 nM staurosporine) or
UV-irradiated (240 mJ). Cell death was determined at 24 h by
trypan blue staining. (mean ± S.E., n = 2).
Similar data were obtained using HT1080 cells (data not shown).
and anti-Fas antibody), but not after
exposure to other stimuli (cisplatin, staurosporine, and UV) that
activate caspases chiefly via an alternative pathway involving
mitochondrial release of cytochrome c. The apical caspases
in the TNF family death receptor and mitochondrion/cytochrome
c pathways for apoptosis are caspase-8 and -9, respectively
(reviewed in Refs. 49 and 67). We therefore compared TRAF1 cleavage in
Jurkat cells and in Jurkat mutant cells containing greatly reduced
amounts of procaspase-8 protein (61). Stimulation of wild-type and
caspase-8-deficient Jurkat cells with TNF-
plus cycloheximide
resulted in comparable amounts of cell death (as measured by trypan
blue dye exclusion assays) and activation of downstream effector
caspases (as measured by hydrolysis of the fluorogenic caspase
substrate acetyl-DEVD-AFC) (Fig. 7). In
contrast, whereas TRAF1 was cleared from the caspase-8-containing cells
in a time-dependent manner after treatment with
TNF-
/cycloheximide, little cleavage of TRAF1 was observed in the
caspase-8-deficient cells. Other proteins such as
-tubulin were not
cleaved during the time frame of these experiments (data not shown),
confirming the specificity of the TRAF1 observations. We therefore
conclude that cleavage of TRAF1 following exposure of cells to TNF-
is largely dependent on caspase-8, implying that this protease is specifically responsible for TRAF1 cleavage in vivo. The
ability of TNF-
to kill these Jurkat mutant cells that contain
greatly reduced amounts of procaspase-8 is consistent with the status of Jurkat T-cells as "Type II" cells, in which small amounts of caspase-8 activation can effectively trigger activation of downstream effector caspases via a mitochondrion-dependent
amplification process (68).
View larger version (30K):
[in a new window]
Fig. 7.
TRAF1 cleavage is impaired in
TNF- -stimulated cells lacking caspase-8.
Wild-type (WT) and caspase-8-deficient (C8-def.)
Jurkat leukemia T-cells (61) were cultured with phorbol 12-myristate
13-acetate (0.1 µM) for 2 days to up-regulate TRAF1.
Subsequently, cells were stimulated with TNF-
(100 ng/ml) in
combination with cycloheximide (CHX; 10 µg/ml) for the
various times as indicated. A, clearance of TRAF1 from
TNF-treated cells was monitored by immunoblotting using cell lysates
normalized for protein content (25 µg/lane) and anti-TRAF1 antibody.
Reprobing the same blot with an antibody recognizing
-tubulin
confirmed loading of equivalent amounts of protein in all lanes (data
not shown). The cleaved fragments of TRAF1 did not accumulate to
detectable levels in these Jurkat cells. B, cell death was
determined by failure of cells to exclude trypan blue dye at various
times after TNF exposure. Data represent percentage trypan
blue-positive cells (mean ± S.D., n = 3).
C, the activity of effector caspases was measured by
hydrolysis of acetyl-DEVD-AFC in cell lysates (normalized for total
protein content) derived from TNF-treated Jurkat cells as described
above. Data are expressed as a percentage relative to the caspase
activity measured in wild-type Jurkat cells after a 24-h treatment with
TNF/cycloheximide (mean ± S.E., n = 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B induction by TNFR1 and TRAF2. In many tumor cell lines and most normal
cell types, TNF-
interactions with TNFR1 result in cytotoxicity only
when protein or mRNA synthesis is blocked (reviewed in Refs. 50 and
78). Transfection experiments using I
B dominant-negative mutants
that are resistant to degradation and use of cells from gene knockout
mice lacking NF-
B components have provided evidence that TNF-induced
NF-
B is responsible for maintaining cell survival in the setting of
TNF-
exposure (79-82). Thus, the observation that TRAF1 Fragment II
(amino acids 164-416) enhances apoptosis induced by TNF-
is
consistent with the finding that this cleavage fragment also blocks
NF-
B induction by TNFR1. We cannot exclude the possibility, however,
that TRAF1 Fragment II also interferes with other aspects of TNFR1
signaling. For example, TRAF1 binds to TRAF2 via its TRAF domain, which
is encompassed in the Fragment II cleavage product of TRAF1 (2).
Although TRAF2 is capable of inducing NF-
B, gene knockout studies
suggest that TRAF2 may be primarily responsible for activation of
MAP3Ks that stimulate JNK rather than for NF-
B induction within the
context of TNFR1 signal transduction (43). Thus, heterodimerization of
cleaved TRAF1 with TRAF2 may interfere with the ability of TRAF2 to
activate both NF-
B as well as JNK. In this regard, a role for JNK in
suppression of apoptosis induction by TNF-
has also been inferred
from studies of transgenic mice expressing a dominant-negative form of
TRAF2 (83).
B induction, the TRAF1 Fragment II cleavage
product hypothetically could interfere with TNFR1-induced apoptosis through a transcription-independent mechanism. For example, it has been
suggested that a tetrameric complex consisting of TRAF1, TRAF2, cIAP1,
and cIAP2 can block procaspase-8 processing and activation by TNFR1
through an unresolved mechanism not requiring induction of NF-
B
(51). The Fragment II cleavage product of TRAF1 could therefore somehow
disturb the anti-apoptotic function of this multiprotein complex. In
this regard, it should be noted that overexpression of non-cleavable,
full-length TRAF1(D163A) versus TRAF1 Fragment II resulted
in different effects on TNFR1- and Fas-induced apoptosis, suggesting
differences in the functions of these proteins. Consequently, by
comparing the structures of full-length TRAF1 (residues 1-415) and
TRAF Fragment II (residues 164-415), it can be deduced that the
N-terminal region of TRAF1 (upstream of the TRAF domain) plays a role
in apoptosis suppression since absence of this domain converts TRAF1
into a pro-apoptotic protein, at least with respect to apoptosis
induced by TNFR1 and Fas. Furthermore, since full-length TRAF1
generally does not induce NF-
B or JNK when overexpressed, this
hypothetical anti-apoptotic function of the TRAF1 N-terminal domain
presumably is unrelated to those signaling pathways.
B (38, 39, 65). In this regard, analysis of the promoter region of the TRAF1 gene
has demonstrated the presence of multiple NF-
B-binding sites and has
revealed that TRAF1 is a NF-
B-inducible gene (65). As
discussed above, full-length TRAF1 has been associated with protection
from apoptosis induced by TNFR1, whereas the cleaved TRAF1 Fragment II
is implicated in sensitization of cells to TNFR1-induced apoptosis. What then dictates whether up-regulation of TRAF1 is cytoprotective versus cytotoxic when cells are exposed to TNF-
? The
answer to this question seems likely to be found in the relative
amounts of full-length TRAF1 versus caspase-cleaved TRAF1
that accumulate in cells. Factors that alter the ratio of these
two versions of the TRAF1 protein presumably would drive the signaling
process either toward or away from an apoptotic result. This ambiguity in the outcome of cellular responses may account for why overexpression of TRAF1 has been associated with protection from apoptosis and enhanced production of active NF-
B in some circumstances
versus induction of apoptosis and reduced NF-
B
induction in others (38-40, 56, 84). Generation of
TRAF1 knockout mice as well as knock-in mice
expressing a non-cleavable TRAF1 mutant protein (D163A) will be
required to reveal the in vivo functions of TRAF1 and
caspase cleavage of TRAF1.
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ACKNOWLEDGEMENTS |
---|
We thank R. Cornell for manuscript preparation and J. Blenis and V. Dixit for cell lines.
![]() |
Note Added in Proof |
---|
Soon after this was originally submitted, a publication by Irmler et al. (FEBS Letters 468, 29-133, 25 February 2000: Caspase-induced inactivation of the anti-apoptotic TRAF1 during Fas ligand-mediated apoptosis) appeared that covers part of the data here.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants CA69381, CA72994, and AG15402.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.
¶ Recipient of Fellowship EL818/1 from the Deutsche Forschungsgemeinschaft.
Recipient of a fellowship from the Leukemia Society of America.
** Recipient of a fellowship from the California Breast Cancer Research Program.
To whom correspondence should be addressed: Burnham Inst.,
10901 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-646-3140; Fax: 858-646-3194; E-mail: jreed@burnham-inst.org.
Published, JBC Papers in Press, November 29, 2000, DOI 10.1074/jbc.M009450200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
TNFR, tumor necrosis factor receptor;
MEKK, mitogen-activated
protein kinase/extracellular signal-regulated kinase kinase kinase;
NF-B, nuclear factor-
B;
MAPK, mitogen-activated protein kinase;
ERKs, extracellular signal-regulated kinases;
JNK, c-Jun N-terminal
kinase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
AFC, amino
fluorocoumarin.
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