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Molecular and Cellular Biology, August 1999, p. 5308-5315, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
B-Raf Inhibits Programmed Cell Death Downstream of Cytochrome
c Release from Mitochondria by Activating the MEK/Erk
Pathway
Peter
Erhardt,
Erin J.
Schremser, and
Geoffrey M.
Cooper*
Department of Biology, Boston University,
Boston, Massachusetts 02215
Received 11 November 1998/Returned for modification 19 January
1999/Accepted 4 May 1999
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ABSTRACT |
Growth factor-dependent kinases, such as phosphatidylinositol
3-kinase (PI 3-kinase) and Raf kinases, have been implicated in the
suppression of apoptosis. We have recently established Rat-1
fibroblast cell lines overexpressing B-Raf, leading to
activation of the MEK/Erk mitogen-activated protein kinase pathway.
Overexpression of B-Raf confers resistance to apoptosis
induced by growth factor withdrawal or PI 3-kinase inhibition. This is
accompanied by constitutive activation of Erk without effects on the PI
3-kinase/Akt pathway. The activity of MEK is essential for
cell survival mediated by B-Raf overexpression, since either treatment
with the specific MEK inhibitor PD98059 or expression of
a dominant inhibitory MEK mutant blocks the antiapoptotic
activity of B-Raf. Activation of MEK is not only necessary but also
sufficient for cell survival because overexpression of
constitutively activated MEK, Ras, or Raf-1, like B-Raf, prevents
apoptosis after growth factor deprivation. Overexpression of B-Raf did not interfere with the release of cytochrome c from mitochondria after growth factor
deprivation. However, the addition of cytochrome c to
cytosols of cells overexpressing B-Raf failed to induce caspase
activation. It thus appears that the B-Raf/MEK/Erk pathway confers
protection against apoptosis at the level of cytosolic
caspase activation, downstream of the release of cytochrome
c from mitochondria.
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INTRODUCTION |
Many types of mammalian cells are
dependent upon growth factors for survival. In a variety of cell types,
growth factors prevent apoptosis by stimulation of
phosphatidylinositol 3-kinase (PI 3-kinase) (52), which
leads to activation of the protein kinase Akt (14, 25-26, 28,
44). Akt directly links growth factor signaling to the central
pathways controlling programmed cell death by phosphorylating the Bcl-2
family member BAD (10-11). In its nonphosphorylated state,
BAD translocates from the cytosol into mitochondria and promotes
apoptotic cell death by inhibiting Bcl-2 or Bcl-xL
through protein-protein interactions (51, 55). Phosphorylation by Akt results in the binding of BAD to cytosolic 14-3-3t, allowing Bcl-2 or Bcl-xL to function as inhibitors
of apoptosis (10-11, 55). Phosphorylation of
glycogen synthase kinase-3 (GSK-3) by Akt has also been reported to
contribute to cell survival (36), but the downstream targets
of GSK-3 that regulate apoptosis remain to be determined.
Signals that induce apoptosis culminate in the activation of
caspases, which are the ultimate effectors of programmed cell death.
Activation of the key apoptotic protease caspase-3 during cell
death induced by a variety of different stimuli, including growth
factor deprivation, is preceded by the release of cytochrome c from mitochondria to the cytosol (32).
Cytochrome c then initiates the formation of a ternary
complex consisting of cytochrome c, the apoptosis
protease-activating factor Apaf-1, and another protease called
caspase-9 (31). In this complex, caspase-9 becomes
proteolytically activated and eventually activates caspase-3, the major
effector protease in apoptosis (19). The release of
cytochrome c from mitochondria, as well as activation of
caspase-3, is blocked by Bcl-2 or Bcl-xL (3, 7, 16,
27, 50).
As with the PI 3-kinase/Akt pathway, members of the Raf family also
function as elements of signaling pathways that are thought to be
involved in the regulation of programmed cell death (37, 46,
48), and may provide an alternative growth
factor-dependent mechanism of cell survival. In
mammals, three members of the Raf family of
protein-serine/threonine kinases have been identified (Raf-1, A-Raf,
and B-Raf), whose activation is linked to receptor protein-tyrosine
kinases by Ras (24). Activation of Raf then leads to
activation of a kinase cascade consisting of MEK and Erk1/2. The Erk
kinases phosphorylate a diverse group of substrate proteins, including
the protein kinase Rsk (pp90 S6 kinase) and transcription factors
(41).
Most studies suggest a functional redundancy among the Raf family,
since all Raf kinases activate Erk through MEK (39). In Raf
knockouts, however, only mice lacking B-Raf, and not mice lacking Raf-1
or A-Raf, showed disturbances in cell survival (40, 47),
raising the possibility that B-Raf may possess specific functions in
cell death regulation. More recently, a specific Rap-1-dependent
activation of B-Raf has been described which is not dependent on Ras
(54). In addition, Raf-1 and A-Raf require tyrosine
phosphorylation for maximal activation, whereas the appropriate tyrosine residues are missing from B-Raf (18, 33). These
data are consistent with the possibility that B-Raf is activated by specific upstream regulators, leading to a unique role for B-Raf in
signaling cell survival.
Two alternative mechanisms have been proposed to account for the
antiapoptotic activity of Raf-1. In some studies, activation of
Erk was found to play a critical role in the prevention of cell death
(48). In contrast, other experiments have shown that Raf-1
targeted to mitochondria by Bcl-2 leads to cell survival without Erk
activation, probably by phosphorylating substrates other than MEK, such
as Bcl-2 family members (46). However, the mechanism of
action of B-Raf in preventing apoptosis has not been investigated.
In the present study, we have used Rat-1 fibroblast cell lines
overexpressing B-Raf to investigate the role of B-Raf in cell survival.
Overexpression of B-Raf conferred resistance to apoptosis induced by either serum deprivation or PI 3-kinase inhibition as a
result of constitutive activation of the MEK/Erk signaling pathway.
This antiapoptotic activity of B-Raf blocked caspase activation
without interfering with the release of cytochrome c from
mitochondria, indicating that B-Raf/MEK/Erk signaling can inhibit
apoptosis at the level of caspase activation, downstream of the
action of Bcl-2 family members in blocking mitochondrial cytochrome
c release.
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MATERIALS AND METHODS |
Cells.
Rat-1 cells and transfected Rat-1 cells
overexpressing B-Raf (Rat-1/B-Raf-1 and -2 cells) were as previously
described (15). Rat-1/control cells were transfected with
the B-Raf expression plasmid but after G418 selection failed to
overexpress B-Raf. Rat-1 cells overexpressing Bcl-2 (Rat-1/Bcl-2 cells)
(35) were a generous gift of J. Yuan. Cells were maintained
in Dulbecco modified Eagle medium (DMEM) supplemented with 10% calf serum.
Assays for apoptosis.
Cells were seeded at
106 cells per 100-mm culture dish (for nuclear morphology
assay) or at 2 × 106 cells per 100-mm culture dish
(for all other apoptosis assays) in DMEM containing 10% calf
serum. After 24 h of incubation, cells were deprived of serum
and/or treated with 50 µM LY294002 (inhibitor of PI 3-kinase) or 50 µM PD98059 (inhibitor of MEK) for an additional 16 to 20 h to
induce apoptosis. For flow cytometry, DNA fragmentation assay,
and caspase-3 cleavage both the adherent and floating cells were then
collected for analysis.
For nuclear morphology assay, cells were fixed with formaldehyde,
permeabilized with 0.5% Triton X-100, and stained with the DNA dye
bisbenzimide (Hoechst 33258), and the number of cells with fragmented
nuclei was then scored.
For flow cytometry, cells were fixed in methanol for at least 1 h
at
20°C, rehydrated in phosphate-buffered saline for at
least an
additional hour at 4°C, and then treated with RNase A
(50 µg/ml)
for 30 min. Propidium iodide (25 µg/ml) was then added
to the cells,
and samples were analyzed in a flow cytometer (FACScalibur;
Becton
Dickinson) by recording the propidium iodide staining in
the red
channel. The percentage of apoptotic cells was determined
by
calculating the fraction of cells with sub-G
1 DNA
content.
For DNA fragmentation assays, soluble cytoplasmic DNA was extracted and
analyzed by electrophoresis in 1.8% agarose gels containing
ethidium
bromide as described previously (
16).
For assays of caspase-3 cleavage, cytoplasmic lysates were
electrophoresed in sodium dodecyl sulfate (SDS)-12% polyacrylamide
gels and analyzed by immunoblotting with an anti-caspase-3 antibody
(Santa Cruz Biotechnology) as described earlier (
16).
Transient transfection and fluorescence microscopy.
Transient transfections were carried out as described previously
(36) with minor modifications. Briefly, 3 × 105 Rat-1 or Rat-1/B-Raf cells were plated on
poly-L-lysine-coated coverslips 24 h before
transfection. Transient transfections were then performed by the
Lipofectamine method as suggested by the manufacturer (Life
Technologies, Inc.). First, 5 µl of Lipofectamine plus 1 µg of
expression vectors (constitutively activated or dominant-negative MEK
[9]; v-raf or v-ras
[20]) and 0.1 µg of a green fluorescent protein
(GFP) expression construct (pEGFP-C1) (Clontech) were incubated with
the cells for 3 h. The medium was then replaced with DMEM
supplemented with 10% calf serum for 16 h. The cells were then
deprived of growth factors by incubation in serum-free medium for an
additional 24 h. Cells were then fixed with formaldehyde, permeabilized with 0.5% Triton X-100, and stained with the DNA dye
bisbenzimide (Hoechst 33258). Transfected cells were identified by GFP
fluorescence and scored for apoptosis by nuclear morphology.
Activation of Erk and Akt.
Cell lysates were analyzed by
SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting as
previously described (15). The membranes were incubated with
one of the following primary antibodies: anti-phospho-Erk (New England
Biolabs), anti-Akt (New England Biolabs), anti-phospho-Akt (New England
Biolabs), or anti-Erk-1 (Santa Cruz Biotechnology). After incubation
with horseradish peroxidase-conjugated secondary antibodies, the
proteins were detected by using the ECL system (Amersham Corp.).
Detection of cytosolic cytochrome c.
Isolation of
cytosolic fraction for detecting cytochrome c was performed
as described previously (6). Briefly, cells were collected
and resuspended in a buffer containing 20 mM HEPES (pH 7.5); 1.5 mM
MgCl2; 10 mM KCl; 1 mM EDTA; 1 mM EGTA; 1 mM
dithiothreitol; 0.1 mM phenylmethyl sulfonyl fluoride; 10 µg of
leupeptin, aprotinin, and pepstatin A per ml; and 250 mM sucrose. The
cells were homogenized with a Dounce homogenizer and centrifuged at
12,000 × g for 5 min at 4°C. Aliquots of the
resulting supernatant containing 10 µg of protein were used as the
soluble cytosolic fraction for immunoblot analysis with anti-cytochrome
c (Pharmingen) and anti-cytochrome oxidase subunit II
(Molecular Probes) antibodies. Approximately equal amounts of the
pellet (mitochondrial fraction) were analyzed in parallel as a positive
control for detection of mitochondrial proteins.
In vitro assay of PARP cleavage in nuclei.
In vitro assays
of poly(ADP-ribose) polymerase (PARP) cleavage in nuclei and
preparation of cytosols and nuclei for these assays were performed
essentially as described earlier (17). Briefly, 5 µg of
cytosolic extracts from untreated cells was combined with intact HeLa
cell nuclei in the presence of 1 mM ATP and an ATP regenerating system
consisting of 1 mM creatine phosphate and 1 µM creatine
phosphokinase. Activation of caspase-3 in control cytosols was induced
by the addition of 0.1 µM horse heart cytochrome c
(Sigma). Reactions were incubated for 1 h at 37°C, and the
cleavage of PARP derived from intact HeLa cell nuclei was analyzed on
immunoblots with an anti-PARP monoclonal antibody (obtained from
G. G. Poirier, Laval University, Ste-Foy, Quebec, Canada). In some
experiments, rather than inducing caspase-3 activation by cytochrome
c, active His6-tagged human caspase-3 purified
from an Escherichia coli expression system (17)
was added to the reaction mixtures.
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RESULTS |
B-Raf possesses antiapoptotic activity.
To test
whether B-Raf promotes cell survival, Rat-1 fibroblast cell lines
overexpressing a wild-type 90-kDa isoform of human B-Raf
(Rat-1/B-Raf cells) (15) were deprived of growth
factors, and apoptosis was quantified by determining the
fraction of cells with fragmented nuclei by Hoechst stain fluorescence.
After 16 h of growth factor deprivation, two independent clones
overexpressing B-Raf were resistant to cell death, whereas
approximately 10% of wild-type Rat-1 cells underwent apoptosis
(Fig. 1A). Like wild-type Rat-1 cells, a
control line of transfected Rat-1 cells without detectable B-Raf
overexpression (Rat-1/control) remained sensitive to growth factor
deprivation-induced apoptosis. We have confirmed with
additional apoptosis assays that overexpression of B-Raf blocks
other characteristic events of programmed cell death, including increase in sub-G1 DNA content with flow cytometry,
oligonucleosomal fragmentation of DNA, and caspase-3 activation (Fig.
1B and 2).
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FIG. 1.
Induction of apoptosis by growth factor
deprivation and PI 3-kinase inhibition. (A) Wild-type Rat-1 cells
(Rat-1), transfected Rat-1 cells overexpressing B-Raf (Rat-1/B-Raf-1
and -2), and transfected Rat-1 cells that fail to overexpress B-Raf
(Rat-1/control) were maintained in normal growth medium with or without
the addition of 50 µM LY294002 or were incubated in serum-free medium
for 16 h. Cells were then fixed, permeabilized, and stained with
the DNA dye bisbenzimide (Hoechst 33258), and the apoptotic
nuclei were scored on the basis of nuclear morphology. Data were
averaged from three experiments, and at least 300 cells were counted
per experiment. The error bars are standard errors of the mean. (B)
Rat-1/B-Raf-1 and Rat-1 cells were maintained and treated as described
in the legend to Fig. 1A. They were then collected and stained with
propidium iodide, and the DNA content was analyzed by flow cytometry.
The bar indicates cells with sub-G1 DNA content, a
characteristic of apoptosis. The percentage of cells with
sub-G1 DNA content is indicated in the upper right corner
of each panel. The results are representative of at least two similar
experiments.
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FIG. 2.
Induction of oligonucleosomal DNA fragmentation and
processing of caspase-3 by growth factor deprivation and PI 3-kinase
inhibition. Rat-1/B-Raf-1 and Rat-1 cells were maintained and treated
as described in the legend to Fig. 1. (A) Oligonucleosomal
fragmentation of DNA. (B) Immunoblot analysis of caspase-3. The
positions of the 32-kDa proenzyme and the 20-kDa active subunit are
indicated by arrows.
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Since the PI 3-kinase/Akt pathway is thought to be the major survival
pathway activated by serum growth factors (
52-53), we
also
treated Rat-1/B-Raf and Rat-1 cells with the PI 3-kinase
inhibitor
LY294002 in the presence of serum rather than inducing
apoptosis by serum deprivation. Consistent with the results
obtained
in growth factor-deprived cells, overexpressed B-Raf blocked
cell
death induced by PI 3-kinase inhibitors (Fig.
1 and
2). These
data
indicate that cells overexpressing B-Raf lose growth factor
dependence
for survival and that the antiapoptotic activity of
B-Raf is
not mediated by PI 3-kinase.
Activation of MEK is essential for cell survival conferred by
B-Raf.
Our previous studies have demonstrated that in Rat-1/B-Raf
cell lines B-Raf is a catalytically active kinase that activates MEK
and Erk (15). We therefore sought to determine whether MEK is a component of the B-Raf-initiated survival pathway. To this end,
Rat-1 and Rat-1/B-Raf cells were treated with the specific MEK
inhibitor PD98059, either alone or in combination with inducers of
apoptosis. Apoptosis was quantified by flow cytometry and by analysis of the proteolytic processing of caspase-3 on immunoblots.
Inhibition of MEK in the presence of serum did not result in
significant cell death in either Rat-1 or Rat-1/B-Raf cells (Fig.
3), suggesting that MEK is not required
for survival in the presence
of serum growth factors. In contrast,
serum-starved Rat-1/B-Raf
cells underwent apoptosis if MEK was
inhibited by treatment with
PD98059, although neither serum starvation
nor MEK inhibition
alone induced apoptosis in these cells (Fig.
3). Similar results
were obtained with the nuclear morphology assay by
scoring the
fragmented apoptotic nuclei in two
independent clones overexpressing
B-Raf (data not shown). Results
obtained with PD98059 were also
confirmed with 10 µM UO126 (Promega),
a structurally different
MEK inhibitor (not shown). It thus appears
that MEK is required
for cell survival mediated by overexpression of
B-Raf but not
for cell survival in the presence of growth factors.
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FIG. 3.
Induction of apoptosis by inhibiting MEK with
PD98059. Cells were maintained in normal growth medium or in serum-free
medium and treated with 50 µM LY294002 or 50 µM PD98059, as
indicated, for 16 h. (A) Fraction of apoptotic cells with
sub-G1 DNA content as determined by flow cytometry. (B)
Immunoblot analysis of caspase-3. The positions of the 32-kDa proenzyme
and the 20-kDa active subunits are indicated. The results are
representative of at least two similar experiments.
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In contrast to Rat-1/B-Raf cells, growth factor deprivation alone
effectively induced apoptosis of normal Rat-1 cells. This
effect was enhanced by treatment with PD98059, leading to a further
increase in both the number of apoptotic cells with
sub-G
1 DNA
content and the formation of active caspase-3
subunits (Fig.
3).
This additional increase in cell death may reflect a
residual
MEK activity persisting in serum-starved cells which is
eliminated
by the MEK inhibitor
treatment.
Similar results were obtained in both Rat-1 and Rat-1/B-Raf cells when
apoptosis was induced by treatment with the PI 3-kinase
inhibitor LY294002 in the presence of serum instead of by serum
deprivation (Fig.
3). This is consistent with the view that serum
growth factors promote survival primarily by activating PI 3-kinase
(
52-53) but that activation of MEK can provide an
alternative cell
survival
pathway.
To further confirm that survival signaling by B-Raf is mediated through
MEK, Rat-1 and Rat-1/B-Raf cells were transiently
transfected with a
catalytically inactive MEK mutant, which acts
as a dominant inhibitor
of MEK activity, and a construct expressing
GFP. Transfected cells were
identified by fluorescence microscopy
to detect GFP expression, and
apoptotic cells were scored by nuclear
morphology after being
stained with Hoechst dye. In control cells
transfected with an empty
vector, ca. 15% of the nuclei showed
apoptotic morphology, a
result apparently due to the Lipofectamine
treatment (Fig.
4). In Rat-1/B-Raf cells, expression of
B-Raf
prevented cell death induced by growth factor deprivation, but
B-Raf was unable to protect cells from apoptosis when growth
factor
deprivation was accompanied by expression of the dominant
inhibitory
MEK (Fig.
4). In contrast, in wild-type Rat-1 cells the
background
level of apoptosis was increased up to 40% upon
serum deprivation,
and approximately 20% more cells underwent
apoptosis when the
cells were also transfected with dominant
inhibitory MEK (Fig.
4).
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FIG. 4.
Induction of apoptosis by ectopic expression of
dominant-negative MEK. Cells were cotransfected with an expression
vector for dominant-negative MEK or an empty vector control (pBABE),
together with an expression construct for GFP. Cells were maintained in
normal growth medium or in serum-free medium for an additional 24 h. Transfected cells were then identified by fluorescence microscopy
and scored for apoptosis on the basis of nuclear morphology.
Data are presented as the percentage of GFP-positive cells with
apoptotic nuclei. Data were averaged from three experiments,
and 100 cells transfected with each vector were counted per experiment.
The error bars are standard errors of the mean. Asterisks indicate a
significant difference (P < 0.05) from controls
maintained in serum (analysis of variance test).
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Constitutively expressed active MEK is sufficient to inhibit cell
death.
Since MEK was required for the antiapoptotic
activity of B-Raf, we next examined whether activation of MEK alone was
sufficient for cell survival. Rat-1 cells were transfected with a
constitutively active MEK mutant by using the GFP cotransfection assay
to identify transfected cells. Expression of activated MEK completely
blocked apoptosis induced by serum withdrawal (Fig.
5). In addition, the expression of active
Ras and Raf-1, which also activate MEK, similarly inhibited cell death
induced by growth factor deprivation (Fig. 5). These results are
consistent with data obtained in other cell types (2, 8, 22, 30,
34, 48) and indicate that MEK activation is sufficient to prevent
apoptosis resulting from growth factor deprivation.
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FIG. 5.
Inhibition of apoptosis by expression of
activated MEK, Raf, or Ras in Rat-1 cells. Rat-1 cells were
cotransfected with the indicated expression vectors and a GFP
expression construct. Transfected cells were maintained in normal
growth medium or in serum-free medium for an additional 24 h. Data
are averaged from three experiments, as in Fig. 4. The extent of
apoptosis in pBABE-transfected cells deprived of serum was
significantly different (P < 0.05) from controls
maintained in serum, whereas apoptosis in serum-deprived cells
transfected with MEK, Raf, or Ras expression constructs did not differ
significantly from controls.
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Overexpression of B-Raf results in increased Erk activity without
effects on the PI 3-kinase pathway.
Since the
antiapoptotic activity of B-Raf was mediated by MEK, we
investigated the activity of the MEK target Erk in Rat-1 and
Rat-1/B-Raf cells in the presence or absence of serum. Activating phosphorylations of Erk (38) were assayed by immunoblotting with an anti-phospho-Erk(202-Thr/204-Tyr) antibody. Overexpression of
B-Raf in Rat-1/B-Raf cells resulted in a constitutively high Erk
activity, even if the cells were deprived of growth factors or treated
with PI 3-kinase inhibitors (Fig. 6). In
normal Rat-1 cells, however, Erk was weakly phosphorylated in the
presence of serum; this was further inhibited by growth factor
withdrawal but not by PI-3 kinase inhibitors (Fig. 6). As expected,
activation of Erk was effectively blocked by treatment with the MEK
inhibitor PD98059 in both Rat-1 and Rat-1/B-Raf cells (Fig. 6). It thus appears that overexpression of B-Raf in Rat-1/B-Raf cells results in an
elevated level of Erk activity which persists after growth factor
deprivation or inhibition of PI 3-kinase and may therefore be
responsible for the antiapoptotic effect of B-Raf.
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FIG. 6.
Overexpression of B-Raf results in increased Erk
activity. Cells were maintained in normal growth medium or in
serum-free medium and were treated with either 50 µM LY294002 or 50 µM PD98059, as indicated, for 16 h. Collected cell lysates were
analyzed by immunoblotting with antibodies to phospho-Erk1/2 or Erk1.
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To test whether PI 3-kinase is activated by B-Raf, we measured the
phosphorylation of the PI 3-kinase target Akt on Ser-473,
which is
thought to reflect Akt activation (
1). In the absence
of
growth factors or in cells treated with LY294002, this phosphorylation
of Akt was strongly diminished in both Rat-1 and Rat-1/B-Raf cells
(Fig.
7). In contrast, inhibition of MEK
did not affect Akt phosphorylation
in either cell type (Fig.
7),
indicating that the B-Raf/MEK pathway
does not have a direct effect on
PI 3-kinase/Akt signaling.
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FIG. 7.
Overexpression of B-Raf does not affect activation of
Akt. Cells were maintained in normal growth medium or in serum-free
medium and were treated with either 50 µM LY294002 or 50 µM
PD98059, as indicated, for 16 h. Collected cell lysates were
analyzed by immunoblotting with antibodies to phospho-Akt or Akt.
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The B-Raf/MEK/Erk pathway inhibits apoptosis downstream of
cytochrome c release from mitochondria.
To position
the interaction between the B-Raf survival pathway and the central
apoptotic machinery, we first sought to determine whether the
release of cytochrome c from the mitochondria was inhibited
by B-Raf overexpression in Rat-1/B-Raf cells. Cytosols prepared from
control and growth factor-deprived Rat-1 and Rat-1/B-Raf cells were
analyzed by immunoblotting to detect cytochrome c release. In parallel blots, cytochrome oxidase served as a marker of
mitochondrial contamination of the extracts. Growth factor deprivation
led to a similar increase in cytosolic cytochrome c, without
detectable release of cytochrome oxidase, both in two independent Rat-1
clones overexpressing B-Raf and in normal Rat-1 cells, as well as in transfected Rat-1 cells without B-Raf overexpression (Fig.
8A). In contrast, overexpression of
Bcl-2, as shown earlier (27, 50), prevented the release of
cytochrome c from the mitochondria. Therefore, it appeared
that overexpression of B-Raf inhibited apoptosis at a later
step than did Bcl-2, downstream of mitochondrial cytochrome
c release.
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FIG. 8.
Effect of B-Raf overexpression on cytochrome
c-dependent caspase activation. (A) Wild-type Rat-1 cells
(Rat-1), transfected Rat-1 cells overexpressing B-Raf (Rat-1/B-Raf-1
and -2) or Bcl-2 (Rat-1/Bcl-2), and transfected Rat-1 cells that fail
to overexpress B-Raf (Rat-1/control) were maintained in the presence of
serum or deprived of growth factors for 24 h. Equal amounts of
cytosolic proteins were then immunoblotted for cytochrome c
(cyt c) and cytochrome oxidase subunit II (cyt ox), along with similar
amounts of mitochondrial fraction (Mit. fr.) of Rat-1 cells maintained
in serum as a positive control. The positions of cytochrome
c and cytochrome oxidase are indicated by arrows. (B)
Cytosols from Rat-1 and Rat-1/B-Raf cells were combined with purified,
active His6-tagged human caspase-3 and intact HeLa cell
nuclei and then incubated for 1 h at 37°C. The reaction mixture
was used for immunoblot analysis to measure the cleavage of the
caspase-3 substrate PARP. Intact PARP (116 kDa) and the cleaved
fragment (85 kDa) are indicated by arrows. (C) Activation of caspase-3
was induced by the addition of cytochrome c to cytosols from
nonapoptotic Rat-1, Rat-1/B-Raf, and Rat-1/Bcl-2 cells
maintained in the presence of serum. Reaction mixtures were combined
with intact HeLa cell nuclei, and the cleavage of PARP was assayed by
immunoblot analysis. Lane 1 is a control in which cytochrome
c was added to nuclei without cytosolic extract. The results
are representative of at least three similar experiments.
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To further test this possibility, we used in vitro assays of nuclear
PARP cleavage to determine whether cytosols of cells
overexpressing
B-Raf contained inhibitors of caspase activation.
HeLa cell nuclei were
added to cytosols of Rat-1 or Rat-1/B-Raf
cells, and the activity of
caspases was assayed by analyzing the
appearance of a PARP cleavage
fragment by immunoblotting. First,
we determined the effect of
Rat-1/B-Raf and Rat-1 cytosols on
the activity of already activated
caspase-3 purified from an
E. coli expression system. Active
caspase-3 added to cytosols prepared
from either Rat-1/B-Raf or Rat-1
cells induced a similar level
of nuclear PARP cleavage (Fig.
8B),
indicating that cytosols of
Rat-1/B-Raf cells did not contain
inhibitors that were effective
against the activated
caspase.
We next tested the possibility that cytosols of Rat-1/B-Raf cells
contained inhibitors of caspase activation mediated by cytochrome
c released from mitochondria. Cytochrome
c was
added to cytosols
of either Rat-1/B-Raf cells or normal Rat-1 cells,
and activation
of caspase-3 was monitored by PARP cleavage in HeLa cell
nuclei.
The addition of cytochrome
c effectively induced
caspase-3 activation
and PARP cleavage in cytosols of Rat-1 cells but
not in cytosols
of Rat-1/B-Raf cells (Fig.
8C). This inhibition of
caspase activation
in cytosols of Rat-1/B-Raf cells contrasted with
Rat-1 cells protected
from apoptosis by overexpression of
Bcl-2. Consistent with the
action of Bcl-2 in preventing
apoptosis by blocking the release
of cytochrome
c
from mitochondria, the addition of cytochrome
c to cytosols
of Rat-1/Bcl-2 cells resulted in effective caspase-3
activation (Fig.
8C). It thus appeared that cytochrome
c-mediated
caspase
activation was blocked in cytosols of Rat1/B-Raf cells,
indicating
that the B-Raf signaling pathway confers protection
against
apoptosis at the level of caspase activation, downstream
of
cytochrome
c release from
mitochondria.
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DISCUSSION |
Growth factor-dependent kinases prevent programmed cell death by
blocking activation of the apoptotic machinery. Thus, upon overexpression of constitutively active growth factor dependent kinases, such as PI 3-kinase/Akt and Raf-1, programmed cell death is
suppressed in a variety of different cell systems (8, 14, 25-26,
28, 44, 46). In the present study, we have demonstrated that
overexpression of B-Raf, another member of the Raf kinase family, is
also a potent inhibitor of apoptosis. This protection by B-Raf
was mediated through MEK and did not involve activation of the PI
3-kinase/Akt pathway.
Consistent with recent results obtained in several different cell
types, such as hematopoietic cells, PC12 rat pheochromocytoma cells,
and fibroblasts (5, 29, 42-43, 49, 52-53), our data with
inhibitors of MEK and PI 3-kinase indicate distinct roles for B-Raf/MEK
and PI 3-kinase/Akt signaling in cell survival. Growth factors maintain
a significant steady state level of PI 3-kinase activity, providing
protection against cell death, whereas the basal activity of MEK is not
sufficient to prevent apoptosis. In contrast, a constitutively
high MEK activity induced by overexpression of B-Raf leads to survival
even in the absence of PI 3-kinase activity. This implies that
overexpression of any oncogene leading to MEK activation will result in
protection from apoptosis, including all three Raf kinases,
Ras, and Rap-1. In agreement with this, we have demonstrated that
overexpression of activated Raf-1 and Ras, as well as activated MEK,
blocks apoptosis resulting from growth factor deprivation.
Similarly, the activated forms of Ras, Raf-1, and MEK have been
reported to prevent cell death in a variety of different cell types,
including PC12 cells, hematopoietic cells, T cells, and fibroblasts
(2, 8, 21-22, 30, 34, 48). Activation of MEK by B-Raf
results in constitutive Erk activity, and the role of this pathway in
cell survival is further supported by results obtained from PC12 and
HeLa cells, where Erk activity appeared to be necessary for survival
(5, 21, 37, 48).
Growth factor deprivation induces a mitochondrion-dependent
apoptosis pathway, ultimately leading to cytochrome
c release from mitochondria and cytochrome
c-dependent activation of caspases (23). The
primary site of action of Bcl-2 and Bcl-xL appears to be in
the mitochondria because their overexpression inhibits the release of
cytochrome c (23, 27, 50) but cannot block caspase activation by microinjected cytochrome c
(13). Since the PI 3-kinase/Akt pathway acts at least in
part by phosphorylating BAD and allowing Bcl-2 and Bcl-xL
to function as inhibitors of apoptosis (10-11), PI
3-kinase/Akt signaling is also expected to inhibit mitochondrial
cytochrome c release.
In contrast, our data indicate that the B-Raf/MEK/Erk pathway
interferes with apoptosis at the level of cytosolic caspase activation, downstream of the release of cytochrome c from
mitochondria. First, cytochrome c accumulated in the cytosol
of growth factor-deprived Rat-1/B-Raf cells without significant
activation of caspase-3 and cell death. In addition, cytochrome
c failed to induce caspase activation in cytosols prepared
from Rat-1/B-Raf cells, suggesting that cytosols from cells
overexpressing B-Raf contained an inhibitor of caspase activation. This
action of B-Raf is also distinct from prevention of apoptosis
by Raf-1 targeted to mitochondria, which was reported to inhibit
apoptosis through Bcl-2 family members without the involvement
of Erk (46).
Such postmitochondrial regulation of apoptosis at the level of
caspase activation has already been described with inhibitors of
caspases, including the human IAP-like proteins (12),
raising the possibility that B-Raf/MEK/Erk signaling may affect the
expression or activity of such caspase inhibitors. Moreover, the
Drosophila gene hid, which interacts with IAPs
(45) and controls caspase activation, has recently been
identified as a direct target of Ras-dependent survival signaling
mediated by Erk (4). Although there are no mammalian
equivalents of Drosophila hid known to date, the existence
of a functional homolog would provide a potential mechanism by which
the B-Raf/MEK/Erk pathway promotes survival in mammalian cells.
| |
ACKNOWLEDGMENTS |
This research was supported by Massachusetts Breast Cancer
Research grant 34088PP1010 (to P.E.), American Cancer Society grant IRG-72-001-24-IRG (to P.E.), and NIH grant RO1 CA18689 (to
G.M.C.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Boston University, 5 Cummington St., Boston, MA 02215. Phone: (617) 353-8735. Fax: (617) 353-8484. E-mail:
gmcooper{at}bu.edu.
| |
REFERENCES |
| 1.
|
Alessi, D. R.,
M. Andjelkovic,
B. Caudwell,
P. Cron,
N. Morrice,
P. Cohen, and B. A. Hemmings.
1996.
Mechanism of activation of protein kinase B by insulin and IGF-1.
EMBO J.
15:6541-6551[Medline].
|
| 2.
|
Arends, M. J.,
A. H. McGregor,
N. J. Toft,
E. J. Brown, and A. H. Wyllie.
1993.
Susceptibility to apoptosis is differentially regulated by c-myc and mutated Ha-ras oncogenes and is associated with endonuclease availability.
Br. J. Cancer
68:1127-1133[Medline].
|
| 3.
|
Armstrong, R. C.,
T. Aja,
J. Xiang,
S. Gaur,
J. F. Krebs,
K. Hoang,
X. Bai,
S. J. Korsmeyer,
D. S. Karanewsky,
L. F. Fritz, and K. J. Tomaselli.
1996.
Fas-induced activation of the cell death-related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors.
J. Biol. Chem.
271:16850-16855[Abstract/Free Full Text].
|
| 4.
|
Bergmann, A.,
J. Agapite,
K. McCall, and H. Steller.
1998.
The Drosophila gene hid is a direct molecular target of ras-dependent survival signaling.
Cell
95:331-341[Medline].
|
| 5.
|
Berra, E.,
M. T. Diaz-Meco, and J. Moscat.
1998.
The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway.
J. Biol. Chem.
273:10792-10797[Abstract/Free Full Text].
|
| 6.
|
Chauhan, D.,
P. Pandey,
A. Ogata,
G. Teoh,
N. Krett,
R. Halgren,
S. Rosen,
D. Kufe,
S. Kharbanda, and K. Anderson.
1997.
Cytochrome c-dependent and -independent induction of apoptosis in multiple myeloma cells.
J. Biol. Chem.
272:29995-29997[Abstract/Free Full Text].
|
| 7.
|
Chinnaiyan, A. M.,
K. Orth,
K. O'Rourke,
H. Duan,
G. G. Poirier, and V. M. Dixit.
1996.
Molecular ordering of the cell death pathway. Bcl-2 and Bcl-xL function upstream of the CED-3-like apoptotic proteases.
J. Biol. Chem.
271:4573-4576[Abstract/Free Full Text].
|
| 8.
|
Cleveland, J. L.,
J. Troppmair,
G. Packham,
D. S. Askew,
P. Lloyd,
M. Gonzalez-Garcia,
G. Nunez,
J. N. Ihle, and U. R. Rapp.
1994.
v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.
Oncogene
9:2217-2226[Medline].
|
| 9.
|
Cowley, S.,
H. Paterson,
P. Kemp, and C. J. Marshall.
1994.
Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.
Cell
77:841-852[Medline].
|
| 10.
|
Datta, S. R.,
H. Dudek,
X. Tao,
S. Masters,
H. Fu,
Y. Gotoh, and M. E. Greenberg.
1997.
Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery.
Cell
91:231-241[Medline].
|
| 11.
|
del Peso, L.,
M. Gonzalez-Garcia,
C. Page,
R. Herrera, and G. Nunez.
1997.
Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt.
Science
278:687-689[Abstract/Free Full Text].
|
| 12.
|
Deveraux, Q. L.,
N. Roy,
H. R. Stennicke,
T. Van Arsdale,
Q. Zhou,
S. M. Srinivasula,
E. S. Alnemri,
G. S. Salvesen, and J. C. Reed.
1998.
IAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases.
EMBO J.
17:2215-2223[Medline].
|
| 13.
|
Duckett, C. S.,
F. Li,
Y. Wang,
K. J. Tomaselli,
C. B. Thompson, and R. C. Armstrong.
1998.
Human IAP-like protein regulates programmed cell death downstream of Bcl-xL and cytochrome c.
Mol. Cell. Biol.
18:608-615[Abstract/Free Full Text].
|
| 14.
|
Dudek, H.,
S. R. Datta,
T. F. Franke,
M. J. Birnbaum,
R. Yao,
G. M. Cooper,
R. S. Segal,
D. R. Kaplan, and M. E. Greenberg.
1997.
Regulation of neuronal survival by the serine-threonine protein kinase Akt.
Science
275:661-665[Abstract/Free Full Text].
|
| 15.
|
Erhardt, P.,
J. Troppmair,
U. R. Rapp, and G. M. Cooper.
1995.
Differential regulation of Raf-1 and B-Raf and Ras-dependent activation of mitogen-activated protein kinase by cyclic AMP in PC12 cells.
Mol. Cell. Biol.
15:5524-5530[Abstract].
|
| 16.
|
Erhardt, P., and G. M. Cooper.
1996.
Activation of the CPP32 apoptotic protease by distinct signaling pathways with differential sensitivity to Bcl-xL.
J. Biol. Chem.
271:17601-17604[Abstract/Free Full Text].
|
| 17.
|
Erhardt, P.,
K. J. Tomaselli, and G. M. Cooper.
1997.
Identification of the MDM2 oncoprotein as a substrate for CPP32-like proteases.
J. Biol. Chem.
272:15049-15052[Abstract/Free Full Text].
|
| 18.
|
Fabian, J. R.,
I. O. Daar, and D. K. Morrison.
1993.
Critical tyrosine residues regulate the enzymatic and biological activity of Raf-1 kinase.
Mol. Cell. Biol.
13:7170-7179[Abstract/Free Full Text].
|
| 19.
|
Faleiro, L.,
R. Kobayashi,
H. Fearnhead, and Y. Lazebnik.
1997.
Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells.
EMBO J.
16:2271-2281[Medline].
|
| 20.
|
Feig, L. A., and G. M. Cooper.
1988.
Inhibition of NIH 3T3 cell proliferation by a mutant Ras protein with preferential affinity for GDP.
Mol. Cell. Biol.
8:3235-3243[Abstract/Free Full Text].
|
| 21.
|
Gardner, A. M., and G. L. Johnson.
1996.
Fibroblast growth factor-2 suppression of tumor necrosis factor alpha-mediated apoptosis requires Ras and the activation of mitogen-activated protein kinase.
J. Biol. Chem.
271:14560-14566[Abstract/Free Full Text].
|
| 22.
|
Gomez, J.,
A. C. Martinez,
B. Fernandez,
A. Garcia, and A. Rebollo.
1996.
Critical role of Ras in the proliferation and prevention of apoptosis mediated by IL-2.
J. Immunol.
157:2272-2281[Abstract].
|
| 23.
|
Green, D. R., and J. C. Reed.
1998.
Mitochondria and apoptosis.
Science
281:1309-1312[Abstract/Free Full Text].
|
| 24.
|
Hunter, T.
1995.
Protein kinases and phosphatases: the yin and yang of protein phosphorylation and signaling.
Cell
80:225-236[Medline].
|
| 25.
|
Kauffmann-Zeh, A.,
P. Rodriguez-Viciana,
E. Ulrich,
C. Gilbert,
P. Coffer,
J. Downward, and G. Evan.
1997.
Suppression of c-Myc-induced apoptosis by Ras signalling through PI(3)K and PKB.
Nature (London)
385:544-548[Medline].
|
| 26.
|
Kennedy, S. G.,
A. J. Wagner,
S. D. Conzen,
J. Jordan,
A. Bellacosa,
P. N. Tsichlis, and N. Hay.
1997.
The PI 3-kinase/Akt signaling pathway delivers an anti-apoptotic signal.
Genes Dev.
11:701-713[Abstract/Free Full Text].
|
| 27.
|
Kluck, R. M.,
E. Bossy-Wetzel,
D. R. Green, and D. D. Newmeyer.
1997.
The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis.
Science
275:1132-1136[Abstract/Free Full Text].
|
| 28.
|
Kulik, G.,
A. Klippel, and M. J. Weber.
1997.
Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.
Mol. Cell. Biol.
17:1595-1606[Abstract].
|
| 29.
|
Kummer, J. L.,
P. K. Rao, and K. A. Heidenreich.
1997.
Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase.
J. Biol. Chem.
272:20490-20494[Abstract/Free Full Text].
|
| 30.
|
Lebowitz, P. F.,
D. Sakamuro, and G. C. Prendergast.
1997.
Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment.
Cancer Res.
57:708-713[Abstract/Free Full Text].
|
| 31.
|
Li, P.,
D. Nijhawan,
I. Budihardjo,
S. M. Srinivasula,
M. Ahmad,
E. S. Alnemri, and X. Wang.
1997.
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
Cell
91:479-489[Medline].
|
| 32.
|
Liu, X.,
C. N. Kim,
J. Yang,
R. Jemmerson, and X. Wang.
1996.
Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c.
Cell
86:147-157[Medline].
|
| 33.
|
Marais, R.,
Y. Light,
H. F. Paterson,
C. S. Mason, and C. J. Marshall.
1997.
Differential regulation of Raf-1, A-Raf, and B-Raf by oncogenic ras and tyrosine kinases.
J. Biol. Chem.
272:4378-4383[Abstract/Free Full Text].
|
| 34.
|
McKenna, W. G.,
E. J. Bernhard,
D. A. Markiewicz,
M. S. Rudoltz,
A. Maity, and R. J. Muschel.
1996.
Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay.
Oncogene
12:237-245[Medline].
|
| 35.
|
Miura, M.,
H. Zhu,
R. Rotello,
E. A. Hartwieg, and J. Yuan.
1993.
Induction of apoptosis in fibroblasts by IL-1 beta-converting enzyme, a mammalian homolog of the C. elegans cell death gene ced-3.
Cell
75:653-660[Medline].
|
| 36.
|
Pap, M., and G. M. Cooper.
1998.
Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway.
J. Biol. Chem.
273:19929-19932[Abstract/Free Full Text].
|
| 37.
|
Parrizas, M.,
A. R. Saltiel, and D. LeRoith.
1997.
Insulin-like growth factor 1 inhibits apoptosis using the phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways.
J. Biol. Chem.
272:154-161[Abstract/Free Full Text].
|
| 38.
|
Payne, D. M.,
A. J. Rossomando,
P. Martino,
A. K. Erickson,
J. H. Her,
J. Shabanowitz,
D. F. Hunt,
M. J. Weber, and T. W. Sturgill.
1991.
Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).
EMBO J.
10:885-892[Medline].
|
| 39.
|
Pritchard, C. A.,
M. L. Samuels,
E. Bosch, and M. McMahon.
1995.
Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.
Mol. Cell. Biol.
15:6430-6442[Abstract].
|
| 40.
|
Pritchard, C. A.,
L. Bolin,
R. Slattery,
R. Murray, and M. McMahon.
1996.
Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase gene.
Curr. Biol.
6:614-617[Medline].
|
| 41.
|
Robinson, M. J., and M. H. Cobb.
1997.
Mitogen-activated protein kinase pathways.
Curr. Opin. Cell. Biol.
9:180-186[Medline].
|
| 42.
|
Roulston, A.,
C. Reinhard,
P. Amiri, and L. T. Williams.
1998.
Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor alpha.
J. Biol. Chem.
273:10232-10239[Abstract/Free Full Text].
|
| 43.
|
Scheid, M. P., and V. Duronio.
1998.
Dissociation of cytokine-induced phosphorylation of Bad and activation of PKB/Akt: involvement of MEK upstream of Bad phosphorylation.
Proc. Natl. Acad. Sci. USA
95:7439-7444[Abstract/Free Full Text].
|
| 44.
|
Songyang, Z.,
D. Baltimore,
L. C. Cantley,
D. R. Kaplan, and T. F. Franke.
1997.
Interleukin 3-dependent survival by the Akt protein kinase.
Proc. Natl. Acad. Sci. USA
94:11345-11350[Abstract/Free Full Text].
|
| 45.
|
Vucic, D.,
W. J. Kaiser, and L. K. Miller.
1998.
Inhibitor of apoptosis proteins physically interact with and block apoptosis induced by Drosophila proteins HID and GRIM.
Mol. Cell. Biol.
18:3300-3309[Abstract/Free Full Text].
|
| 46.
|
Wang, H. G.,
U. R. Rapp, and J. C. Reed.
1996.
Bcl-2 targets the protein kinase Raf-1 to mitochondria.
Cell
87:629-638[Medline].
|
| 47.
|
Wojnowski, L.,
A. M. Zimmer,
T. W. Beck,
H. Hahn,
R. Bernal,
U. R. Rapp, and A. Zimmer.
1997.
Endothelial apoptosis in B-Raf deficient mice.
Nat. Genet.
16:293-297[Medline].
|
| 48.
|
Xia, Z.,
M. Dickens,
J. Raingeud,
R. J. Davis, and M. E. Greenberg.
1995.
Opposing effects of Erk and JNK-p38 MAP kinases on apoptosis.
Science
270:1326-1331[Abstract/Free Full Text].
|
| 49.
|
Yan, C. Y. I., and L. A. Greene.
1998.
Prevention of PC12 cell death by N-acetylcysteine requires activation of the Ras pathway.
J. Neurosci.
18:4042-4049[Abstract/Free Full Text].
|
| 50.
|
Yang, J.,
X. Liu,
K. Bhalla,
C. N. Kim,
A. M. Ibrado,
J. Cai,
T. I. Peng,
D. P. Jones, and X. Wang.
1997.
Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked.
Science
275:1129-1132[Abstract/Free Full Text].
|
| 51.
|
Yang, E.,
J. Zha,
J. Jockel,
L. H. Boise,
C. B. Thompson, and S. J. Korsmeyer.
1995.
Bad, a heterodimeric partner for Bcl-xL and Bcl-2, displaces Bax and promotes cell death.
Cell
80:285-291[Medline].
|
| 52.
|
Yao, R., and G. M. Cooper.
1995.
Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor.
Science
267:2003-2006[Abstract/Free Full Text].
|
| 53.
|
Yao, R., and G. M. Cooper.
1996.
Growth factor-dependent survival of rodent fibroblasts requires phosphatidylinositol 3-kinase but is independent of pp70S6K activity.
Oncogene
13:343-351[Medline].
|
| 54.
|
York, R. D.,
H. Yao,
T. Dillon,
C. L. Ellig,
S. P. Eckert,
E. W. McClesey, and P. J. S. Stork.
1998.
Rap1 mediates sustained MAP kinase activation induced by nerve growth factor.
Nature (London)
392:622-626[Medline].
|
| 55.
|
Zha, J.,
H. Harada,
E. Yang,
J. Jockel, and S. J. Korsmeyer.
1996.
Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not Bcl-xL.
Cell
87:619-628[Medline].
|
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-
Sun, Y., Sinicrope, F. A.
(2005). Selective inhibitors of MEK1/ERK44/42 and p38 mitogen-activated protein kinases potentiate apoptosis induction by sulindac sulfide in human colon carcinoma cells. Molecular Cancer Therapeutics
4: 51-59
[Abstract]
[Full Text]
-
He, Y.-Y., Huang, J.-L., Chignell, C. F.
(2004). Delayed and Sustained Activation of Extracellular Signal-regulated Kinase in Human Keratinocytes by UVA: IMPLICATIONS IN CARCINOGENESIS. J. Biol. Chem.
279: 53867-53874
[Abstract]
[Full Text]
-
Parikh, N., Sade, H., Kurian, L., Sarin, A.
(2004). The Bax N Terminus Is Required for Negative Regulation by the Mitogen-Activated Protein Kinase Kinase and Akt Signaling Pathways in T Cells. J. Immunol.
173: 6220-6227
[Abstract]
[Full Text]
-
Tsukamoto, H., Irie, A., Nishimura, Y.
(2004). B-Raf Contributes to Sustained Extracellular Signal-regulated Kinase Activation Associated with Interleukin-2 Production Stimulated through the T Cell Receptor. J. Biol. Chem.
279: 48457-48465
[Abstract]
[Full Text]
-
Gardai, S. J., Whitlock, B. B., Xiao, Y. Q., Bratton, D. B., Henson, P. M.
(2004). Oxidants Inhibit ERK/MAPK and Prevent Its Ability to Delay Neutrophil Apoptosis Downstream of Mitochondrial Changes and at the Level of XIAP. J. Biol. Chem.
279: 44695-44703
[Abstract]
[Full Text]
-
Baker, K, Zhang, Y, Jin, C, Jass, J R
(2004). Proximal versus distal hyperplastic polyps of the colorectum: different lesions or a biological spectrum?. J. Clin. Pathol.
57: 1089-1093
[Abstract]
[Full Text]
-
Cheung, E. C. C., Slack, R. S.
(2004). Emerging Role for ERK as a Key Regulator of Neuronal Apoptosis. Sci Signal
2004: pe45-pe45
[Abstract]
[Full Text]
-
Flaherty, D. M., Hinde, S. L., Monick, M. M., Powers, L. S., Bradford, M. A., Yarovinsky, T., Hunninghake, G. W.
(2004). Adenovirus vectors activate survival pathways in lung epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol.
287: L393-L401
[Abstract]
[Full Text]
-
Hausenloy, D. J, Mocanu, M. M, Yellon, D. M
(2004). Cross-talk between the survival kinases during early reperfusion: its contribution to ischemic preconditioning. Cardiovasc Res
63: 305-312
[Abstract]
[Full Text]
-
Murayama, Y., Miyagawa, J.-i., Oritani, K., Yoshida, H., Yamamoto, K., Kishida, O., Miyazaki, T., Tsutsui, S., Kiyohara, T., Miyazaki, Y., Higashiyama, S., Matsuzawa, Y., Shinomura, Y.
(2004). CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells. J. Cell Sci.
117: 3379-3388
[Abstract]
[Full Text]
-
Higuchi, T, Jass, J R
(2004). My approach to serrated polyps of the colorectum. J. Clin. Pathol.
57: 682-686
[Abstract]
[Full Text]
-
Schulze, A., Nicke, B., Warne, P. H., Tomlinson, S., Downward, J.
(2004). The Transcriptional Response to Raf Activation Is Almost Completely Dependent on Mitogen-activated Protein Kinase Kinase Activity and Shows a Major Autocrine Component. Mol. Biol. Cell
15: 3450-3463
[Abstract]
[Full Text]
-
Hausenloy, D. J, Yellon, D. M
(2004). New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway. Cardiovasc Res
61: 448-460
[Abstract]
[Full Text]
-
Li, L., Ren, C. H., Tahir, S. A., Ren, C., Thompson, T. C.
(2003). Caveolin-1 Maintains Activated Akt in Prostate Cancer Cells through Scaffolding Domain Binding Site Interactions with and Inhibition of Serine/Threonine Protein Phosphatases PP1 and PP2A. Mol. Cell. Biol.
23: 9389-9404
[Abstract]
[Full Text]
-
Pardo, O. E., Lesay, A., Arcaro, A., Lopes, R., Ng, B. L., Warne, P. H., McNeish, I. A., Tetley, T. D., Lemoine, N. R., Mehmet, H., Seckl, M. J., Downward, J.
(2003). Fibroblast Growth Factor 2-Mediated Translational Control of IAPs Blocks Mitochondrial Release of Smac/DIABLO and Apoptosis in Small Cell Lung Cancer Cells. Mol. Cell. Biol.
23: 7600-7610
[Abstract]
[Full Text]
-
Sorensen, B. B., Rao, L. V. M., Tornehave, D., Gammeltoft, S., Petersen, L. C.
(2003). Antiapoptotic effect of coagulation factor VIIa. Blood
102: 1708-1715
[Abstract]
[Full Text]
-
Xu, X., Quiros, R. M., Gattuso, P., Ain, K. B., Prinz, R. A.
(2003). High Prevalence of BRAF Gene Mutation in Papillary Thyroid Carcinomas and Thyroid Tumor Cell Lines. Cancer Res.
63: 4561-4567
[Abstract]
[Full Text]
-
Yaglom, J., O'Callaghan-Sunol, C., Gabai, V., Sherman, M. Y.
(2003). Inactivation of Dual-Specificity Phosphatases Is Involved in the Regulation of Extracellular Signal-Regulated Kinases by Heat Shock and Hsp72. Mol. Cell. Biol.
23: 3813-3824
[Abstract]
[Full Text]
-
Furukawa, T., Sunamura, M., Motoi, F., Matsuno, S., Horii, A.
(2003). Potential Tumor Suppressive Pathway Involving DUSP6/MKP-3 in Pancreatic Cancer. Am. J. Pathol.
162: 1807-1815
[Abstract]
[Full Text]
-
Ostrovsky, O., Bengal, E.
(2003). The Mitogen-activated Protein Kinase Cascade Promotes Myoblast Cell Survival by Stabilizing the Cyclin-dependent Kinase Inhibitor, p21WAF1 Protein. J. Biol. Chem.
278: 21221-21231
[Abstract]
[Full Text]
-
Tsutsumi, S., Hogan, V., Nabi, I. R., Raz, A.
(2003). Overexpression of the Autocrine Motility Factor/Phosphoglucose Isomerase Induces Transformation and Survival of NIH-3T3 Fibroblasts. Cancer Res.
63: 242-249
[Abstract]
[Full Text]
-
Martin, A. G., Fearnhead, H. O.
(2002). Apocytochrome c Blocks Caspase-9 Activation and Bax-induced Apoptosis. J. Biol. Chem.
277: 50834-50841
[Abstract]
[Full Text]
-
Quinn, J. C., Johnson-Farley, N. N., Yoon, J., Cowen, D. S.
(2002). Activation of Extracellular-Regulated Kinase by 5-Hydroxytryptamine2A Receptors in PC12 Cells is Protein Kinase C-Independent and Requires Calmodulin and Tyrosine Kinases. J. Pharmacol. Exp. Ther.
303: 746-752
[Abstract]
[Full Text]
-
Boldt, S., Weidle, U. H., Kolch, W.
(2002). The role of MAPK pathways in the action of chemotherapeutic drugs. Carcinogenesis
23: 1831-1838
[Abstract]
[Full Text]
-
Nowak, G.
(2002). Protein Kinase C-alpha and ERK1/2 Mediate Mitochondrial Dysfunction, Decreases in Active Na+ Transport, and Cisplatin-induced Apoptosis in Renal Cells. J. Biol. Chem.
277: 43377-43388
[Abstract]
[Full Text]
-
Grana, T. M., Rusyn, E. V., Zhou, H., Sartor, C. I., Cox, A. D.
(2002). Ras Mediates Radioresistance through Both Phosphatidylinositol 3-Kinase-dependent and Raf-dependent but Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase-independent Signaling Pathways. Cancer Res.
62: 4142-4150
[Abstract]
[Full Text]
-
Gu, J., Fujibayashi, A., Yamada, K. M., Sekiguchi, K.
(2002). Laminin-10/11 and Fibronectin Differentially Prevent Apoptosis Induced by Serum Removal via Phosphatidylinositol 3-Kinase/Akt- and MEK1/ERK-dependent Pathways. J. Biol. Chem.
277: 19922-19928
[Abstract]
[Full Text]
-
Milella, M., Estrov, Z., Kornblau, S. M., Carter, B. Z., Konopleva, M., Tari, A., Schober, W. D., Harris, D., Leysath, C. E., Lopez-Berestein, G., Huang, Z., Andreeff, M.
(2002). Synergistic induction of apoptosis by simultaneous disruption of the Bcl-2 and MEK/MAPK pathways in acute myelogenous leukemia. Blood
99: 3461-3464
[Abstract]
[Full Text]
-
Hindley, A., Kolch, W.
(2002). Extracellular signal regulated kinase (ERK)/mitogen activated protein kinase (MAPK)-independent functions of Raf kinases. J. Cell Sci.
115: 1575-1581
[Abstract]
[Full Text]
-
Tsuruta, F., Masuyama, N., Gotoh, Y.
(2002). The Phosphatidylinositol 3-Kinase (PI3K)-Akt Pathway Suppresses Bax Translocation to Mitochondria. J. Biol. Chem.
277: 14040-14047
[Abstract]
[Full Text]
-
Yu, X., Guo, Z. S., Marcu, M. G., Neckers, L., Nguyen, D. M., Chen, G. A., Schrump, D. S.
(2002). Modulation of p53, ErbB1, ErbB2, and Raf-1 Expression in Lung Cancer Cells by Depsipeptide FR901228. JNCI J Natl Cancer Inst
94: 504-513
[Abstract]
[Full Text]
-
Gabai, V. L., Sherman, M. Y.
(2002). Molecular Biology of Thermoregulation: Invited Review: Interplay between molecular chaperones and signaling pathways in survival of heat shock. J. Appl. Physiol.
92: 1743-1748
[Abstract]
[Full Text]
-
Lu, L., Holmqvist, K., Cross, M., Welsh, M.
(2002). Role of the Src Homology 2 Domain-containing Protein Shb in Murine Brain Endothelial Cell Proliferation and Differentiation. Cell Growth Differ.
13: 141-148
[Abstract]
[Full Text]
-
Perkins, D., Pereira, E. F. R., Gober, M., Yarowsky, P. J., Aurelian, L.
(2002). The Herpes Simplex Virus Type 2 R1 Protein Kinase (ICP10 PK) Blocks Apoptosis in Hippocampal Neurons, Involving Activation of the MEK/MAPK Survival Pathway. J. Virol.
76: 1435-1449
[Abstract]
[Full Text]
-
Carson, J. P., Behnam, M., Sutton, J. N., Du, C., Wang, X., Hunt, D. F., Weber, M. J., Kulik, G.
(2002). Smac Is Required for Cytochrome c-induced Apoptosis in Prostate Cancer LNCaP Cells. Cancer Res.
62: 18-23
[Abstract]
[Full Text]
-
Yan, F., John, S. K., Polk, D. B.
(2001). Kinase Suppressor of Ras Determines Survival of Intestinal Epithelial Cells Exposed to Tumor Necrosis Factor. Cancer Res.
61: 8668-8675
[Abstract]
[Full Text]
-
Xiao, R.-P.
(2001). {beta}-Adrenergic Signaling in the Heart: Dual Coupling of the {beta}2-Adrenergic Receptor to Gs and Gi Proteins. Sci Signal
2001: re15-re15
[Abstract]
[Full Text]
-
Qiao, L., Studer, E., Leach, K., McKinstry, R., Gupta, S., Decker, R., Kukreja, R., Valerie, K., Nagarkatti, P., Deiry, W. E., Molkentin, J., Schmidt-Ullrich, R., Fisher, P. B., Grant, S., Hylemon, P. B., Dent, P.
(2001). Deoxycholic Acid (DCA) Causes Ligand-independent Activation of Epidermal Growth Factor Receptor (EGFR) and FAS Receptor in Primary Hepatocytes: Inhibition of EGFR/Mitogen-activated Protein Kinase-Signaling Module Enhances DCA-induced Apoptosis. Mol. Biol. Cell
12: 2629-2645
[Abstract]
[Full Text]
-
Hsu, E. H., Lochan, A. C., Cowen, D. S.
(2001). Activation of Akt1 by Human 5-Hydroxytryptamine (Serotonin)1B Receptors Is Sensitive to Inhibitors of MEK. J. Pharmacol. Exp. Ther.
298: 825-832
[Abstract]
[Full Text]
-
Ballif, B. A., Blenis, J.
(2001). Molecular Mechanisms Mediating Mammalian Mitogen-activated Protein Kinase (MAPK) Kinase (MEK)-MAPK Cell Survival Signals. Cell Growth Differ.
12: 397-408
[Full Text]
-
Chen, J., Fujii, K., Zhang, L., Roberts, T., Fu, H.
(2001). Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc. Natl. Acad. Sci. USA
10.1073/pnas.141224398v1
[Abstract]
[Full Text]
-
Schulze, A., Lehmann, K., Jefferies, H. B.J., McMahon, M., Downward, J.
(2001). Analysis of the transcriptional program induced by Raf in epithelial cells. Genes Dev.
15: 981-994
[Abstract]
[Full Text]
-
von Gise, A., Lorenz, P., Wellbrock, C., Hemmings, B., Berberich-Siebelt, F., Rapp, U. R., Troppmair, J.
(2001). Apoptosis Suppression by Raf-1 and MEK1 Requires MEK- and Phosphatidylinositol 3-Kinase-Dependent Signals. Mol. Cell. Biol.
21: 2324-2336
[Abstract]
[Full Text]
-
Hansen, M. R., Zha, X.-M., Bok, J., Green, S. H.
(2001). Multiple Distinct Signal Pathways, Including an Autocrine Neurotrophic Mechanism, Contribute to the Survival-Promoting Effect of Depolarization on Spiral Ganglion Neurons In Vitro. J. Neurosci.
21: 2256-2267
[Abstract]
[Full Text]
-
Whitlock, B. B., Gardai, S., Fadok, V., Bratton, D., Henson, P. M.
(2000). Differential Roles for {alpha}M{beta}2 Integrin Clustering or Activation in the Control of Apoptosis via Regulation of Akt and ERK Survival Mechanisms. JCB
151: 1305-1320
[Abstract]
[Full Text]
-
Chesley, A., Lundberg, M. S., Asai, T., Xiao, R.-P., Ohtani, S., Lakatta, E. G., Crow, M. T.
(2000). The {beta}2-Adrenergic Receptor Delivers an Antiapoptotic Signal to Cardiac Myocytes Through Gi-Dependent Coupling to Phosphatidylinositol 3'-Kinase. Circ. Res.
87: 1172-1179
[Abstract]
[Full Text]
-
Zhou, H., Li, X.-M., Meinkoth, J., Pittman, R. N.
(2000). Akt Regulates Cell Survival and Apoptosis at a Postmitochondrial Level. JCB
151: 483-494
[Abstract]
[Full Text]
-
Nagahara, Y., Ikekita, M., Shinomiya, T.
(2000). Immunosuppressant FTY720 Induces Apoptosis by Direct Induction of Permeability Transition and Release of Cytochrome c from Mitochondria. J. Immunol.
165: 3250-3259
[Abstract]
[Full Text]
-
Li, J., Yang, S., Billiar, T. R.
(2000). Cyclic Nucleotides Suppress Tumor Necrosis Factor alpha -Mediated Apoptosis by Inhibiting Caspase Activation and Cytochrome c Release in Primary Hepatocytes via a Mechanism Independent of Akt Activation. J. Biol. Chem.
275: 13026-13034
[Abstract]
[Full Text]
-
Le Gall, M., Chambard, J.-C., Breittmayer, J.-P., Grall, D., Pouysségur, J., Van Obberghen-Schilling, E.
(2000). The p42/p44 MAP Kinase Pathway Prevents Apoptosis Induced by Anchorage and Serum Removal. Mol. Biol. Cell
11: 1103-1112
[Abstract]
[Full Text]
-
Depre, C., Taegtmeyer, H.
(2000). Metabolic aspects of programmed cell survival and cell death in the heart. Cardiovasc Res
45: 538-548
[Abstract]
[Full Text]
-
Rossig, L., Haendeler, J., Hermann, C., Malchow, P., Urbich, C., Zeiher, A. M., Dimmeler, S.
(2000). Nitric Oxide Down-regulates MKP-3 mRNA Levels. INVOLVEMENT IN ENDOTHELIAL CELL PROTECTION FROM APOPTOSIS. J. Biol. Chem.
275: 25502-25507
[Abstract]
[Full Text]
-
McGuire, T. F., Trump, D. L., Johnson, C. S.
(2001). Vitamin D3-induced Apoptosis of Murine Squamous Cell Carcinoma Cells. SELECTIVE INDUCTION OF CASPASE-DEPENDENT MEK CLEAVAGE AND UP-REGULATION OF MEKK-1. J. Biol. Chem.
276: 26365-26373
[Abstract]
[Full Text]
-
Chen, J., Fujii, K., Zhang, L., Roberts, T., Fu, H.
(2001). Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK-ERK independent mechanism. Proc. Natl. Acad. Sci. USA
98: 7783-7788
[Abstract]
[Full Text]
-
Tashker, J. S., Olson, M., Kornbluth, S.
(2002). Post-Cytochrome c Protection from Apoptosis Conferred by a MAPK Pathway in Xenopus Egg Extracts. Mol. Biol. Cell
13: 393-401
[Abstract]
[Full Text]
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Abstract
Introduction
