From the Department of Molecular Medicine, Veterinary
Medical Center and the § Department of Chemistry and
Chemical Biology, Baker Laboratory, Cornell University,
Ithaca, New York 14853
Received for publication, October 27, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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Fas-mediated apoptosis results in the activation
of caspases, which subsequently cleave cellular substrates that are
essential for normal cell viability. In the present study, we show that the Ras-related GTP-binding protein Cdc42 is susceptible to
caspase-catalyzed proteolysis in a number of cell lines, including
NIH3T3 fibroblasts, human breast cancer cells (e.g. T47D),
and COS-7 cells. Both caspase-3 and caspase-7 were able to
catalyze the cleavage of Cdc42, whereas caspase-6 and caspase-8 were
without effect. The susceptibility to the caspase-stimulated
degradation is specific; although Rac can also serve as a caspase
substrate, neither Rho nor Ras is degraded. Caspase sensitivity is
conferred by a consensus sequence (DXXD) that lies
immediately upstream of the Rho insert regions (residues 122-134) of
Cdc42 and Rac. The removal of a stretch of residues (120) that
includes the insert region or site-directed mutagenesis of either
aspartic acid 118 or 121 within a constitutively active background
(i.e. Cdc42(F28L)) as well as a wild-type Cdc42 background
yields Cdc42 molecules that provide a marked protection against Fas
ligand-induced apoptosis. Overall, these results are consistent with a
model in which Cdc42 acts downstream of Fas, perhaps to influence the
rate of apoptosis, with the ultimate caspase-mediated degradation of
Cdc42 then allowing for a maximal apoptotic response.
Cdc42, a Rho-related member of the Ras superfamily, acts as a
GTP-binding protein/molecular switch to control a diversity of cellular
processes, including the actin cytoskeletal architecture, cell polarity
and motility, cell cycle progression, and gene transcription (1).
Recently Cdc42 has also been implicated in membrane trafficking (2)
with this role being linked to its ability to induce malignant transformation. Slight perturbations in the GTP-binding/GTPase cycle of
Cdc42 can give rise to a transformed phenotype (3, 4), and it appears
that multiple target/effectors are required. Recently Cdc42 and the
related GTP-binding protein Rac have been suggested to play a role in
suppressing programmed cell death (5, 6). Therefore, it is an
intriguing possibility that at least part of the ability of Cdc42 to
transform cells is linked to a circumvention of apoptosis.
A possible link between Cdc42 and the suppression of apoptotic signals
may be the serine/threonine kinase p21-activated kinase (PAK).1 It has been well
established that PAK is a primary target for the Cdc42 and Rac
GTP-binding proteins with its serine/threonine kinase activity being
markedly stimulated by activated versions of the GTP-binding proteins
(7, 8). It has also recently been shown that PAK1 phosphorylates Bad, a
proapoptotic member of the Bcl-2 family (5, 6). In addition, PAK1 is
known to stimulate NF Thus, in the present study, we examined whether Cdc42 might influence
apoptotic signaling pathways, perhaps by performing some type of
survival function. We show here that Cdc42 serves as a caspase
substrate with the cleavage site lying just upstream of the Rho insert
region. Deletion of residues 120-139 or specific point mutations at
sites immediately upstream of the insert yields a Cdc42 molecule that
significantly retards the ability of apoptotic stimuli (e.g.
Fas ligand) to give rise to programmed cell death.
Cell Culture and Preparation of Cell Extracts--
COS-7 and
NIH3T3 cells were maintained in a humidified 7% CO2
environment in Dulbecco's modified medium supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc.). The media for COS-7 and NIH3T3 cells were supplemented with 10%
fetal bovine serum. The human breast cancer cell line T47D was cultured
in RPMI 1640 supplemented with 100 units/ml penicillin and 100 µg/ml
streptomycin (Life Technologies, Inc.). The media for T47D cells were
supplemented with 10% calf serum. COS-7 or T47D cells were washed with
ice-cold phosphate-buffered saline and then resuspended in lysis buffer
(20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet
P-40, 10% glycerol, 10 mM MgCl2, 1 mM EDTA, 10 µg/ml leupeptin, and 10 µg/ml aprotinin)
for 15 min at room temperature prior to use.
Fas Activation--
T47D cells (1-2 × 106)
were suspended in 1 ml of RPMI 1640 containing 10% calf serum to
minimize incubation volumes. The cells were incubated without serum for
4 h at 37 °C. After the incubation, 50 ng/ml of activating Fas
ligand (FasL) (obtained from Upstate Biotechnology Inc.) were added,
and cells were incubated for various periods of time. After 1 h, 2 ml of RPMI 1640 without serum were added to cells.
COS-7 or NIH3T3 cells were seeded in 6-well plates in 2 ml of DMEM
containing 10% fetal bovine serum. The cells were then incubated
without serum for 10 h at 37 °C. After the incubation, 100 ng/ml of activating FasL were added, and cells were incubated for
varying periods of time. After 2 h, 1 ml of DMEM without serum was
added to adherent cells.
Molecular Constructs--
Point mutants were generated using the
polymerase chain reaction from a cDNA-encoding Cdc42 that
had been subcloned into the BamHI/EcoRI site of
pcDNA3. The expression of recombinant proteins in
Escherichia coli was performed as described previously (11). For transient expression in COS-7 cells, the cDNAs encoding the GTP-binding proteins were subcloned into the hemagglutinin (HA)-tagged pcDNA3 vector by using the
BamHI/EcoRI restriction sites. For stable
expression in NIH3T3 cells, constructs were subcloned into the
HA-tagged pLex vector using the same restriction sites.
Assessing Cellular Apoptosis--
Stable cell lines were
cultured on dual-chamber microscope slides (Nunc) for 1 day in normal
media and then serum-starved for 8 h. Cells were then treated with
100 ng/ml of FasL as described previously (12). The media were removed,
and the cells were placed at 4 °C and gently washed with 10 ml of
phosphate-buffered saline. Trypsin (1 ml) was then added to remove
adherent cells. The cells were assayed for apoptosis using the Annexin
V FITC apoptosis detection kit (Oncogene Research Products)
according to the manufacturer's procedures. Annexin V and propidium
iodine (Molecular Probes) staining were used to test for membrane
blebbing as described previously (13).
Assays of Caspase-catalyzed Cleavage of Cdc42 and Related
Proteins--
Typically ~20 µg of lysate proteins from cells
expressing HA-tagged Cdc42, Rac1, or RhoA were mixed with 50 ng of
caspase-3, caspase-6, caspase-7, or caspase-8 in a total volume of 100 µl. Caspases were purchased from PharMingen. Reactions were carried out in a final concentration of 20 mM HEPES, pH 7.4, 100 mM NaCl, 4 mM EDTA, 20% sucrose, and 0.1%
CHAPS. The reactions were performed at room temperature for varying
times in the presence or absence of the tetrapeptide inhibitor
Ac-DEVD-CHO (200 ng). Aliquots (25 µl) were removed from the reaction
mixture, and the reaction was stopped by boiling for 5 min in 6×
SDS-polyacrylamide gel electrophoresis sample buffer. Cdc42 and
other GTP-binding proteins were detected by Western blot analysis using
a 1:1000 dilution of anti-HA (HA 1.11) monoclonal antibody purchased
from Covance Research Products.
Measurement of Caspase Activities in Cell Lysates--
Cells
(1 × 105) were plated onto 6-well plates and starved
for 12 h in 1% DMEM. The cells were then lysed in 50 mM Tris, pH 7.2, 1 mM EDTA, 10 mM
EGTA, and 1% Nonidet P-40. Fifty µg of lysate proteins were
incubated with 10 µM
Ac-DEVD-(7-amino-4-trifluoromethylcoumarin) (purchased from PharMingen)
in 1 ml of 20 mM PIPES, pH 7.2, 100 mM NaCl, 10 mM dithiothreitol, 1 mM EDTA, 0.1% CHAPS, and
10% sucrose. Fluorescence was then monitored using a PerkinElmer Life Sciences LS-5 fluorescence spectrophotometer with an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Caspase-8 activity was measured in an identical fashion but using 10 µM Ac-IETD-(7-amino-4-trifluoromethylcoumarin)
(purchased from PharMingen).
Assaying the Activation of Cdc42 in Cells Using the Limit
Cdc42/Rac-binding Domain from PAK--
The activation of
cellular Cdc42 was assayed as described previously (14) based on a
procedure originally developed for Ras (15). COS-7 cells were
transiently transfected with the cDNA for wild-type Cdc42 in the
pcDNA3 vector. Cells were allowed to grow in the presence of 10%
fetal bovine serum for 24 h and then starved for 4 h followed
by stimulation with 100 ng/ml epidermal growth factor or 100 ng/ml FasL. Typically ~20 min after stimulation, cells were lysed in
20 mM HEPES, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 20 mM NaF, 20 mM Effects of Cdc42 on FasL-induced Apoptosis--
It has recently
been shown that the proapoptotic Bad protein can be phosphorylated by
PAK1 (5, 6), thereby preventing its interaction with Bcl-2 or
Bcl-xL. Given that Cdc42 is a potent activator of PAK (7,
8, 16), we set out to examine whether wild-type Cdc42 or different
Cdc42 mutants might be capable of influencing apoptotic responses.
Several different constructs of Cdc42 were stably transfected into
NIH3T3 cells that were subsequently treated with the proapoptotic
ligand FasL. We found that a Cdc42 construct (designated Cdc42 Is Susceptible to Degradation in Response to Apoptotic
Stimuli--
We have found that Cdc42 is subject to degradation when
cells are exposed to a variety of types of apoptotic treatments. For example, upon the transient expression of HA-tagged Cdc42 in NIH3T3 fibroblasts, COS-7 cells, and human breast cancer T47D cells followed by treatment with FasL, the HA-tagged Cdc42 underwent a
time-dependent degradation. Fig.
2A, left panel,
shows the results obtained upon treating cells with FasL and then
monitoring Cdc42 by Western blotting with an anti-HA antibody, whereas
the right panel shows the corresponding controls (no FasL
treatment). There was significant variability in the time period for
degradation, depending on the cell type, with COS-7 cells requiring a
much greater time period for achieving complete degradation as compared
with T47D or NIH3T3 cells. This most likely reflects differences in the
relative amount of caspase activity in the individual cell types.
Similar results were obtained when exposing the different cell types to
ultraviolet radiation. Cdc42 was significantly degraded in irradiated
NIH3T3 cells (Fig. 2B), and this was also the case in T47D
cells (data not shown). Rac is also susceptible to the degradation
induced by apoptotic stimuli, although typically it appears to be a
less effective substrate than Cdc42 (e.g. see Fig.
2B). On the other hand, we did not find either Ras or RhoA to be degraded in any of the cell types examined following treatment with apoptotic stimuli. An example is shown for the treatment of NIH3T3
cells with FasL (Fig. 2C), and similar results were obtained
with COS-7 and T47D cells (data not shown). Thus, there appears to be a
rather striking selectivity in terms of the small GTP-binding proteins
that are sensitive to degradation.
Cdc42 Is a Substrate for Caspases--
We next examined which
specific caspases cleave Cdc42 and Rac. Lysates from COS-7 cells
expressing HA-tagged Cdc42 or Rac were treated with a number of
different purified recombinant caspases (e.g.
caspase-3, caspase-6, caspase-7, and caspase-8). We found that
both Cdc42 and Rac were effectively degraded by caspase-3 and
caspase-7, whereas neither GTP-binding protein was susceptible to
caspase-6 or caspase-8 (Fig.
3A). The addition of a caspase inhibitor peptide, DEVD, to the lysates blocked the degradation of both
Cdc42 and Rac (see Fig. 3B; in these experiments, tissue transglutaminase was examined as a negative control). Overall, these
findings are consistent with the notion that caspase-3 and caspase-7
are effector caspases (i.e. downstream participants in
caspase cascades), whereas caspase-6 and caspase-8 typically serve as initiator caspases (18). We also examined whether the cleavage of Cdc42 was nucleotide-dependent and thus
transiently expressed wild-type (i.e. predominantly
GDP-bound) Cdc42 in COS-7 cells as well as a nucleotide-depleted Cdc42
mutant (Cdc42(T17N)) and a GTPase-defective, dominant-active
Cdc42 mutant (Cdc42(Q61L)). We found that each of these Cdc42 proteins
was cleaved at essentially the same rate (Fig.
4). Thus, the caspase susceptibility of
Cdc42 must involve a site (or sites) that is distinct from those
influenced by the GTP-dependent conformational changes that
underlie the molecular switch function of GTP-binding proteins.
We have also performed experiments examining the ability of recombinant
caspases to directly catalyze the degradation of recombinant Cdc42.
GST-Cdc42, -Rac, and -RhoA were incubated with different purified
recombinant caspases and then analyzed by Western blot analysis with
anti-GST. We found that caspase-3 was able to effectively degrade Cdc42
as read out either by using an anti-GST antibody (Fig.
5) or by using an anti-Cdc42
carboxyl-terminal antibody (data not shown). Under these conditions,
GST-Rac was weakly degraded by caspase-3 (relative to the degradation
observed for Cdc42), and GST-RhoA showed no detectable degradation.
Other purified recombinant caspases, such as caspase-6 or caspase-8,
were ineffective against all of the GTP-binding proteins tested. The
necessity of adding caspase-7 to cellular lysates to catalyze Cdc42
degradation, coupled with the finding that purified caspase-3
effectively degrades Cdc42, suggests that caspase-7 may be acting
upstream of caspase-3 in a pathway leading to Cdc42 proteolysis. Other
caspases, aside from caspase-3, may also be acting downstream from
caspase-7 given that both caspase-7 and caspase-3 are able to strongly
catalyze the degradation of Rac in cellular lysates, whereas the direct addition of purified caspase-3 to Rac shows a significantly slower degradation.
Identification of the Cleavage Sites on Cdc42--
Sequence
analysis of Cdc42 and Rac revealed a caspase consensus sequence
(DXXD motif) located between residues 118 and 121 just
upstream of the Rho insert region (Fig.
6). Neither Rho nor Ras appears to
contain a caspase consensus sequence, which is consistent with our
findings that these GTP-binding proteins do not serve as substrates for
proteolysis. We then used site-directed mutagenesis to alter the
putative caspase cleavage sites on Cdc42 with the following point
mutants being generated: Cdc42(D118N), Cdc42(D121N), and Cdc42(D122N).
The HA-tagged wild-type Cdc42 and the HA-tagged Cdc42 mutant proteins
were stably expressed in NIH3T3 cells and then analyzed for
FasL-induced degradation. Both wild-type Cdc42 and the Cdc42(D122N)
mutant were effectively degraded under conditions of FasL-induced
apoptosis (Fig. 7), whereas the
Cdc42(D118N) and Cdc42(D121N) mutants were resistant to
degradation.
Similar results were obtained when purified caspases were added to
lysates from NIH3T3 cells that stably expressed the different HA-tagged
Cdc42 proteins. We found that wild-type Cdc42 and the Cdc42(D122N)
mutant were susceptible to degradation stimulated by either caspase-7
or caspase-3 (Fig. 8), whereas the
Cdc42(D118N) and Cdc42(D121N) mutants were resistant to degradation
under each condition. The same was true when examining the ability of
recombinant caspase-3 to catalyze the degradation of the different
purified recombinant GST-Cdc42 proteins (data not shown). Thus, we
conclude that aspartic acid residues 118 and 121 within Cdc42 are
essential sites for caspase-catalyzed proteolysis.
Caspase-insensitive Cdc42 Mutants Block Fas-mediated
Apoptosis--
Having identified the essential sites on Cdc42 for
caspase-mediated degradation, we then examined whether these
caspase-insensitive mutants could influence the ability of FasL to
induce apoptosis. We first established NIH3T3 cell lines that stably
expressed the activated Cdc42(F28L) mutant and different Cdc42
molecules that were point-mutated within an F28L background
(i.e. Cdc42(F28L/D118N), Cdc42(F28L/D121N), and
Cdc42(F28L/D122N)) as we have done in the past to assess the role of
Cdc42(F28L) in cell growth and actin cytoskeletal rearrangements (2-4,
17). Fig. 9A shows the results obtained when the different stable cell lines were treated with 100 ng/ml FasL and then scored for apoptosis using dye-binding assays (as
described under "Experimental Procedures"). Consistent with other
results (e.g. Fig. 1), cells expressing the constitutively active Cdc42(F28L) mutant showed essentially the same susceptibility to
FasL-induced apoptosis as control cells expressing vector alone. Cells
expressing the Cdc42(F28L/D122N) double mutant also showed little
protection against FasL-mediated cell death. However, cells expressing
either of the caspase-insensitive mutants, Cdc42(F28L/D118N) or
Cdc42(F28L/D121N), showed significant resistance against programmed cell death. It is interesting that similar results were obtained when
the point mutations were generated within a wild-type Cdc42 background.
The stable expression of either Cdc42(D118N) or Cdc42(D121N) conferred
resistance to FasL-induced cell death in NIH3T3 cells, whereas the
Cdc42(D122N) mutant was essentially ineffective (Fig. 9B).
This was also the case when human breast cancer cells (T47D) were
transiently transfected with the corresponding cDNAs for these
different Cdc42 mutants (Fig. 9C).
Fig. 10A shows the
dose-response profiles for FasL-induced apoptosis in NIH3T3 cells that
stably express the various point mutants in the Cdc42(F28L) background,
whereas Fig. 10B shows the corresponding profiles for
fibroblasts expressing the different point mutants in the wild-type
Cdc42 background. We see that in both cases, a substantial protection
is provided by Cdc42 molecules containing either the D118N or the D122N
mutation at FasL levels
It has been reported that Ras has the ability to inhibit FasL-mediated
apoptosis through the down-regulation of Fas expression (21). However,
as shown in Fig. 11, there is no
detectable effect on Fas expression in NIH3T3 cells that express those
Cdc42 mutants that give rise to protection against FasL-mediated
apoptosis. Likewise, no detectable effects on the expression of the
Fas-signaling participants Fas-associated death domain,
caspase-3, or caspase-7 were found.
We also examined whether the expression of the Cdc42 mutants that
exhibited protection against FasL-induced apoptosis had any effect on
the cellular activities of caspases. Fig.
12, A and B,
shows that FasL stimulated the activation of caspase-3 and caspase-8 in
NIH3T3 cells with maximal activation occurring in 3-4 h followed by a
steady decline in activity, consistent with previous reports (22). The
FasL-stimulated caspase-3 activity in cells expressing each of the
Cdc42 mutants tended to be higher (sometimes by as much as 50-60%)
rather than lower than the activity measured in control NIH3T3 cells
(Fig. 12A). When assaying caspase-8, which is upstream of
caspase-3, we did observe minor reductions in the activity compared
with control cells (Fig. 12B). However, in some experiments
we found no detectable differences under conditions in which
significant protection against apoptosis was provided by the D118N and
D121N mutants, thus indicating that the protective effects cannot be
attributed to a change in the expression or function of caspase-8.
The fact that caspase-insensitive mutations in a wild-type Cdc42
background were still able to confer protection against FasL-induced apoptosis was somewhat surprising. Thus, we hypothesized that treatment
with FasL is able to promote Cdc42 activation. In fact, various Rho
family members including Cdc42 have been reported to be activated by
FasL (19, 20). We have examined this under conditions in which Cdc42 is
able to protect against apoptosis and found that FasL was able to
induce a weak but consistent stimulation of Cdc42 activation as read
out by the interaction of GTP-bound Cdc42 with the limit-binding domain
of one of its primary targets PAK (Fig.
13). However, the overall extent of
activation of Cdc42 by FasL was significantly reduced compared with
other activating ligands (e.g. epidermal growth factor (Fig.
13)). Thus, it appears that FasL may only activate a small percentage
of the total cellular pool of Cdc42 but that this may be sufficient to
have an impact on FasL-induced apoptosis.
It is becoming increasingly clear that the signaling pathways
responsible for programmed cell death are every bit as complex as those
signaling activities that stimulate cell cycle progression and
mitogenesis. Moreover, there is every reason to believe that apoptotic
ligands/factors will initiate a diversity of signaling outputs, some of
which will have the expected stimulatory effects on apoptosis, whereas
others will exert opposing effects, perhaps providing some modulation
to the overall rate at which cell death proceeds. This is similar to
what has emerged from studies of the signaling pathways responsible for
mitogenesis. For example, the growth factor-dependent
activation of Ras stimulates cell cycle progression but also leads to
the expression of genes that negatively regulate cell growth and even
contribute to apoptosis (reviewed in Ref. 23). Thus, for these reasons,
we felt that it was of interest to examine how Cdc42, a Rho-related
member of the Ras superfamily that has been implicated in a number of fundamental cellular processes including cell cycle progression, cell
shape, and motility, influences programmed cell death.
We show here that the deletion of a stretch of residues that included
the Rho insert region yielded a Cdc42 molecule that was able to
significantly retard the rate of apoptotic progression. We have gone on
to show that immediately upstream from the insert region, Cdc42
contains an essential site for caspase-catalyzed proteolysis. The
degradation of Cdc42 in cells occurs under conditions of apoptotic
stimulation and appears to be specific. Rac, which is highly related to
Cdc42, can also serve as a caspase substrate, but neither Rho nor Ras
was degraded during Fas-mediated apoptosis in any of the cell lines
examined. Mutating the essential sites for caspase-catalyzed
proteolysis yields Cdc42 molecules that can markedly reduce the
susceptibility of cells to apoptosis, most likely by slowing their rate
of progression toward cell death. What is especially interesting is
that Cdc42 molecules, which are not constitutively active but
nonetheless are mutated at the caspase-sensitive sites, exhibit strong
antiapoptotic effects.
This effect then points to apoptotic ligands such as FasL as
potentially having the capability for promoting the activation of
Cdc42. Various reports have appeared suggesting that FasL and related
ligands (e.g. tumor necrosis factor- Fig. 14 depicts a working model that is
consistent with the results presented here. Fas activation is
shown to initiate at least two types of pathways, one that leads
to the activation of caspases and a classical apoptotic response and a
second that serves to oppose the apoptotic signal (i.e.
similar to a survival activity). This is similar to what has been
proposed for the related apoptotic ligand tumor necrosis factor-
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activity, thus protecting cells against
apoptosis (9, 10). Given the increasing indications that PAK activation leads to cell survival and evasion from apoptosis, it becomes attractive to consider a role for its upstream activator, Cdc42, in
similar processes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glycerol
phosphate, 20 µM GTP, 1 mM sodium vanadate,
10 µg/ml leupeptin, and 10 µg/ml aprotonin and then incubated with
50 µg of GST·PBD (for p21-binding domain from PAK). The cells were
rocked at 4 °C for 3 h. GST·PBD was then precipitated with
glutathione-agarose beads, washed three times with lysis buffer, and
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting
using the indicated antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
L8) in
which loop 8 from H-Ras (residues 121-127) was substituted for amino
acids 120-139 in Cdc42, including a stretch of amino acids unique to
Rho-related proteins but missing in Ras (the Rho insert, residues
122-134), protected cells against FasL-induced apoptosis (Fig.
1). Even under conditions in which a
constitutively active form of Cdc42 (Cdc42(F28L)) that is potently transforming (3) did not show significant protective effects, the
L8
deletion in a constitutively active background (designated Cdc42(F28L)
L8), which yielded an activated but
transformation-defective Cdc42 molecule (17), showed strong protection.
Moreover, the
L8 deletion in a wild-type background also showed
substantial protection against apoptosis. The quantitation of apoptosis
using Annexin V FITC/propidium iodine staining showed that under
conditions in which at least 80% of the (vector) control cells
underwent apoptosis following treatment with FasL, typically only
20-30% of the cells died when expressing the Cdc42
L8 constructs.
These results suggested that the
L8 deletion somehow provided Cdc42 with a significant protective capability. Among the possible
explanations for these findings was that Cdc42 might itself be a target
for apoptotic factors (e.g. caspases) such that the
L8
deletion reduced the susceptibility of Cdc42 and thereby slowed the
overall time course for programmed cell death.
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Fig. 1.
Protection by Cdc42 against Fas-mediated
apoptosis. NIH3T3 cells that stably express either a
constitutively active Cdc42 mutant or wild-type Cdc42 lacking residues
120-139 (designated Cdc42(F28L L8) and Cdc42
L8, respectively)
were treated with 100 ng/ml FasL for 8 h. Apoptosis was assayed by
staining Annexin V/propidium iodine as described under "Experimental
Procedures."
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Fig. 2.
Fas induces the degradation of Cdc42 and
Rac. A, NIH3T3 fibroblasts, human breast cancer T47D
cells, and COS-7 cells that transiently express HA-tagged Cdc42 were
serum-starved for 12 h in DMEM supplemented with 1% calf serum
and then treated with 100 ng/ml FasL for the indicated times. The
degradation of Cdc42 was assessed by Western blotting with anti-HA
antibody. B, NIH3T3 cells that transiently express HA-tagged
Cdc42 or HA-tagged Rac1 were serum-starved and then irradiated with
ultraviolet light for 5 min at room temperature. The degradation of
Cdc42 or Rac was assessed by Western blotting with anti-HA antibody.
C, NIH3T3 fibroblasts that transiently express HA-tagged
Ras or HA-tagged Rho were treated with FasL and analyzed as
described in panel A.
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Fig. 3.
Recombinant caspases catalyze the degradation
of Cdc42 and Rac. A, lysates from COS-7 cells that
transiently express HA-tagged Cdc42, HA-Rac1, HA-Ras, or HA-RhoA were
treated with 50 ng of the indicated purified (recombinant) caspases.
The degradation of the different GTP-binding proteins was analyzed by
Western blot analysis using anti-HA antibody. B, COS-7 cells
that transiently express HA-tagged Cdc42 or HA-tagged Rac1 were lysed
and treated with 100 ng of purified recombinant caspase-7 in the
presence or absence of 100 ng of the caspase-peptide inhibitor
(DEVD-CHO). Endogenous tissue transglutaminase
(TG) served as a negative control for caspase-catalyzed
degradation.
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Fig. 4.
The caspase sensitivity of Cdc42 is
independent of nucleotide occupancy. Lysates from COS-7 cells
transiently expressing wild-type Cdc42 (Cdc42-wt),
GTPase-defective and dominant-active Cdc42 (Cdc42-L61), or
nucleotide-depleted, dominant-negative Cdc42 (Cdc42-N17)
were treated with 50 ng of purified, recombinant caspase-3. The
degradation of the Cdc42 proteins was assessed at the indicated time
periods by Western blotting with anti-HA antibody.
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Fig. 5.
Purified Cdc42 is degraded by purified
caspase-3. E. coli-expressed, purified GST-Cdc42 (1 µg), GST-Rac1, and GST-RhoA were incubated with 100 ng of purified
recombinant caspase-3 for the indicated time periods. The GTP-binding
proteins were then subjected to SDS-polyacrylamide gel electrophoresis
and Western blot analysis using an anti-GST antibody.
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Fig. 6.
Depiction of essential sites on Cdc42 for
caspase-catalyzed proteolysis. G1 and G2
refer to boxes of residues essential for guanine nucleotide binding.
Effector Loop and Switch II represent
regions thought to undergo conformational transitions upon GDP-GTP
exchange. The Rho Insert region represents residues
122-134.
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Fig. 7.
FasL induces the degradation of Cdc42.
NIH3T3 fibroblasts were stably transfected with wild-type HA-tagged
Cdc42 or with the different indicated Cdc42 mutants. The cells were
serum-starved for 10 h and then treated with FasL (100 ng/ml) for
the indicated periods of time. The degradation of Cdc42 was then
analyzed by Western blotting with anti-HA antibody.
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Fig. 8.
Point mutations of a consensus caspase
cleavage site in Cdc42 protect against degradation. Lysates from
NIH3T3 fibroblasts that stably express HA-tagged (wild-type) Cdc42 or
the indicated Cdc42 point mutants were treated with 100 ng of
recombinant, purified caspase-3 or caspase-7 for the indicated periods
of time. The degradation of the different Cdc42 molecules was assessed
by Western blotting with an anti-HA antibody.
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Fig. 9.
Cdc42 point mutants that are
caspase-insensitive inhibit apoptosis. A, NIH3T3
fibroblasts stably expressing Cdc42(F28L), Cdc42(F28L/D118N),
Cdc42(F28L/D121N), or Cdc42(F28L/D122N) were serum-starved in DMEM with
1% calf serum and then treated with 100 ng/ml FasL for 4 h.
Apoptosis was assayed by staining with Annexin V/propidium iodine. The
results represent the analysis of 500 cells (n = 6).
B, an identical set of experiments as shown in panel
A except that the NIH3T3 cells were stably expressing
Cdc42(D118N), Cdc42(D121N), and Cdc42(D122N). In all cases, the control
represents cells transfected with vector alone. C, human
breast cancer cells T47D transiently expressing Cdc42(D118N),
Cdc42(D121N), or Cdc42(D122N) were serum-starved in DMEM with 1% calf
serum and then treated with 50 ng/ml FasL. After 12 h, the cells
were analyzed for apoptosis by staining with Annexin V/propidium
iodine. wt, wild type.
100 ng/ml. However, at higher levels
of FasL (200 ng/ml), the protection effects are greatly diminished.
Similarly, in time course experiments, the D118N and D122N mutants show
substantial protection up to 24 h of treatment with 100 ng/ml
FasL, whereas at longer times (
36 h), the protective effects
by the Cdc42 mutants were significantly diminished (data not shown).
These data indicate that the protective effects provided by Cdc42 are
not irreversible and can be overcome by increasing the strength and the
time duration of the apoptotic stimuli.
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Fig. 10.
Dose-response profiles for FasL-induced
apoptosis. A, NIH3T3 fibroblasts that stably express
Cdc42(F28L/D118N), Cdc42(F28L/D121N), Cdc42(F28L/D122N), or empty
vector (controls) were serum-starved in DMEM with 1% calf serum and
then treated with the indicated doses of FasL for 24 h. Apoptosis
was assayed by staining with Annexin V/propidium iodine. The results
represent the analysis of 500 cells (n = 6).
B, an identical set of experiments as shown in panel
A except using cells that express Cdc42(D118), Cdc42(D121N), and
Cdc42(D122N). Conc, concentration.
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Fig. 11.
Relative expression of Fas and other
apoptotic signaling components. NIH3T3 fibroblasts stably
expressing wild-type HA-tagged Cdc42(D118N), Cdc42(D121N),
Cdc42(D122N), or empty vector were subjected to Western blot analysis
to assess the relative levels of expression of Fas, Fas-associated
death domain (FADD), caspase-7, and caspase-3.
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Fig. 12.
Caspase-3 and caspase-8 activities in cells
expressing Cdc42. NIH3T3 fibroblasts stably expressing wild-type
HA-tagged Cdc42(D118N), Cdc42(D121N), Cdc42(D122N), or empty vector
were treated with 100 ng/ml FasL for the indicated times. Cells were
then lysed, and 50 µg of lysates were added to 1 ml of reaction
buffer (see "Experimental Procedures"). The reactions were
performed at 37 °C for 1 h. DEVD and IETD-directed caspase
activity were then measured as described under "Experimental
Procedures."
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Fig. 13.
Activation of Cdc42 in cells. NIH3T3
fibroblasts transiently expressing wild-type HA-tagged Cdc42 were
treated with 100 ng/ml FasL or epidermal growth factor
(EGF). After 30 min, the cells were lysed and the
limit Cdc42/Rac-binding domain from PAK (GST·PBD) was added.
Lysates were incubated with GST·PBD for 4 h, washed six times in
lysis buffer, and then subjected to Western blot analysis using an
anti-HA antibody.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) are able to
stimulate the activation of Cdc42 (18, 24). We have carefully examined this under conditions in which Cdc42 influences apoptotic responses and
find that FasL is able to increase the levels of activated Cdc42 in
cells, but this appears to be a rather modest effect, apparently
representing a small percentage of the total cellular pool of Cdc42.
Therefore, in order for Cdc42 to influence Fas-mediated apoptosis, some
type of signal amplification may be required.
(25-27). The activation of Cdc42 and/or one of its downstream
effectors would be essential for the survival pathway. It appears that
the effects of Cdc42 are not directed at the level of caspase
expression or activation. Rather, both caspase-3 and its upstream
activator caspase-8 were strongly stimulated by FasL under conditions
in which caspase-insensitive Cdc42 mutants were able to provide strong
antiapoptotic effects. Thus, the protective/survival effects mediated
by Cdc42 must be directed at events occurring downstream from caspase
activation, perhaps influencing the susceptibility of key substrates,
the proteolysis of which is necessary for a full commitment to
apoptosis. The ability of FasL to both initiate caspase activation and
promote Cdc42-mediated survival signals might then provide for a
certain time frame for the apoptotic response to occur. As caspase
activation becomes maximal and the Cdc42 degradation ensues, the
apoptotic signaling pathway(s) would in effect take over and allow the
completion of the cell death program.
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Fig. 14.
Depiction of Fas-induced signaling pathways
leading to caspase activation and Cdc42-mediated survival signals.
The activation of Fas is suggested to initiate at least two pathways,
one that leads to caspase activation and a second that triggers a
Cdc42-mediated signaling response that has the potential of slowing the
overall rate of apoptosis. The caspase-catalyzed degradation of Cdc42
then shuts down this pathway and allows a maximal apoptotic
response.
The expression of the constitutively active Cdc42(F28L) mutant did not
cause a significant slowing of the Fas-induced apoptotic response.
However, clearly an advantage is gained by expressing Cdc42 molecules
that are not susceptible to caspase-catalyzed degradation. At the
present time, we have not identified the downstream Cdc42 targets that
are responsible for providing a survival signal. However, two
attractive candidates are PAK and NFB, both of which have been
demonstrated to participate in Cdc42-mediated signaling events and to
contribute to antiapoptotic responses (5, 6, 9). Future work will be
directed toward delineating the Cdc42 signaling pathways operating in
Fas-induced responses and to establish whether any connection exists
between Cdc42-mediated survival responses and the ability of
Cdc42 to cause the malignant transformation of cells.
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FOOTNOTES |
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* This work was supported by Grants GM61762 and GM47458 from the National Institutes of Health.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.: 607-253-3888; Fax: 607-253-3659; E-mail: rac1@cornell.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M009838200
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ABBREVIATIONS |
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The abbreviations used are: PAK, p21-activated kinase; FasL, Fas ligand; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HA, hemagglutinin; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; PBD, p21-binding domain from PAK.
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