From the Cancer Research Center and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118
Received for publication, October 4, 2002, and in revised form, November 27, 2002
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ABSTRACT |
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Bcl-2 protects cells against Ras-mediated
apoptosis; this protection coincides with its binding to Ras.
However, the protection mechanism has remained enigmatic. Here,
we demonstrate that, upon apoptotic stimulation, newly
synthesized Bcl-2 redistributes to mitochondria, interacts there with
activated Ras, and blocks Ras-mediated apoptotic signaling. We also
show, by employing bcl-2 mutants, that the BH4 domain of
Bcl-2 binds to Ras and regulates its anti-apoptotic activity.
Experiments with a C-terminal-truncated Ras or a farnesyltransferase inhibitor demonstrate that the CAAX motif of Ras is
essential for apoptotic signaling and Bcl-2 association. The
results indicate a potential mechanism by which Bcl-2 protects cells
against Ras-mediated apoptotic signaling.
Bcl-2 suppress apoptosis elicited by various pro-death stimuli
(1-3). Bcl-2 family members include anti- and pro-apoptotic factors
that possess hydrophobic stretches of amino acids at the C termini that
allow them to bind to intracellular membranes, including the membranes
of ER1 and of mitochondria
(4-6). Bcl-2 and most of its homologs share four conserved regions:
Bcl-2 homology domains (BH1-4), through which the proteins dimerize
with each other or with other proteins (7-11). The BH1 and BH2 domains
of Bcl-2 are required for homodimerization with the pro-apoptotic Bcl-2
family member Bax. The BH3 domain of some members of the Bcl-2 family
(BH3-only proteins, such as Bad, Bik, or Bid) interacts with other
Bcl-2 proteins to initiate apoptosis (12, 13). The BH4 domain, a
heterodimerization region, is responsible for Bcl-2-directed targeting
of Raf-1 to mitochondria and the cooperation between Bcl-2 and Raf-1 in
the suppression of apoptosis (50). The loop region of Bcl-2 regulates
its protective function (14). Bcl-2 that lacks its C-terminal domain
inefficiently associates with cellular membranes and is incapable of
preventing apoptosis (15, 16). We previously demonstrated that
overexpressed Bcl-2 prevents cells that express v-Ha-ras
from undergoing apoptosis in response to protein kinase C (PKC)
down-regulation. This protection coincides with the binding of Bcl-2 to
Ras (17, 18). However, the molecular mechanism by which the interaction
of Bcl-2 and Ras regulates apoptosis has remained unknown.
The biological effects of Ras on cell growth or apoptosis depend
strongly upon the kind of stimulus, cell type, or regulatory environment (19). Human or mouse lymphocytes expressing activated ras undergo apoptosis in response to PKC down-regulation and
this apoptotic process is blocked by overexpression of bcl-2
(17, 18, 20). Ras activity is involved in Fas-regulated, multiple signaling pathways (13, 21). However, Bcl-2 partially protects lymphocytes from Fas-induced apoptosis, possibly through decreasing the
permeability of the mitochondrial membrane (22, 23). It is well known
that upon activation or mitogenic stimulation a series of
post-translational modifications, including prenylation, -AAX proteolysis, and carboxyl methylation, occur on the C
terminus of Ras. This process allows Ras to associate with membrane
compartments, especially with plasma membrane (24-26). Ras family
proteins (Ki-, Ha-, and N-Ras) have also been demonstrated to associate
with mitochondria in murine lymphokine-dependent TS1 In Fas-induced apoptosis, endogenous Ras in Jurkat cells is activated
via the ceramide signaling pathway (22, 23, 29). Jurkat cells have been
reported to be sensitive to Fas-engagement, which may be due to very
low expression of Bcl-2 (18, 30). In order to explore further the
mechanism(s) of Bcl-2 protection against Ras-mediated apoptotic
signaling, we introduced either bcl-2 or ras, as
well as both genes, into Jurkat cells, and then examined the
consequences in the setting of Fas-engagement or PKC suppression. We
demonstrate that, under apoptotic conditions, newly synthesized Bcl-2
is preferentially expressed in mitochondria, and subsequently binds to
activated, mitochondrial Ras. Such regulation was confirmed in
EBV-positive Burkitt's lymphoma Akata cells that express increased
levels of endogenous Bcl-2 or mouse lung cancer LKR cells that contain
activated ras. Also, by using various bcl-2 mutants, we identified the BH4 domain of Bcl-2 as the interaction site
with Ras, and this domain is crucial for protection against Ras-mediated apoptotic signaling. Furthermore, we established that the
integrity of the CAAX motif of Ras and its prenylation are
necessary for apoptotic activity, and regulate the ability of Ras to
associate with Bcl-2.
Cell Lines and Transfection--
Jurkat cells were obtained from
ATCC. Fas/FADD-defective Jurkat cells were generated by random
mutagenesis and obtained from Dr. J. Blenis (Harvard Medical School,
MA). The EBV-positive Burkitt's lymphoma Akata cell line and the
EBV-negative Burkitt's lymphoma Ramos cell line were obtained from Dr.
S. Ghosh (Boston University School of Medicine). The lung cancer LKR
cells derived from the lung foci of v-Ki-ras transgenic
mouse were given by Dr. T. Jack (MIT). v-Ha-ras or
ras with a C-terminal deletion was inserted into a
MSCV retroviral vector containing the neo gene
(Invitrogen). The expression of ras was examined by Northern
blot (17). bcl-2 and various bcl-2 mutants were
generously provided by Dr. T. Parslow (University of California at San
Francisco). An ER marker and a c-myc epitope
(pCMV/myc/ER) were inserted into a pShooter vector for the
determination of ER location (Invitrogen).
Preparation of Various Cellular Fractions--
Cells (50 × 106), after the treatments, were washed twice with 1×
phosphate-buffered saline and resuspended in 1 ml of 1% Triton X-114
lysis buffer (31). The cell suspensions were transferred to a 1-ml
syringe and sheared by being passed 40 times through a 25-gauge needle.
The lysates were centrifuged at 280 × g for 10 min to
precipitate nuclei, and the supernatants were collected. One-third of
the whole cell extract was saved, and the remainder was centrifuged at
16,000 × g for 30 min. The supernatant (cytosol) was
collected, and the pellet was washed in the lysis buffer containing 1%
Nonidet P-40 for 1 h on ice and centrifuged again at 100,000 × g. The supernatant (P100) was saved (50). For the ER
fraction, an OptiPrepTM kit (Invitrogen) was used. For the
mitochondrial fraction, 3 × 109 cells were
resuspended in buffer A (50 mM Tris, pH 7.5, 1 mM EGTA, 5 mM 2-mercaptoethanol, 0.2% bovine
serum albumin, 10 mM KH2PO4, pH
7.6, 0.4 M sucrose), and allowed to swell on ice for 40 min. After differential centrifugation, the resulting pellets were
resuspended in buffer B (10 mM
KH2PO4, pH 7.2, 0.3 mM mannitol, 0.1% bovine serum albumin). Mitochondria were subsequently separated on a sucrose step gradient (32).
Immunoprecipitation and Immunoblot--
After treatment with
anti-Fas Ab (1.5 µg/ml for 60 min for human cells (PanVera Corp.) or
HMG
(1-O-hexadecyl-2-O-methyl-rac-glycerol, 150 nM for 24 h, Calbiochem), cell fractions were
isolated and total protein concentrations in each fraction were
normalized. Subsequently, the fractions were adjusted to 0.4 M NaCl, 0.5% deoxycholate, and 0.05% SDS (31). Each
sample was divided into two aliquots for reciprocal immunoblotting. The
samples were immunoprecipitated with either anti-pan-Ras Ab (Oncogene
Science) or anti-human Bcl-2 Ab (BD PharMingen) for 4 h at
4 °C. Immunoprecipitates were collected with protein A-Sepharose and
separated on a 12% SDS-PAGE gel. Bcl-2 or Ras were then detected with
anti-Ras Ab or anti-Bcl-2 Ab. Anti-cytochrome c, inositol
trisphosphate receptor (IP3R) or tubulin Abs (Santa Cruz
Biotechnology Inc.) were used as immunoblot controls.
Flow Cytometry Analysis--
For cell surface staining, the
cells were incubated with anti-Fas Ab for 2 h and stained with a
second Ab conjugated with fluorescein. For DNA fragmentation assay,
after the treatments with anti-Fas Ab [1.5 µg/ml for 60 min for
human cells (PanVera Corp.) or HMG
(1-O-hexadecyl-2-O-methyl-rac-glycerol,
150 nM for 24 h, Calbiochem), cells (1 × 106/ml) were washed with phosphate-buffered saline twice,
fixed with 70% ethanol, and stained with phosphate-buffered saline
containing 10 ng/ml RNase and 50 ng/ml propidium iodide.
We previously demonstrated that oncogenic ras elicits
apoptosis once the activity of endogenous PKC is suppressed, and
that the overexpression of Bcl-2 blocks this process, possibly through its association with Ras (18). The mechanism of Bcl-2-mediated protection against Ras apoptotic signaling is unclear. Because Ras is
involved in Fas-induced apoptosis and Bcl-2 partially protects cells
from Fas-induced apoptosis (20, 22, 23, 39, 33), we tested whether
Bcl-2 interacts with Ras during Fas-induced apoptosis.
v-Ha-ras, bcl-2, or both genes were introduced
into Jurkat cells (designated PH1, Jurkat/bcl-2, or
PH1/bcl-2, respectively), and a DNA fragmentation assay was
conducted. The cells were treated with anti-Fas antibody (Ab) for 6 or
15 h to engage Fas signaling, and the percentages of the cells
with fragmented DNA were analyzed by flow cytometry (Fig.
1A). Jurkat cells that
overexpress v-Ha-ras (PH1 cells) were more susceptible to
Fas-induced apoptosis than the other three cell lines. The percentage
of PH1 cells with fragmented DNA was dramatically increased at 6 h
after addition of anti-Fas Ab (about 35%), and reached more than 40%
after 15 h, indicating that overexpressed, activated Ras
accelerates Fas-induced apoptosis. However, DNA fragmentation in
Jurkat/bcl-2 cells did not increase 6 h after
Fas-ligation and was only about 12% at 15 h. The level of DNA
fragmentation in PH1/bcl-2 cells was intermediate between the levels observed in Jurkat/bcl-2 and PH1 cells. It
appears that overexpressed Bcl-2, in the initial period (up to 15 h after Fas-ligation), protects cells (Jurkat/bcl-2) from
Fas-mediated cell death, or delays the onset of the apoptotic process.
A control experiment was also conducted using an unrelated,
isotype-matched Ab. An unrelated Ab did not cause apoptosis (data not
shown). Indirect immunofluorescence staining of Fas was also conducted to exclude the possibility that introduction of the exogenous gene(s)
affects surface expression of Fas antigen. The levels of Fas did not
change if bcl-2, ras or both were overexpressed (Fig. 1A, inset).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells (27). The function of mitochondrial Ras has not been fully
investigated yet. Furthermore, the CAAX motif of Ras may
regulate its N-terminal conformation and further affect its ability to
interact with other proteins (2, 26, 28). It is, so far, unclear
whether prenylation on the CAAX motif of Ras is required for
its apoptotic signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The expression of Bcl-2 or Ras upon apoptotic
stimulation. A, effect of overexpressed Bcl-2 on
Fas-mediated apoptosis. The cells were untreated or treated with
anti-Fas Ab for 6 or 15 h, and stained with propidium iodide. The
percentages of the cells with fragmented DNA were determined by flow cytometry. The error bars
represent S.D. of five independent experiments. Inset, cells
were stained with anti-Fas Ab and subsequently with anti-mouse IgG Ab
conjugated to fluorescein for determination of surface Fas Ag
expression. Unshaded profile, cells stained with the second
Ab alone; dark profile, cells stained with anti-Fas Ab plus
the second Ab. B, Bcl-2 or Ras expression in whole cell
extract. The cells were untreated or treated with anti-Fas Ab for 60 min, and whole cell extracts were then prepared. Samples containing
equal amounts of total proteins were separated on a 12% SDS-PAGE gel
and then probed with anti-Bcl-2 or Ras Ab. C, Bcl-2 or
Ras expression in various subcellular compartments. The mitochondrial,
ER, cytosolic, or plasma membrane fractions from
Jurkat/bcl-2 or PH1/bcl-2 cells, with or without
Fas-ligation, were prepared. Expression of Bcl-2 or Ras in these
subcellular fractions was determined by immunoblotting.
D, relative purity of subcellular fractions, with or
without Fas-ligation, was determined with anti-cytochrome c,
-IP3R, -tubulin, or -CD4 Ab. E, subcellular
localization of Bcl-2 in PKC/Ras-mediated apoptosis. After exposure of
cells to HMG (150 nM) for 24 h, the mitochondrial or
ER fractions were prepared. Expression of Bcl-2 was examined by
immunoblotting with anti-Bcl-2 Ab (left panel). Relative
purity of subcellular fractions was determined with the corresponding
antibodies (right panels).
We then examined whether Fas-engagement alters Bcl-2 or Ras protein expression using immunoblotting analysis. Jurkat or PH1 cells expressed very low amounts of Bcl-2. The levels of Bcl-2 in a whole cell extract from untreated Jurkat/bcl-2 and PH1/bcl-2 cells were higher (about 6.6-fold than Jurkat or PH1 cells), and Fas-ligation did not cause further induction of the protein (Fig. 1B, upper panels). A Ras immunoblot showed a similar result, in which the ras transfectants express 5-fold higher levels of the protein compared with the parental cell lines (Fig. 1B, lower panels). Again, Fas-ligation did not alter protein expression. Because the parental cells express a very low amount of Bcl-2 or Ras, we used Jurkat/bcl-2 and PH1/bcl-2 cells to examine whether Fas-ligation alters the subcellular distribution of Bcl-2 and Ras. The subcellular fractions from these two cell lines, with or without Fas-ligation, were prepared for immunoblotting (Fig. 1C). Bcl-2 was detectable in the mitochondrial fraction of untreated cells, and the protein level increased 4-6-fold in response to Fas-engagement. Significant Bcl-2 was seen in the ER fraction under normal growth conditions. After Fas-ligation, however, the level of Bcl-2 in ER was dramatically reduced (about 3-4-fold). Bcl-2 was not detected in the cytosol fractions from these cells, as expected. The expression of Ras in the subcellular fractions was also examined by immunoblot. A basal level of endogenous Ras protein was detected in various subcellular membrane fractions from Jurkat/bcl-2 cells under normal growth conditions, and Fas-ligation did not alter the protein expression. In contrast, moderate and high levels of Ras protein were detected in the mitochondrial and plasma membrane fractions from untreated PH1/bcl-2 cells, respectively, and the expression did not change upon Fas-engagement. The data suggest that apoptotic stimulation by Fas-ligation alters the expression of Bcl-2 in the subcellular compartments, but not of Ras. The relative purity of the subcellular fractions from the cells, with or without Fas-engagement, was confirmed by immunoblotting for the expression of cytochrome c (a mitochondrial marker), inositol trisphosphate receptor (IP3R, an endoplasmic reticulum marker), tubulin (a cytosol marker), and CD4 (a plasma membrane marker) respectively (Fig. 1D). After the apoptotic stimulation, equal amounts of the proteins were detected in their corresponding subcellular fractions in comparison to the untreated controls. Bcl-2 expression in various subcellular fractions was also examined in different bcl-2 or bcl-2/ras clones, and similar results were obtained, which excludes the possibility of clonal variation (data not shown).
We next examined whether changes of Bcl-2 expression levels in different subcellular fractions occur in PKC/Ras-mediated apoptosis. Jurkat/bcl-2 and PH1/bcl-2 cells were exposed to HMG (150 nM, a PKC inhibitor) for 24 h, and then the mitochondrial and ER fractions were prepared for immunoblotting (Fig. 1E, left panel). In response to PKC inhibition, the level of Bcl-2 in the ER fraction from PH1/bcl-2 cells was dramatically reduced, which coincided with increased protein in mitochondria. Interestingly, because PKC suppression is not a death signal to Jurkat/bcl-2 cells, the levels of the protein in the subcellular fractions from the cells did not change, which suggests that the altered expression of Bcl-2 in mitochondria of PH1/bcl-2 cells after PKC suppression is related to apoptosis. The relative purity of the subcellular fractions from the cells, with or without HMG treatment, was confirmed by the immunoblotting (Fig. 1E, right panels).
It is known that Bcl-2 is induced in some Epstein-Barr virus
(EBV)-positive Burkitt's lymphoma cells (34). Akata (a EBV-positive) and Ramos (a EBV-negative) Burkitt's cell lines were used to test whether endogenous Bcl-2 could provide the same protection. We also
employed mouse LA4 cells (a mouse lung epithelial-like cell line) and
LKR (a mouse lung cancer cell line derived from the lung foci of
v-Ki-ras transgenic mouse). The expression of Bcl-2 or Ras
in these cells was examined by immunoblotting (Fig.
2A, upper panels).
Increased amounts of Bcl-2 or Ras were detected in Akata or LKR cells,
respectively. The amount of total proteins in each sample was monitored
by re-probing the blots with anti-actin Ab (Fig. 2A,
lower panels). We then introduced v-Ha-ras or
bcl-2 into Akata (Akata/ras) or LKR
(LKR/bcl-2) cells to examine their susceptibility to
PKC/Ras-mediated apoptosis, using a DNA fragmentation assay. After
treating the cells with HMG, the percentages of the cells with
fragmented DNA were analyzed by flow cytometry (Fig. 2B).
The inhibitor did not induce apoptosis in Akata cells, which is
consistent with our previous observation in which normal cells (without
oncogenic ras) do not die in response to PKC suppression (17, 18). However, the introduction of v-ras did not render the cells (Akata/ras) susceptible to HMG-mediated cell
killing, indicating that overexpression of endogenous Bcl-2 blocks the apoptotic process. In comparison, about 40% of mouse lung cancer LKR
cells had fragmented DNA after HMG treatment. Overexpressed bcl-2 interfered with this apoptotic process in
LKR/bcl-2 cells. The expression of Bcl-2 in different
subcellular compartments of the cells was also examined (Fig.
2C, left panels). The ER and mitochondrial
fractions from the cells with or without HMG treatment were prepared
and subsequently immunoblotted with an anti-Bcl-2 Ab. A high level of
Bcl-2 was detected in the ER fraction from untreated Akata,
Akata/ras, and LKR/bcl-2 cells, and the protein
was moderately expressed in the ER fraction from LKR cells. Bcl-2 in
the ER was significantly reduced after HMG treatment in
Akata/ras, LKR/bcl-2, and LKR cells, but not in
Akata cells. In mitochondria, a very low amount of the protein was
detected in the untreated cells. However, the protein level in those
cells (except Akata cells) was dramatically increased in response to HMG treatment. The data, again, indicate that endogenous Bcl-2 redistributes and protects the cells against PKC/Ras-mediated apoptosis. The relative purity of the subcellular fractions from the
cells, with or without Fas-engagement, was confirmed by immunoblotting (Fig. 2C, right panels).
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We then tried to determine whether the altered expression of Bcl-2 in
the subcellular fractions during apoptosis is due to the translocation
or redistribution of the protein. After Fas-ligation, immunoblotting of
ER or mitochondrial Bcl-2 in PH1/bcl-2 cells, with or
without cycloheximide (a protein synthesis inhibitor) treatment, was
performed (Fig. 3A). Bcl-2 was
visualized in the ER fraction under normal growth conditions, and only
a small amount of the protein was detected in mitochondria. After
Fas-ligation, the level of Bcl-2 in ER was dramatically reduced, and,
in contrast, the protein expression in mitochondria was augmented in
the cells. After blocking protein synthesis by cycloheximide, the
amount of ER Bcl-2, with or without Fas-engagement, was reduced to more than half in comparison to the control. In the mitochondrial fraction, in the presence of cycloheximide, the protein was decreased to an
almost undetectable level. The data suggest that the increased amount
of mitochondrial Bcl-2 induced by Fas-ligation (seen in Fig.
1C) is from the newly synthesized protein, but not from
pre-existing Bcl-2 via translocation. To confirm this further,
35S pulse-chasing analysis was conducted (Fig.
3B). After labeling PH1/bcl-2 cells with
[35S]methionine, the ER and mitochondrial fractions were
prepared. Each sample was divided into two portions for 35S
pulse-chasing or co-immunoprecipitation and immunoblotting. Two hours
after terminating the pulse, 35S-labeled Bcl-2 in ER, with
or without Fas Ab treatment, was reduced (about 0.7-fold reduction)
(Fig. 3B, upper panels). In the mitochondrial fraction, a similar decay pattern of labeled Bcl-2 was also observed. In a co-immunoprecipitation and immunoblotting experiment, Bcl-2 expression in the ER fraction was dramatically reduced after
Fas-ligation, and the mitochondrial Bcl-2 expression was increased
(Fig. 3B, lower panels). Therefore, the results
provide the same conclusion that newly synthesized Bcl-2, upon
Fas-engagement, is preferentially expressed in mitochondria.
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We previously demonstrated that, in PH1/bcl-2 cells, Bcl-2
bound to Ras in response to PKC down-regulation (18). Here, we tried to
confirm further this association by using another apoptotic setting
(Fas-engagement) and to determine the location of the interaction. The
reciprocal co-immunoprecipitation of Bcl-2 and Ras was performed (Fig.
4). There was no co-precipitation of
these two molecules in the whole cell lysate from untreated Jurkat, Jurkat/bcl-2, PH1, or PH1/bcl-2 cells (Fig.
4A, first row). After Fas-ligation, an anti-Bcl-2
immunoblot visualized Bcl-2 associated with the anti-Ras
co-immunoprecipitate from PH1/bcl-2 and
Jurkat/bcl-2 cells. The association was not seen in either
PH1 or Jurkat cells, which is probably due to the fact that Jurkat
cells express very low amounts of Bcl-2 and Ras. The similar result was
obtained from the co-immunoprecipitation and immunoblotting of the
mitochondrial fraction, in which a high level of Bcl-2 was recovered in
the anti-Ras co-immunoprecipitate from PH1/bcl-2 cells after
Fas-ligation, and a lower level of the protein was detected in
Jurkat/bcl-2 cells. The interaction was not detected in the
ER fractions from any of the four cell lines after Fas-engagement. In a
reciprocal experiment, wherein the initial immunoprecipitation was
carried out with anti-Bcl-2 Ab, an anti-Ras immunoblot detected an
increased amount of Ras in the co-immunoprecipitate from the whole cell extract or mitochondrial fraction of PH1/bcl-2 cells, and a
detectable level of co-bound Ras in the same subcellular fractions of
Jurkat/bcl-2 cells, in response to Fas-ligation (Fig.
4A, third row). Again, Ras did not
co-immunoprecipitate with the anti-Bcl-2 Ab in ER fractions from any of
the four cell lines in response to Fas-engagement. The presence of Ras
(Fig. 4A, second row) or Bcl-2 (Fig.
4A, fourth row) in the immunoprecipitates from
these subcellular fractions was also confirmed by re-probing the
corresponding blots with either the anti-Ras Ab or the anti-Bcl-2 Ab.
As a control, preimmune serum was used for immunoprecipitation in whole
cell extracts from the cells, followed by immunoblotting with
anti-Bcl-2 or anti-Ras Ab to eliminate the possibility of nonspecific
binding of these two proteins (Fig. 4B). There was no
co-immunoprecipitation of either Bcl-2 or Ras with preimmune serum. The
same results were obtained from the mitochondrial fraction (data not
shown). The co-immunoprecipitation and immunoblotting of Bcl-2 and Ras, in response to PKC suppression, was also performed in Akata,
Akata/ras, LKR, and LKR/bcl-2 cells (Fig.
4C, upper panels). The strong interaction between
Bcl-2 and Ras, after HMG treatment, was detected in
Akata/ras or LKR/bcl-2 cells. A low level of
Bcl-2 was precipitated by the anti-Ras Ab in LKR cells. The association
was not detected in Akata cells in response to the treatment (because
PKC inhibition is not a death signal to the cells). The existence of
Ras in the co-precipitates was confirmed by re-probing the same blot
with the anti-Ras Ab (Fig. 4C, lower panels).
Overall, the data suggest that the physical association of Bcl-2 with
Ras occurs only under apoptotic conditions and in mitochondria.
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Cytochrome c release indicates an increase in mitochondrial
permeability, and Bcl-2 regulates the permeability transition to block
apoptosis (35-37). We tested if overexpressing ras,
bcl-2, or both genes affects mitochondrial cytochrome
c release to cytosol after Fas-engagement (Fig.
5A). Cytoplasmic fractions
from PH1, PH1/bcl-2, Jurkat and Jurkat/bcl-2
cells at various times after Fas-engagement were prepared for
immunoblot analysis. Cytochrome c was detected in the
cytosol fraction from PH1 cells 6 h after Fas-ligation. Release of
the protein was persistent during the apoptotic process. In
Jurkat/bcl-2 cells, the protein was undetectable in the
cytosol 9 h after addition of anti-Fas Ab, and cytosolic cytochrome c did appear in PH1/bcl-2 cells. The
pattern of cytochrome c release in Jurkat cells was similar
to PH1/bcl-2 cells. The data suggest that the anti-apoptotic
effects of bcl-2 and the pro-apoptotic effect of
ras both contribute to the degree of mitochondrial damage
resulting from Fas-induced apoptotic signaling. The relative purity of
the cytosolic fractions from the cells, with or without Fas-engagement,
was confirmed by immunoblotting (data not shown).
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The formation of the death-inducing signaling complex (DISC) via recruitment of FADD and caspase 8 is the initial event in Fas signaling (38). To confirm that the redistribution of Bcl-2 and its interaction with Ras indeed depend upon an intact DISC, v-Ha-ras and bcl-2 were co-introduced into Fas or FADD mutant Jurkat cells (Fasm or FADDm cells). The defect in the formation of DISC (which is due to Fas or FADD mutation) impaired the redistribution of Bcl-2 mediated by Fas-ligation (Fig. 5B, left panels). Ras expression in the mutant cells did not change and was the same as in the non-mutant cells (PH1/bcl-2), as expected. The relative purity of the subcellular fractions from the cells, with or without Fas-engagement, was confirmed by immunoblotting for cytochrome c or IP3R, respectively (Fig. 5B, right panels). Reciprocal co-immunoprecipitation and immunoblotting experiments, after Fas-ligation, were also performed with whole cell extracts from the mutant cells as well as PH1/bcl-2 cells (as a positive control) (Fig. 5C, upper panels). Bcl-2 did not co-precipitate with Ras in the mutant cells during the Fas-mediated apoptotic process, indicating that the interaction requires the intact, apoptotic signaling. The existence of Ras in the co-precipitates was confirmed by re-probing the same blots with the anti-Ras Ab (Fig. 5C, lower panels).
Next, we determined the region of Bcl-2 that is responsible for the
interaction. bcl-2 mutant genes (encoding Bcl-2 proteins that contain deletions at BH4 (1), BH3 (
2), BH1 (
3), and BH2 (
4) regions) were introduced into PH1 cells, and then the expression of the mutant proteins was examined by immunoblotting (Fig.
6A). Under normal growth
conditions, Bcl-2 mutant proteins were not expressed in mitochondria
(data not shown). After Fas-ligation, all Bcl-2 mutant proteins smaller
than wt-Bcl-2 were present in mitochondria. The ability of Bcl-2
mutants to bind to Ras after Fas-engagement was examined by
co-immunoprecipitation and immunoblotting (Fig. 6B,
upper panels). Under normal growth conditions, there was no
interaction of Bcl-2 (wild-type or mutants) with Ras in mitochondria.
After Fas-ligation, wt-,
2-,
3-, and
4-Bcl-2 proteins
co-immunoprecipitated with Ras in this subcellular compartment. However, the
1-Bcl-2 protein did not interact with Ras under the
same conditions. A similar result was obtained from the reciprocal experiment (data not shown). The existence of Ras protein in the immunoprecipitates was determined by re-probing the same blots with the
anti-Ras Ab (Fig. 6B, lower panels). To confirm
the hypothesis that the BH4 domain of Bcl-2 is crucial for its
interaction with Ras, co-immunoprecipitation and immunoblotting was
conducted under PKC/Ras-mediated apoptotic conditions instead. The
association of Ras with wt- or mutant-Bcl-2s in mitochondria was not
seen in the untreated cells (Fig. 6C, upper
panels). However, after HMG treatment, wt-Bcl-2 and smaller
2
were visualized in the Ras co-immunoprecipitate. Once again, the
-1
mutant protein did not co-immunoprecipitate with Ras in
PKC/Ras-mediated apoptosis. The presence of Ras protein in the
immunoprecipitates was also examined by re-probing the same blots with
the anti-Ras Ab (Fig. 6C, lower panels). These
data confirm that the BH4 domain of Bcl-2 is the binding region for its
interaction with Ras. However, Bcl-2 that lacks the BH4 domain is still
capable of redistributing to mitochondria upon induction of the death
pathway.
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It is known that the anchor CAAX domain of Ras regulates its signaling
in response to mitogenic stimulation (24-26). A C-terminal deleted
Ha-ras (C-ras) was introduced into
Jurkat/bcl-2 cells. The truncated Ras was detected in whole
cell extract from Jurkat/bcl-2/
C-ras cells,
with faster mobility on the gel (Fig.
7A). The expression of the
mutant Ras in mitochondria was also examined, and the mutant protein
could not be seen in this subcellular membrane fraction, with or
without Fas-ligation. To test whether the deletion of the C terminus of
Ras affects its interaction with Bcl-2 during apoptosis, whole cell
extracts from untreated or Fas Ab-treated Jurkat/bcl-2/
C-ras cells were prepared for
co-immunoprecipitation and immunoblotting. The C-terminal-deleted Ras
did not bind to Bcl-2 after Fas-ligation (Fig. 7B,
left panels). We then tested if the inhibition of the
prenylation ablates the interaction of Ras with Bcl-2, using farnesyl
transferase inhibitor (FTI). PH1/bcl-2 cells were treated
with FTI (100 nM) for 12 h prior to Fas-ligation, in
order to block continuous farnesylation of activated Ras. Subsequently, the co-immunoprecipitation and immunoblotting was conducted in the
whole cell extracts. The association of Ras with Bcl-2 in the cells was
blocked by FTI (Fig. 7B, right panels). The
existence of the mutant Ras or Ras was confirmed by re-probing the same blots with the anti-Ras Ab (data not shown). To exclude the possibility that FTI may cause a conformational change in Bcl-2 or in Ras and
generate a form that can not be recognized by the corresponding Abs,
co-immunoprecipitation and blotting with the same Ab was performed in
PH1/bcl-2 cells, treated or untreated with FTI prior to
Fas-ligation (Fig. 7C). FTI did not alter the ability of the proteins to be immunoprecipitated. Overall, the data suggest that the
prenylation of Ras is required for its interaction with Bcl-2 during
the apoptotic process.
|
The ability of the bcl-2 mutants to suppress Fas-induced
apoptosis was examined with a DNA fragmentation assay (Fig.
7D). After 9 h of Fas-engagement, the -1 mutant of
Bcl-2 did not protect PH1/
1 cells from apoptosis. The
percentage of DNA fragmentation in the
1 mutant cells was
comparable to that in PH1 cells, which express Ha-ras alone.
In contrast, the BH1 (
3), BH3 (
4), and BH2 (
5) deletion of
Bcl-2 provided similar resistance to apoptosis as that provided by
wt-Bcl-2, which is in good agreement with the observation that the
pro-apoptotic factor Bax is not directly involved in Fas-induced
apoptosis (39, 40). The protection provided by Bcl-2 mutants was also
examined in Jurkat cells, and similar results were obtained (data not
shown). We also tested whether Ras without prenylation could still
transmit apoptotic signals. After addition of FTI to suppress
continuous prenylation, the percentage of PH1 cells with fragmented
DNA, in response to Fas-ligation, was dramatically reduced (about
3-fold). The introduction of
C-ras did not increase the
susceptibility of Jurkat or Jurkat/bcl-2 cells to
Fas-induced apoptosis. We conclude that Bcl-2 may block Ras apoptotic
signaling pathway in Fas-activated cells via its BH4 domain. Similar to
its role in mitogenic stimulation, Ras must be prenylated to transmit
its apoptotic signal.
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DISCUSSION |
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Jurkat cells undergo apoptosis in response to Fas-ligation and overexpressed Bcl-2 partially protects the cells from this apoptotic process (20, 22, 23, 29, 33). Ras activity is involved in the regulation of Fas-induced apoptosis (20, 29). We previously demonstrated that overexpressed Bcl-2 in Jurkat cells blocks PKC/Ras-mediated apoptosis, possibly through its association with Ras (17, 18). Experiments reported provide further insight into the mechanism by which Bcl-2 interferes with apoptotic signaling elicited by activated ras. The results showed that newly synthesized Bcl-2, upon Fas-ligation or PKC suppression, preferentially redistributes in the mitochondrial compartment, interacts there with Ras, and blocks Ras-mediated apoptotic signaling. In contrast, activated Ras potentiates the apoptotic process. When both Bcl-2 and Ras are overexpressed simultaneously, apoptosis once again becomes a balanced process. bcl-2 mutants that encode the protein products lacking different BH domains demonstrate that Bcl-2 binds to activated, mitochondrial Ras through its BH4 domain, and this region is also required for its suppression of Ras-mediated apoptotic signaling. However, the deletions of the homo- or heterodimerization domains of Bcl-2, including BH4, have no effect on its redistribution mediated by either Fas-engagement or PKC inhibition. Experiments with C-terminal-truncated Ras or FTI show that deletion of the prenylation motif or inhibition of prenylation abrogate not only the apoptotic activity of Ras, but also its association with Bcl-2.
The reciprocal roles of Fas and Bcl-2 family proteins have been studied in cell lines, as well as in transgenic and mutant mice (2, 9, 22, 41). Studies suggest that Ras activity is elicited via ceramide signaling in the multiple apoptotic pathways mediated by Fas, and that overexpressed ras accelerates the Fas-induced death process (17, 20, 29). Conversely, as a negative regulator of apoptosis, Bcl-2 blocks cell death induced by a wide variety of effectors, and partially suppresses Fas-induced cell death (2, 22, 23, 41). In order to protect cells from apoptosis, Bcl-2 interacts with various factors and functions in several subcellular locations, particularly in mitochondria (42, 43). It is possible that, through binding to Ras, Bcl-2 neutralizes the apoptotic signaling mediated by Ras, but not other pathways, during the Fas-mediated apoptotic process. Thus, although Ras activity is involved in only one of the multiple Fas-induced death pathways, the interaction of these two proteins may affect the threshold of the sensitivity of the cells to Fas stimulation.
It has been suggested that apoptotic signaling suppresses pro-survival
mechanism(s) to ensure the execution of the cell death program (44,
45). For example, cross-linking of Fas antigen inhibits some of the
TcR/CD3-mediated signals, as well as the activation of PKC and
PKC
(44, 45). In LKR cells, although endogenous Bcl-2 redistributes
to mitochondria, there is little protection against Ras-mediated
apoptosis. Furthermore, in PH1 cells, endogenous Bcl-2 does not
co-immunoprecipitate with Ras during Fas-induced apoptosis, and further
protects these cells. Because Bcl-2 requires modification (such as
phosphorylation) to function (18, 46), it is possible that Ras-mediated
apoptotic signaling activates a phosphatase that regulates Bcl-2
phosphorylation status, and further inhibits the anti-apoptotic
activity of Bcl-2.
The C terminus of Bcl-2 contains a stretch of hydrophobic amino acids that anchors the protein in the lipid bilayer of the membrane compartments (4-6). Bcl-2 that lacks the C-terminal domain loses the ability to associate with cellular membranes and to suppress cell death (47, 48). Some Bcl-2 family members, such as Bcl-XL or Bax, undergo subcellular redistribution in response to various apoptotic stimuli (38). Here, we demonstrate that overexpressed Bcl-2 is located in subcellular membrane compartments, mainly in the ER. Upon Fas-ligation or PKC suppression, Bcl-2 or its BH domain deletion mutants preferentially express in mitochondria. The results suggest that subcellular redistribution of Bcl-2 is an early event during the apoptotic process. This process only requires the C-terminal anchor region of Bcl-2, not the protein/protein interaction domains. Given the fact that the mitochondrial membrane has been identified as one of the Bcl-2 targets and that increases in mitochondrial free radicals have been observed in apoptosis mediated by oncogenic Ras (10, 42, 43, 49), it is reasonable to propose that apoptotic signals redirect Bcl-2 to damaged sites. In this case, Bcl-2 mediated by Fas signaling or PKC inhibition, is preferentially expressed mitochondria, and subsequently reduces or blocks an increase in mitochondrial permeability, thereby preventing the Ras-triggered release of cytochrome c or free radicals.
It is well established that subcellular targeting of Ras protein requires multiple steps of post-translational modification at its C-terminal CAAX motif in response to growth or differentiation stimulation, such as prenylation (24-26). Upon mitogenic or growth stimulation, prenylated Ras translocates from cytosol to subcellular membrane compartments, mainly to plasma membrane. However, it is not clear whether Ras requires this modification for its apoptotic activity. We show here that inhibition of the prenylation of activated Ras interferes with its binding to Bcl-2, as well as its apoptotic activity. We also demonstrate that overexpressed, activated Ras proteins localize normally in various intracellular membrane compartments and do not undergo redistribution or translocation upon apoptotic stimulation. Therefore, we speculate that activated Ras in different locations may have different roles in biological processes, depending upon the nature of the stimulation or the context of the signaling transmitters. Instead of its traditionally known effectors (such as Raf-1) that transmit growth or differentiation signals from cytoplasmic membrane to nucleus, after apoptotic stimulation, mitochondrial Ras may interact with a different set of signal transducers to execute the death program.
Many studies have suggested the involvement of multiple pathways in Fas-mediated apoptosis (17, 20, 29). In response to Fas-ligation, FADD binds and recruits caspase 8 to form the receptor complex, which ultimately results in the activation of Fas apoptotic signaling (50-53). Experiments from Fas or FADD mutant cells or from knockout mice show the importance of these molecules in the induction of cell death (54-56). Studies have also demonstrated that upon Fas-engagement, molecules such as DAXX bind to Fas and mediate FADD-independent apoptotic signaling (57). The results of experiments with Fas- and FADD-deficient mutant cell lines indicate that intact Fas and FADD are required for the interaction between Ras and Bcl-2, and that Ras is a downstream effector of Fas and FADD during Fas-induced apoptosis. However, these results do not rule out the existence of parallel signaling pathways that may originate from Fas-ligation, but that are not sufficient to elicit the interaction or are independent of Ras activity.
During activation-induced T lymphocyte death, a correlation has been observed between the expression of bcl-2 and fas genes. Upon priming, the expression of bcl-2 in a T cell progressively falls and the expression of Fas reciprocally increases (58, 59). Our study demonstrates that oncogenic Ras accelerates Fas-mediated apoptosis in Jurkat cells, whereas overexpressed Bcl-2 confers resistance to the death process. Neither the overexpression of Bcl-2 nor oncogenic Ras changes cell surface levels of Fas antigen expression; therefore, facilitation of Fas-induced apoptosis by activated Ras (and resistance to the death process by forced Bcl-2 expression) is likely to operate at an essential step in the Fas-mediated signal transduction pathway that is distal to Fas ligand expression.
Our study provides evidence for a model of the dynamic
interrelationship between Bcl-2 and Ras molecules: in response to
mitogenic stimuli, activated Ras mainly located at the plasma membrane
transmits signals to downstream effectors and mobilizes the growth
program. Upon death stimulation, however, apoptotic factors elicit
mitochondrial Ras activity, a process which Bcl-2 blocks. We define
specific domains of Bcl-2 and Ras that mediate this interaction. In
particular, the BH4 domain of Bcl-2 controls both its association with
Ras and protection against Ras-induced apoptotic signals. We speculate therefore that naturally occurring BH4 domain mutants of Bcl-2 may be
linked to certain defects in T lymphocyte cell death. An increased
understanding of the pathways regulated individually or collectively by
these mediators may guide approaches to Fas- or Ras-based tumor immunotherapy.
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ACKNOWLEDGEMENTS |
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We thank T. Parslow (University of San Francisco), J. Blenis (Harvard University Medical School), and J. Yuan (Harvard University Medical School) for the generous gifts of various bcl-2 mutants, Jurkat mutant cell lines, and expression vectors.
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FOOTNOTES |
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* This work was supported by Grant RPG-00-111-01-MGO from the American Cancer Society (to C. Y. C.).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.
These authors contributed equally to the current work.
§ To whom correspondence should be addressed: Cancer Research Center, Boston University School of Medicine, 80 E. Concord St., Rm. R908, Boston, MA 02118. Tel.: 617-638-4128; Fax: 617-638-4176; E-mail: yanyan@bu.edu.
Published, JBC Papers in Press, December 10, 2002, DOI 10.1074/jbc.M210202200
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; PKC, protein kinase C; Ab, antibody; wt, wild type; FTI, farnesyl transferase inhibitor; CAAX, motif where A is an aliphatic residue and X is any amino acid; HMG, 1-O-hexadecyl-2-O-methyl-rac-glycerol; DISC, death-inducing signaling complex; BH, Bcl-2 homology; FADD, Fas-associated death domain.
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