From the Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
Received for publication, February 27, 2001
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ABSTRACT |
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Upon stimulation, CD95 (APO-1/Fas) recruits the
adapter molecule FADD/MORT1, procaspase-8, and the cellular
FLICE-inhibitory proteins (c-FLIP) into the death-inducing signaling
complex (DISC). According to the induced proximity model, procaspase-8
is activated in the DISC in an autoproteolytic manner by two subsequent
cleavage steps. c-FLIP proteins exist as a long
(c-FLIPL) and a short (c-FLIPS) splice
variant, both of them capable of protecting cells from death
receptor-mediated apoptosis. In stably transfected BJAB cells, both
c-FLIPS and c-FLIPL block procaspase-8
activation at the DISC. However, cleavage is blocked at different
steps. c-FLIPL allows the first cleavage step of
procaspase-8, leading to the generation of the p10 subunit. In
contrast, c-FLIPS completely inhibits cleavage of
procaspase-8. Interestingly, p43-c-FLIPL lacking the p12
subunit also prevents cleavage of procaspase-8. In contrast, a
nonprocessable mutant of c-FLIPL allows the first cleavage
of procaspase-8. In conclusion, both c-FLIP proteins prevent caspase-8
activation at different levels of procaspase-8 processing at the DISC.
Our results indicate that c-FLIPL induces a conformation of
procaspase-8 that allows partial but not complete proteolytical
processing, whereas in contrast c-FLIPS even prevents partial procaspase-8 activation at the DISC.
Apoptosis plays an important role in tissue homeostasis. In the
immune system, apoptosis is used for negative and positive selection of
T and B cells in the thymus and bone marrow, respectively, and to
maintain immune homeostasis (1). Apoptosis can be induced by death
receptors, a subgroup of the
TNF1/nerve growth factor
receptor superfamily (2). The best characterized member of the death
receptor subfamily is CD95, also known as APO-1 or Fas. Stimulation of
CD95 with its cognate ligand, CD95L, leads to clustering of the
monomeric receptor or, as recently suggested, to conformational changes
of preformed receptor complexes (3). This enables binding of the
adapter molecule FADD/MORT1 (4, 5) and of procaspase-8 (FLICE, MACH,
Mch5) (6-8) to CD95 via homophilic death domain and death effector
domain (DED) interactions, respectively, forming the death-inducing
signaling complex (DISC) (9). Recruitment of procaspase-8 to the DISC leads to its proteolytic activation through several cleavage steps. Free p18 and p10 subunits form the active caspase-8 heterotetramer. Active caspase-8 then initiates a cascade of caspase activation finally
leading to cell death (10).
Sensitivity toward CD95-mediated apoptosis can be modulated at
different levels in the CD95 signaling pathways (e.g. by the viral caspase inhibitors CrmA or p35 (11-14) or in certain cells (type
II cells)) by Bcl-2/Bcl-xL overexpression inhibiting
mitochondrial changes during apoptosis (15). Viral FLICE-inhibitory
proteins (v-FLIP), which are components of the class of
A human homolog of v-FLIP is called
c-FLIP/FLAME-1/I-FLICE/Casper/CASH/MRIT/CLARP/Usurpin (17-24). On the
mRNA level, several c-FLIP splice variants exist. On the protein
level, however, only two endogenous forms, c-FLIPL and
c-FLIPS, could be detected so far (17, 20, 24, 25).
c-FLIPL is structurally similar to procaspase-8, since it
contains two death effector domains and a caspase-like domain. However,
this domain lacks residues that are important for the catalytic
activity of caspase-8, most notably the cysteine within the active
site. The short form of c-FLIP, c-FLIPS, structurally
resembles v-FLIP.
Despite the analysis of mice deficient for c-FLIP, which indicates a
role of c-FLIP in cardiac development (26), the definitive physiological role of this molecule still remains controversial. Recent
reports show that high expression of FLIP promotes tumor growth and
facilitates immune escape of tumors (27, 28). In addition, mouse
embryonic fibroblasts deficient of c-FLIP clearly display an increased
sensitivity toward death receptor-mediated apoptosis (26).
Several reports suggest an involvement of c-FLIP in the modulation of
the immune response (17, 29-33). We recently demonstrated a potential
physiological role for c-FLIPS and found that it is up-regulated upon restimulation of the T cell receptor in primary human
T cells. This finding correlates with rescue of these cells from
activation-induced cell death (34). In addition, we demonstrated up-regulation of c-FLIPS after CD3/CD28 costimulation,
which might contribute to protection toward activation-induced cell
death (35).
The mechanism of c-FLIPL function has only been partly
elucidated (25). Both procaspase-8 and c-FLIPL are
recruited to the DISC. After initial cleavage of both molecules, the
cleavage intermediates remain bound to the DISC and can no longer be
replaced by procaspase-8. This prevents activation of the cytoplasmic
procaspase-8 pool and renders the cell resistant to CD95-induced
apoptosis (25). So far, nothing is known about the mechanism of
c-FLIPS function.
In a previous study, we found that c-FLIPS, but not
c-FLIPL, contributes to rescue from activation-induced cell
death in T cells (34, 35). Therefore, we further clarify the mechanisms of apoptosis inhibition mediated by c-FLIPS to detect
possible differences compared with c-FLIPL action. To
investigate this issue, we generated BJAB clones stably expressing
c-FLIPS or c-FLIPL or both. Overexpression of
either c-FLIPS or c-FLIPL results in resistance
toward death receptor-mediated cell death. The two splice variants,
however, cause this effect in a distinct fashion. Thus, procaspase-8
activation is inhibited at two different cleavage steps at the DISC by
c-FLIPS and c-FLIPL, respectively, the two splice variants of c-FLIP. A detailed analysis of the domains of
c-FLIPL revealed that full-length c-FLIPL, but
not a mutant lacking the p12 subunit, contributes to the first cleavage
step of caspase-8.
Cell Lines--
The B lymphoblastoid cell line BJAB and the T
cell line H9 were maintained in RPMI 1640 (Life Technologies, Inc.), 10 mM HEPES (Life Technologies), 2 mg/ml Gentamycin (Life
Technologies), 10% fetal calf serum (Life Technologies) in 5%
CO2.
Antibodies and Reagents--
Monoclonal antibodies against FADD
(mouse IgG1) and the FLAG epitope (MDYKDDDDK, clone M2, mouse IgG1)
were purchased from Transduction Laboratories (Lexington, KY) and
Sigma, respectively. The C15 monoclonal antibody (mouse IgG2b)
recognizes the p18 subunit of caspase-8 (36), the C5 monoclonal
antibody (mouse IgG2a) recognizes the p10 subunit of caspase-8 (36),
the anti-c-FLIP monoclonal antibody NF6 (mouse IgG1) was described in
(25), and anti-APO-1 is an agonistic monoclonal antibody (IgG3, Isolation of the c-FLIPS Coding Region--
The
coding sequence of c-FLIPS was isolated after total RNA
isolation of H9 cells, reverse transcriptase-polymerase chain reaction,
and subsequent polymerase chain reaction, using the following
primer pair: c-FLIP sense,
5'-ggcgaattcATGcccgggTCTGCTGAAGTCATCCATCCAGG-3'; c-FLIPS
antisense, 5'-cgtctagaTCACATGGAACAACAATTTCCAAG-3'. The sequences shown in capital letters are homologous to the coding sequence of c-FLIPS. The isolated polymerase chain reaction
fragment was cloned into pcDNA3 (Invitrogen); the correct sequence
was confirmed by DNA sequencing.
Generation of Expression Vectors--
Expression vectors coding
for epitope-tagged proteins were cloned by ligating double-stranded
oligonucleotides comprising the sequences of the FLAG epitope
(pcDNA3-FLAG) or the Myc epitope (pcDNA3-Myc), respectively,
into the pcDNA3 expression vector (Invitrogen).
FLAG-c-FLIPS and Myc-c-FLIPS expression
constructs were generated by ligating the coding sequence of
c-FLIPS in frame into pcDNA3-FLAG or pcDNA3-Myc,
respectively. The FLAG-p43mu expression construct coding
for amino acids 1-376 of c-FLIPL was generated by standard
polymerase chain reaction and cloning techniques using the
following primers: 5'-ggcgaattcATGcccgggTCTGCTGAAGTCATCCATCCAGG-3' and
5'-gattctagaTCAATCCACCTCCAAGAGGCTGCTGTCC-3'. The FLAG-D376N expression
construct was generated using the primer
5'-CTTGGAGGTGAATGGGCCAGCGATG-3' and the corresponding
complementary primer applied in the QuikChange Mutagenesis Kit
(Stratagene, La Jolla, CA).
Stable Transfections of BJAB Cells--
Stable transfection of
BJAB cells was performed with expression vectors coding
c-FLIPS C-terminally fused to the FLAG or Myc epitope tag
(pcDNA3-FLAG-c-FLIPS,
pcDNA3-Myc-c-FLIPS), FLAG epitope-tagged c-FLIPL (pEFrsFLAG-FLIPL) (25), or the
respective empty vectors (control) by electroporation (960 microfarads,
200 V). Selection pressure was added 48 h after transfection (4000 µg/ml G418 and/or 1 µg/ml puromycin; Sigma), and high expressing
clones were identified by Western blot analysis using NF6 monoclonal antibody.
DISC Analysis by Immunoprecipitation--
The composition of the
CD95 DISC was determined as follows. 5 × 107 cells
(if not otherwise indicated) were either treated with 10 µg of
LZ-CD95L-containing supernatant for 5 min at 37 °C and then lysed in
lysis buffer (30 mM Tris/HCl, pH 7.5, 150 mM
NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl
fluoride, small peptide inhibitors (Sigma), 1% Triton X-100 (Serva),
and 10% glycerol) (stimulated condition) or lysed without treatment
(unstimulated condition). The CD95 DISC was then precipitated for
2 h or overnight at 4 °C with 2 µg of anti-APO-1 coupled to
protein A-Sepharose (Sigma) or 2 µg of anti-FLAG (M2) coupled to
protein G-agarose (Roche Molecular Biochemicals). After
immunoprecipitation, the beads were washed four times with 20 volumes
of lysis buffer.
Western Blot--
For Western blotting, immunoprecipitates or
cytosolic proteins equivalent to 106 cells or 20 µg of
protein were separated by 12% SDS-polyacrylamide gel electrophoresis
and transferred to Hybond nitrocellulose membrane (Amersham Pharmacia
Biotech), blocked with 5% nonfat dry milk in PBS/Tween (PBS plus
0.05% Tween 20) for at least 1.5 h, washed with PBS/Tween, and
incubated with the primary antibody in PBS/Tween for 16 h at
4 °C. Blots were developed with horseradish peroxidase-conjugated secondary antibody diluted 1:20,000 in PBS/Tween. After washing with
PBS/Tween, the blots were developed with a chemiluminescence method
following the manufacturer's protocol (PerkinElmer Life Sciences).
Cytotoxicity Assay--
For assaying apoptosis, 5 × 105 cells were incubated in 24-well plates with or without
the indicated amounts of anti-APO-1 plus 10 ng/ml protein A, LZ-TRAIL,
LZ-CD95L, or TNF c-FLIPS Inhibits CD95-mediated Apoptosis in BJAB
Cells--
Recently, c-FLIPS and c-FLIPL were
described as inhibitors of death receptor-mediated apoptosis (17).
However, the molecular mechanisms of c-FLIP action remained largely
elusive. To study the mechanisms of apoptosis inhibition mediated by
c-FLIPS and c-FLIPL in detail, we generated
BJAB cells stably transfected with expression constructs coding either
for N-terminally FLAG-tagged c-FLIPS, N-terminally
Myc-tagged c-FLIPS, or N-terminally FLAG-tagged c-FLIPL alone or in combination with
Myc-tagged-c-FLIPS (FL/MS) (see
Fig. 1A for expression
constructs). BJAB cell clones expressing low, intermediate or high
amounts of FLAG-tagged c-FLIPS were identified by Western
blotting (Fig. 1B, left) as well as clones expressing high concentrations of FLAG-tagged-c-FLIPL or
Myc-tagged c-FLIPS alone or in combination
(FL/MS; Fig. 1B, right).
To exclude clonal effects due to different death receptor expression,
we determined cell surface expression of TRAILR1, TRAILR2, TRAILR3, TRAILR4, TNFR1, and CD95. We also determined expression of procaspase-8 and FADD. The expression patterns of these proteins were comparable (data not shown). To analyze the antiapoptotic function of the transfected c-FLIP proteins, we investigated the sensitivity of these
BJAB cell clones toward death receptor-mediated apoptosis. Overexpression of c-FLIPS resulted in cells resistant
toward CD95-mediated apoptosis, induced by LZ-CD95L, as well as
resistance toward TNF c-FLIPS Completely Blocks Cleavage of
Procaspase-8--
To investigate the molecular mechanisms of
c-FLIPS-mediated apoptosis inhibition, we determined
activation of procaspase-8 as one of the first detectable events upon
CD95 triggering. In vector-transfected cells, procaspase-8 was
completely processed, and the p10 and p18 active subunits were
detectable after 10-30 min (Fig.
2B, lane
3; for the procaspase-8 cleavage pattern, also compare Fig.
2A). In contrast, the generation of active subunits was
completely inhibited in cells overexpressing c-FLIPS.
Neither the p18 nor the p10 subunits were detectable, and uncleaved
procaspase-8 was detectable over a period of up to 3 h (Fig.
2B, lane 18). In cells expressing
c-FLIPL, the generation of the p18 subunit was blocked, and
only small amounts of the p10 subunit of procaspase-8 were generated
(Fig. 2B, lanes 7-12). In the latter
case, c-FLIPL only allowed the p43/41 cleavage product of
caspase-8 to be generated. The p43/41 cleavage product stays at the
DISC, thereby preventing recruitment and activation of cytosolic
procaspase-8. Similar to procaspase-8, c-FLIPL is also
cleaved at the DISC by either procaspase-8 or its p43/41 cleavage
product (Fig. 2B, lanes 8-12). Thus,
both c-FLIP proteins block the generation of active caspase-8. Our
results suggest that c-FLIPS completely prevents
procaspase-8 cleavage, whereas c-FLIPL allows the first
cleavage step of procaspase-8.
c-FLIPS Completely Blocks DISC Activity--
Since
both procaspase-8 and c-FLIPS are recruited into the DISC
(25), we assumed that in the presence of c-FLIPS
procaspase-8 cleavage is blocked at the DISC or that its recruitment is
completely inhibited. Therefore, we performed a DISC analysis by
immunoprecipitation of either CD95-unstimulated or -stimulated BJAB
cells that were either transfected with c-FLIP or vector alone, and we
determined DISC-associated caspase-8 cleavage products by Western blot
analysis. In cells overexpressing c-FLIPS, only unprocessed
procaspase-8 was detectable in the DISC (Fig.
3, lane 6). In
addition, cleavage of c-FLIPL was also significantly
reduced (Fig. 3, lane 6). That was also found in
cellular lysates (compare Fig. 2B). In contrast, the
presence of c-FLIPL did not prevent the generation of the p43/41 cleavage product of caspase-8. This finding explains the detection of the p10 caspase-8 subunit in cellular lysates. FADD recruitment to the DISC was not modulated in the presence of
c-FLIPL or c-FLIPS. In summary,
c-FLIPS blocks cleavage of procaspase-8 at the DISC,
whereas c-FLIPL prevents further processing of the p43/41
cleavage product.
The First Cleavage Step of Procaspase-8 Activation Is Driven by
Full-length c-FLIPL--
The fact that the p43 cleavage
form of c-FLIPL is the predominant form at the DISC upon
CD95 triggering (see Ref. 25 and Fig. 3), suggested that cleavage of
c-FLIPL is important for blocking caspase-8 activation. To
analyze the mechanism of procaspase-8 cleavage inhibition by
c-FLIPL in more detail, we generated expression constructs
coding for N-terminally FLAG-tagged mutants of c-FLIPL which either resemble the p43 cleavage form lacking the p12 subunit of
c-FLIPL entirely (FLAG-p43mu) or contain a
defective cleavage site (FLAG-D376N) (20) (compare Fig.
4A and scheme in Fig. 6). BJAB
cells stably transfected with these expression constructs or empty
vector were identified by Western blotting (Fig. 4B), and
the sensitivity toward CD95-mediated apoptosis was investigated. Overexpression of either FLAG-tagged p43-c-FLIPL
(p43mu) or FLAG-tagged unprocessable c-FLIPL
(D376N) resulted in cells resistant toward LZ-CD95L-induced apoptosis
(Fig. 4C). Therefore, both the full-length and the p43
cleavage form of c-FLIPL act in an antiapoptotic manner and
block CD95-mediated apoptosis. Thus, cleavage of c-FLIPL is not required for its antiapoptotic function.
To determine the step in apoptosis signaling at which the mutated
c-FLIPL molecules inhibit cleavage of procaspase-8, we
looked for cleavage products after CD95 triggering. In vector
(control)-transfected cells, procaspase-8 was completely processed, and
the p10 and p18 active subunits were detectable (Fig. 4D,
lanes 1-6). In cells transfected with an
expression construct coding for the FLAG-tagged p43 cleavage product of
c-FLIPL (p43mu), the generation of the p10
subunit was delayed and not detectable after 10 min (Fig. 4D, lane 4). In contrast, in cells
overexpressing FLAG-tagged uncleavable c-FLIPL (D376N),
procaspase-8 was cleaved into its p43/41 and p10 subunits within the
first 10 min of stimulation (Fig. 4D, compare
lanes 8 and 14). The p18
subunit was not generated in the presence of both c-FLIPL
mutants (Fig. 4D). Thus, blocking of procaspase-8 cleavage
by p43mu and the D376N mutant occurred at different steps.
To study this phenomenon in more detail, we performed DISC analysis of
vector- or c-FLIP mutant-transfected BJAB cells and tested for
associated caspase-8 cleavage products by Western blot analysis. In
vector-transfected cells, both the full-length caspase-8 and the
p43/p41-caspase-8 form were detectable in the DISC. In contrast, in
D376N-expressing cells, only the p43/41 cleavage products of caspase-8
could be detected, thus resembling the composition of the DISC in the
presence of high amounts of wild-type c-FLIPL. The presence
of p43mu inhibited the generation of the active caspase-8
subunits, which explains our findings presented in Fig. 4D
and resembles the DISC in c-FLIPS high expressing cells.
These results indicate that the presence of full-length
c-FLIPL but not cleavage of c-FLIPL is
necessary for the initial cleavage of procaspase-8 in the presence of
high amounts of c-FLIPL.
c-FLIPL and c-FLIPS Coexist in the Same
DISC--
Since high expression of either c-FLIPL or
c-FLIPS blocks procaspase-8 processing at different
cleavage steps, we asked whether the presence of either
c-FLIPL or c-FLIPS in one DISC would exclude the respective other splice variant from recruitment. Therefore, we
precipitated FLAG-tagged c-FLIPL in LZ-CD95L-triggered BJAB cells to isolate the DISCs containing FLAG-tagged c-FLIPL
and looked for recruitment of Myc-tagged c-FLIPS to the
DISC. Precipitation of the DISC upon CD95 triggering via FLAG-tagged
c-FLIPL resulted in coimmunoprecipitation of Myc-tagged
c-FLIPS in the double-transfected cells (Fig.
5, lane 6). In
addition, we isolated the DISC via precipitation of CD95 in the
double-transfected BJAB cells as described above (Fig. 5,
lanes 3 and 4). Both
c-FLIPL and c-FLIPS were detected in the DISC,
and FADD recruitment was not impaired when compared with DISC formation
in the vector-transfected cells. These results suggest that upon CD95
triggering, c-FLIPS and c-FLIPL can coexist in
the same DISC. However, in view of the techniques used, our data do not
exclude DISC species containing only one form of c-FLIP.
The apoptosis-inducing signal transduction pathways of the CD95
system have been well characterized (39). In contrast, much less is
known about mechanisms of inhibition of CD95-mediated apoptosis. One
protein that has an inhibitory function on the DISC by reducing the
generation of active caspase-8 is c-FLIPL. However, its
mechanism of inhibition is not well understood. A second cellular
splice variant of c-FLIP, c-FLIPS, also has antiapoptotic effects. Both c-FLIP proteins contain two DEDs and are recruited into
the DISC and thereby block death receptor-mediated apoptosis. In this
report, we provide important insight into the molecular mechanisms of
c-FLIP-mediated apoptosis inhibition. BJAB cells that express high
amounts of either c-FLIPL or c-FLIPS are
protected against death receptor-mediated apoptosis. The presence of
c-FLIPS prevents the initial cleavage step of procaspase-8,
and therefore, its full-length form can be detected at the DISC. In
contrast, c-FLIPL allows the initial cleavage step but
blocks further processing and thus the generation of the p18 subunit.
The difference in processing of procaspase-8 in the presence of the two
c-FLIP splice variants is also reflected in cell lysates and
sheds new light on caspase-8 activation at the DISC. From these
results, we suggest the following model of c-FLIP proteins mediated
inhibition of death receptor initiated apoptosis. In the presence of
low concentrations of c-FLIP proteins, procaspase-8 represents the
majority of tandem DED-containing proteins at the DISC and is activated
by trans- and autocatalytical cleavage due to the close proximity of
several procaspase-8 molecules (Fig.
6B; Ref 40).
c-FLIPL that is recruited into the DISC is cleaved by
caspase-8. High amounts of c-FLIPL in the DISC abolish the
close proximity of procaspase-8 molecules, instead leading to proximity
of c-FLIPL and procaspase-8, resulting in the first but not
the second step of procaspase-8 processing. In this conformation, only
the p10 subunit of caspase-8 and the p12 subunit of c-FLIPL
are generated (Fig. 6C). Since c-FLIPL itself
has no intrinsic catalytic activity, the generation of the p10 subunit
of caspase-8 proceeds autocatalytically, whereas the generation of the
p18 subunit would require transcatalytical actvity (Fig.
6C). High amounts of c-FLIPS in the DISC totally prevent procaspase-8 cleavage (Fig. 6D). This indicates that
c-FLIPL, in contrast to c-FLIPS, still induces
a conformation of the DISC that leads to autocatalytic activity of
procaspase-8 and the first cleavage step. This hypothesis is supported
by the cleavage pattern of procaspase-8 in the presence of
c-FLIPL mutants. The uncleavable c-FLIPL
mutant, like the wild-type c-FLIPL, allows the
generation of the p10 subunit of caspase-8. Therefore the full-length
protein of c-FLIPL, but not its cleavage, facilitates the
first cleavage step of procaspase-8 (Fig. 6F). Our results
are further supported by the observation that the deletion mutant of
c-FLIPL that does not contain the p12 subunit
(p43mu) prevents the first cleavage step of procaspase-8,
similar to c-FLIPS (Fig. 6E). Both the p43 cleavage product of c-FLIPL and the uncleaved form block
caspase-8 activation and, therefore, inhibit apoptosis. Given that
procaspase-8 interacts with c-FLIP proteins in the DISC in a dimeric
manner, our results suggest that the generation of the p10 subunit of caspase-8 occurs autocatalytically. In contrast, the second cleavage step leading to release of the p18 subunit requires transcatalytic activity. However, our results do not exclude interactions of dimers
with dimers due to the multimeric nature of death receptor complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-herpesviruses, form another family of apoptosis-inhibitory
molecules. These molecules are composed of two death effector domains,
a structure resembling the N-terminal half of procaspase-8. Via
DED-DED-interaction, v-FLIP proteins are recruited to the CD95-DISC,
preventing procaspase-8 recruitment and processing and thereby
CD95-induced apoptosis (16).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
recognizing an epitope on the extracellular part of CD95 (APO-1/Fas)
(37). The horseradish peroxidase-conjugated goat anti-mouse IgG1 and IgG2b were from Southern Biotechnology Associates (Birmingham, AL).
Leucine zipper (LZ)-TRAIL and LZ-CD95L were produced as described (38).
TNF
was purchased from Biomol (Plymouth Meeting, PA). All other
chemicals used were of analytical grade and purchased from Merck or Sigma.
plus 10 µg/ml cycloheximide in medium for 16 h at 37 °C. Cells were centrifuged briefly in a minifuge (Heraeus)
at 4000 rpm for 5 min, washed once with PBS, and resuspended in a
buffer containing 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100,
and 50 µg/ml propidium iodide (Sigma). After incubation at 4 °C in
the dark for at least 16 h, apoptotic nuclei were quantified by
FACScan (Becton Dickinson). Specific apoptosis was calculated as
follows: (% experimental apoptosis - % spontaneous
apoptosis)/(100
% spontaneous apoptosis) × 100.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and TRAIL-mediated apoptosis. Resistance
directly correlated with the amounts of c-FLIPS (Fig.
1C). However, the cells were still sensitive toward
staurosporine-induced apoptosis (data not shown (17)). Overexpression
of c-FLIPL and c-FLIPS simultaneously resulted
in increased inhibition toward CD95-mediated apoptosis in an additive
manner as compared with cells expressing c-FLIPL or
c-FLIPS alone (Fig. 1D). TRAIL- and
TNF
-mediated apoptosis were completely inhibited. We conclude from
these results that the CD95 DISC was not saturated with c-FLIP proteins
in the single transfected cells, and therefore an additive effect in
cells expressing both c-FLIP proteins was observed. In contrast, c-FLIP
expression levels in the single transfected BJAB cells were sufficient
for blocking of TRAIL- and TNF
-mediated apoptosis in BJAB cells, which might be due to lower expression levels of receptors for TRAIL
and TNF
as compared with CD95.
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Fig. 1.
c-FLIP proteins inhibit CD95-mediated
apoptosis in BJAB cells. A, overview of tagged
c-FLIP expression constructs used for stable expression in BJAB cells.
The numbers indicate amino acids. B,
Western blot analysis of lysates of BJAB cells stably transfected with
expression vectors coding for the indicated FLIP proteins or vector
control. Lanes 1 and 5,
vector-transfected cells (Control); lanes
2-4, FLAG-tagged c-FLIPS; lane
6, FLAG-tagged c-FLIPL; lane
7, Myc-tagged c-FLIPS; lane
8, Myc-tagged c-FLIPS and FLAG-tagged
c-FLIPS (FL/MS). The tagged c-FLIP
proteins migrate with reduced mobility compared with the endogenous
ones. C, BJAB cells described in B were incubated
with the indicated concentrations of LZ-CD95L, TNF , or LZ-TRAIL for
16 h. Apoptosis was measured by the amount of DNA-fragmentation.
D, BJAB cells described for B were incubated with
the indicated concentrations of LZ-CD95L, TNF
, or LZ-TRAIL for
16 h. Apoptosis was measured as described for C. One
representative experiment out of five is shown.
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Fig. 2.
Different procaspase-8 cleavage products upon
CD95 triggering in the presence of c-FLIPS or
c-FLIPL. A, simplified model for
procaspase-8 processing. Procaspase-8 processing occurs in two
consecutive steps: the generation of the p10 and the p43/41 subunits
(1) and processing of the p43/41 cleavage product into the
prodomain and the p18 subunit (2). B, time course
of procaspase-8 and c-FLIPL processing in BJAB cells stably
expressing FLAG-tagged c-FLIPS (lanes
13-18) or FLAG-tagged c-FLIPL (lanes
7-12) demonstrated in Fig. 1B.
Control, BJAB cells tranfected with empty vector
(lanes 1-6).
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Fig. 3.
Differences in procaspase-8 processing in the
presence of c-FLIPS or c-FLIPL at the
DISC. DISC analysis of BJAB cells stably transfected with empty
vector (Control, lanes 1 and
2), FLAG-tagged c-FLIPL (lanes
3 and 4), or Myc-tagged c-FLIPS
(lanes 5 and 6) encoding expression
plasmids, triggered with 10 µg of LZ-CD95L (+) or left untriggered
( ).
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Fig. 4.
Full-length c-FLIPL is required
for procaspase-8 processing. A, overview of FLAG-tagged
c-FLIPL expression constructs containing the sequence of
unprocessable c-FLIPL (D376N) or the truncated
c-FLIPL (p43mu) used for stable expression in
BJAB cells. The numbers indicate the amino acids in the
resulting proteins. B, Western blot analysis of lysates of
BJAB cells stably transfected with expression vectors coding for the
indicated FLIP proteins. Lane 1,
vector-transfected cells (Control); lane
2, FLAG-tagged p43-c-FLIPL (p43mu);
lane 3, FLAG-tagged unprocessable
c-FLIPL (D376N). C, BJAB cells described for
B were incubated with the indicated concentrations of
LZ-CD95L. Apoptosis was measured by the amount of DNA degradation.
D, time course of procaspase-8 and c-FLIPL
processing in BJAB cells stably expressing D376N (lanes
7-12) or p43mu (lanes
13-18) demonstrated in A and B. Control, BJAB cells tranfected with empty vector
(lanes 1-6). E, DISC analysis of BJAB
cells shown in A-D triggered with 10 µg of LZ-CD95L (+)
or left untriggered ( ).
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Fig. 5.
c-FLIPL and c-FLIPS
coexist in the same DISC. DISC analysis of BJAB cells stably
transfected with empty vector (Control, lanes
1 and 2) or FLAG-tagged c-FLIPL and
Myc-tagged c-FLIPS (FL/MS,
lanes 3 and 4) expression plasmids,
triggered with 10 µg of LZ-CD95L (+) or left untriggered ( ). DISC
was precipitated (I.P.) either by using anti-APO-1
(lanes 1-4) or anti-FLAG antibodies
(lanes 5 and 6). BJAB cells containing
the empty vector were used as the control (lanes
1 and 2).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Model for c-FLIP mediated inhibition of
procaspase-8 processing at the DISC. A, triggering of
CD95 leads to the recruitment of FADD, procaspase-8, and c-FLIP
proteins into the DISC. Binding of procaspase-8 results in its
activation by auto- and transproteolytic cleavage. The remaining
caspase-8 prodomain is replaced by uncleaved procaspase-8, which is
processed in the same manner as described above. B-F,
depending on the ratio of procaspase-8 and c-FLIP proteins at the DISC
(gray box), different products are released from
the DISC upon receptor triggering. B, low amounts of c-FLIP
proteins allow processing of procaspase-8, leading to formation of the
active caspase-8 heterotetramer composed of the p18 and p10 subunits.
C, in the presence of high amounts of c-FLIPL,
procaspase-8 is recruited into the DISC, and cleavage is blocked after
the generation of the p43 cleavage products of both caspase-8 and
c-FLIPL. D, in the presence of high amounts of
c-FLIPS, procaspase-8 is recruited into the DISC but
remains unprocessed. In each case, modulation of caspase-8 cleavage
renders cells resistant to CD95-mediated cell death. E, high
amounts of truncated c-FLIPL-p43mu prevent
procaspase-8 processing completely. F, expression of
unprocessable c-FLIPL-D376N allows initial cleavage of
procaspase-8 but prevents further processing, leading to accumulation
of p43/41-caspase-8 in the DISC.
High expression of c-FLIPS also prevents cleavage of c-FLIPL at the DISC. This phenomenon was also detected in restimulated primary T cells (34). It may be due to spatial interference of c-FLIPS with the interaction of procaspase-8 and c-FLIPL. Another possibility is that initial cleavage of procaspase-8, blocked by c-FLIPS, is required for its transcatalytic activity and, thus, c-FLIPL is not cleaved at the DISC upon high expression of c-FLIPS.
Transfectants expressing FLAG-tagged c-FLIPL and Myc-tagged c-FLIPS showed an additive effect with respect to protection against CD95-mediated apoptosis. This indicates that DISCs in single transfectants are not saturated for c-FLIP binding. In addition, p43/41-caspase-8 could be detected in the DISC, which was not the case in c-FLIPS single transfectants. This could be explained by either a dominant function of c-FLIPL over c-FLIPS or, alternatively, by caspase-8 cleavage in DISC species containing c-FLIPL alone.
Since both c-FLIP splice variants act as antiapoptotic proteins, the question arises why certain physiological stimuli exclusively induce either c-FLIPL or c-FLIPS, like BCR/CD40 (32, 33) or TCR/CD28 (34, 35), respectively. Therefore, the biological function of different procaspase-8 cleavage patterns remains to be elucidated. Since c-FLIPS totally prevents cleavage of both caspase-8 and c-FLIPL, one might speculate that the generation of these cleavage products is blocked because they are necessary for the recruitment of yet to be identified molecules into the DISC or, alternatively, they prevent the association of unidentified molecules.
Recently, it was reported that certain, but not all, viral and cellular
FLIP proteins enhance activation of the NF-B and AP-1 pathways upon
stimulation of death receptors, possibly via recruitment of RIP, TRAF1,
and TRAF2 into the DISC (41-43). However, one study also shows
suppressive effects of DED-containing proteins, such as c-FLIP, on the
NF-
B pathway (44). Therefore, the role of c-FLIP in activation of
NF-
B by death receptors needs to be addressed in more detail.
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ACKNOWLEDGEMENTS |
---|
We thank A. Strasser for providing the pEFrs-FLAG expression vector and H. Walczak for providing LZ-TRAIL. We are grateful to Marcus E. Peter for helpful discussions in the initial phase of this study. We thank Wendelin Wolf for technical assistance and Heidi Sauter for expert secretarial assistance.
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FOOTNOTES |
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* This work was supported by Grants from Sonderforschungsbereich der deutschen Forschungsgemeinschatz 601 and 405, Deutsches Krebsforschungszentrum/Israeli Minister of Science (DKFZ/MOS) (Ca 86), Deutsch-Israelische Projektkooperation, and the Sander Stiftung.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.
To whom correspondence should be addressed. Tel.: 49 6221 423765;
Fax: 49 6221 411715; E-mail: S.Kirchhoff@dkfz-heidelberg.de.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M101780200
1
The abbrevations used are: TNF, tumor necrosis
factor; CD95L, CD95 ligand; FLICE, FADD-like
interleukin-1-converting enzyme; FADD, Fas-associated death domain;
DED, death effector domain; DISC, death-inducing signaling complex;
FLIP, FLICE-inhibitory protein; TRAIL, TNF-related apoptosis-inducing
ligand; LZ, leucine zipper; PBS, phosphate-buffered saline.
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