From the Laboratorio Glaxo Wellcome-Consejo Superior de
Investigaciones Científicas de Biología Molecular y
Celular, Centro de Biología Molecular "Severo Ochoa"
(Consejo Superior de Investigaciones Científicas-Universidad
Autónoma de Madrid), Universidad Autónoma, Canto
Blanco, 28049 Madrid, Spain
Considerable attention has recently been focused
on the role played by different kinase cascades in the control of
apoptosis. The triggering of stress-activated kinases concomitant with
the inhibition of the extracellular signal-regulated kinase (ERK) pathway has been observed in a number of cell systems undergoing programmed cell death. In addition, the activation of the
phosphoinositide 3-kinase (PI 3-kinase)-Akt signaling cascade has been
shown to protect from apoptosis. Here we have explored the potential
role played by the inhibition of ERK in the activation of the stress kinases as well as the possible cross-talk with the PI 3-kinase pathway
in HeLa cells. We show that the simple inhibition of ERK basal activity
is sufficient to trigger apoptosis and p38 activation with no changes
in Jun N-terminal kinase/stress-activated protein kinase. This is a
process dependent on the caspases and is completely abrogated by serum.
The incubation with wortmannin or the transfection of dominant negative
mutants of p85 or Akt block the inhibitory function of serum,
suggesting the involvement of the PI 3-kinase-Akt system. Consistent
with this, expression of active mutants of PI 3-kinase and Akt inhibits
p38 activation and apoptosis. We also show here that the inhibition of
ERK triggers the caspase system, which is abolished by serum in a
wortmannin-dependent manner. Collectively, these results
demonstrate a link between ERK and the p38 apoptotic pathway that is
modulated by the survival PI 3-kinase-Akt module, acting upstream the
caspase system.
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INTRODUCTION |
Signal transduction pathways controlled by kinase modules regulate
critical cellular functions such as cell growth, differentiation and
apoptosis. Three major kinase cascades have recently been identified
that culminate in the activation of three different sets of
mitogen-activated protein kinases: the extracellular signal-related kinase (ERK),1 JNK/SAPK, and
p38 (1, 2). The three have been implicated in the control of apoptosis.
ERK is activated by mitogens and survival factors, whereas JNK/SAPK and
p38 are stimulated by stress signals (1, 2). A number of findings
indicate that the activation of JNK/SAPK and p38 may play decisive
roles in the control of cell death. Thus, the JNK/SAPK pathway is
critical during ceramide and stress-induced apoptosis (3-5) as well as
in the Daax-mediated Fas cascade (6). Also in favor of a role of
JNK/SAPK and p38 in apoptosis are the results reported by Xia et
al. (7) who demonstrated that the transfection of a constitutively
active mutant of MKK3/6 (the physiological activator of p38) along with p38 is sufficient to induce apoptosis in PC-12 cells. In addition, transfection of dominant negative mutants of MKK3/6 in PC-12 cells or
of p38 in NIH-3T3 fibroblasts dramatically inhibits apoptosis by nerve
growth factor withdrawal (7) and UV irradiation (8), respectively.
Consistent with this notion is the lack of excitotoxicity-induced apoptosis in the hipocampus of JNK3 knock-out mice (9). However, the
simple transient activation of the stress kinase cascades may not
always be sufficient to induce apoptosis, because, for example, tumor
necrosis factor
promotes a significant induction of JNK/SAPK but
does not invariably induce apoptosis (10). In this regard, the
concomitant inactivation of survival signals may be a prerequisite for
JNK/SAPK and p38 to induce cell death (11). Interestingly, deprivation
of neurotrophic factors in PC-12 or UV-irradiation of NIH-3T3 cells not
only activates the stress kinase cascades but also leads to a dramatic
inhibition of the ERK pathway (7, 8). This is particularly relevant because ERK has been shown to be required for survival signaling in
response to fibroblast growth factor (12) and insulin-like growth
factor 1 (13). Furthermore, the overexpression of ERK in NIH-3T3 cells
impairs a large part of the UV-induced apoptotic response (8).
Thus, it seems that the ability of a cell to die or survive may be
dictated by a critical balance between the ERK and the JNK/SAPK and p38
pathways (11).
Recently, considerable attention has been focused on the role of PI
3-kinase as a source of survival signals. Yao and Cooper (14) have
shown that inhibition of PI 3-kinase in PC-12 cells is sufficient to
induce apoptosis. PI 3-kinase also plays a critical role in anoikis and
apoptosis induced by myc and UV irradiation (15-17). Interestingly,
the expression of permanently active mutants of PI 3-kinase or Akt (a
target of PI 3-kinase) is sufficient to inhibit apoptosis (15, 17-19).
Collectively, these results suggest that the PI 3-kinase-Akt pathway is
required and sufficient for cell survival. However, this does not seem
to always be the case. Thus, for example, inhibition of PI 3-kinase
does not induce apoptosis in cerebellar granule neurons maintained in
serum and K+-rich medium (18). The inhibition of PI
3-kinase only has an impact in this system when cells are incubated in
serum-free low K+ medium in the presence of insulin-like
growth factor 1, which partially supports neuron survival (18). In
addition, and consistent with these observations, the inhibition of PI
3-kinase is not sufficient to induce apoptosis in superior cervical
neurons (20), although the overexpression of active mutants of PI
3-kinase or Akt can support survival of these cells in the absence of
nerve growth factor (20).
Together these results suggest that multiple pathways are important for
cell survival and that the comprehension of the cross-talk existing
between these cascades will greatly help in the understanding of this
important pathophysiological process. In the present study, we have
addressed the role that the inhibition of ERK plays in the activation
of the JNK/SAPK and p38 pathways as well as the possible cross-talk
between this and the PI 3-kinase-Akt signaling cascade.
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MATERIALS AND METHODS |
Plasmids and Expression of Recombinant Fusion Protein--
The
pCDNA3 and pCMV-
Gal have previously been described (Diaz-Meco
et al. (21)). The p38 expression vectors (pCMV5-Flag-p38 and
the dominant negative pCMV5-Flag-p38AGF) were provided by
Dr. Roger Davis. pRK5rCD2p110myc, containing a permanently active
mutant of PI 3-kinase was a gift from Dr. Cantrell (ICRF, London). The
pCDNA3-myc-
p85 is a myc-tagged version of plasmid pRS
p85,
generously provided by Dr. Kasuga. The FADD dominant negative
(pCDNA3AU1FADDDN) was provided by Dr. Dixit (Genenteck,
San Francisco). The expression vectors for the permanently active and
the kinase defective mutants of Akt were a gift from Dr. Julian
Downward (ICRF, London). The pGEX-2TK-Elk-1 construct was transformed
into Escherichia coli JM101, and the expression and
purification of the glutathione S-transferase fusion protein
on glutathione-Sepharose was carried out according to the
manufacturer's procedures.
Cell Culture and Transfections--
HeLa cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal calf
serum, penicillin G (100 µg/ml), and streptomycin (100 µg/ml) (Life
Technologies, Inc.) in a CO2 incubator (5%
CO2) at 37 °C. Subconfluent cells were transfected with
different plasmid DNAs by the calcium phosphate method
(CLONTECH, Inc.). Some experiments were performed
in the presence of the MEK inhibitor PD98059 (Calbiochem), the
interleukin 1
converting enzyme (ICE) inhibitor z-VAD-fmk (Bachem),
actinomycin D (Sigma), or wortmannin (Sigma).
Immunoblot Analysis--
HeLa cells either untreated or
incubated with different stimuli were extracted with lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton
X-100, 2 mM EDTA, 1 mM EGTA, protease
inhibitors), and cellular extracts (20 µg) were resolved in
SDS-polyacrylamide (10%) gels. Afterward, they were
electrophoretically transferred into a nitrocellulose membrane (Hybond
ECL, Amersham Pharmacia Biotech) and incubated with the specific
antibodies: ERK (Pharmingen); p38, phospho-specific ERK,
phospho-specific p38, and JNK/SAPK (New England Biolabs); anti-Fas
ligand (Santa Cruz Biotechnology); anti-FADD (Santa Cruz
Biotechnology); anti-Flag (Eastman Kodak Co.); anti-CPP32 (Tansduction
Laboratories); or anti-myc. The bands were visualized with the ECL
system (Amersham).
p38 Activity--
For the p38 assay, HeLa cells transfected with
Flag-p38 were extracted with lysis buffer (20 mM Tris (pH
7.5), 10% glycerol, 1% Triton X-100, 137 mM NaCl, 2 mM EDTA, protease inhibitors) and immunoprecipitated with
anti-Flag antibody (2 µg/mg of protein extract). The activity was
determined by phosphorylation of 3 µg of glutathione
S-transferase-Elk-1 in 25 mM Hepes (pH 7.5), 25 mM
-glycerophosphate, 0.5 mM dithiothreitol,
0.1 mM sodium orthovanadate. The kinase reaction was
terminated after 30 min at 30 °C by the addition of Laemmli sample
buffer. The phosphorylation of the substrate protein was examined after
SDS-polyacrylamide gel electrophoresis followed by exposure and
quantitation in an InstantImager (Packard).
Apoptosis Assay--
Terminal
deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling
(TUNEL) analysis was performed by using an in situ cell
death detection kit (Boehringer Mannheim). The
-galactosidase co-transfection assays for determination of cell death was performed as
described previously (21).
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RESULTS |
The Inhibition of ERK Activates p38 but Not JNK/SAPK--
We
initially determined whether the simple inhibition of ERK leads to the
activation of p38 and JNK/SAPK. HeLa cells cultured either in the
presence or in the absence of serum were incubated with the MEK
inhibitor PD98059, and the activity of ERK, JNK/SAPK, and p38 was
determined by immunoblot analysis with antibodies that recognized the
activated phosphorylated forms of the three kinases in cell extracts
obtained at different times. The levels of phospho-ERK were very
similar in cells that were incubated either with or without serum (Fig.
1), indicating that the basal levels of
ERK are not dramatically affected by the presence of serum when cells
are cultured asynchronously. The addition of the MEK inhibitor rapidly
and profoundly reduced the phospho-ERK signal with no effect on ERK
protein levels (Fig. 1). This is consistent with the reported ability
of this drug to inhibit the MEK-ERK pathway. This inhibition was
detected in cells maintained both in serum-containing and in serum-free
media (Fig. 1). Interestingly, subsequent to the inhibition of ERK,
there is a dramatic activation of p38 in cell cultures kept in
serum-free media, as determined by immunoblot analysis with an
anti-phospho-p38 antibody (Fig. 1) or in anti-p38 immunoprecipitates
(not shown). This effect takes place with no changes in p38 protein
levels (Fig. 1). This increase in p38 activity is detectable at 2 h after ERK inhibition and remained elevated at least until 12 h,
the maximum time point measured (Fig. 1). These changes in ERK and p38
activities took place with little or no effect on the activity or
levels of JNK/SAPK (not shown). Therefore, the inhibition of ERK leads
to the activation of p38 in cell cultures maintained in serum-free
media. However, the presence of serum, although unable to affect the
inhibition of ERK by the MEK inhibitor, completely abrogated the
stimulation of p38 (Fig. 1). This indicates that signals activated by
the presence of serum blocked, in an ERK-independent manner, the
ability of p38 to be activated by the inhibition of the ERK
cascade.

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Fig. 1.
The inhibition of the ERK pathway activates
p38. HeLa cells were incubated for 24 h either in the absence
or in the presence of serum, after which they were treated with 50 µM MEK inhibitor PD98059 for different times. Cell
lysates were analyzed by immunoblotting with antibodies to phospho-ERK,
phospho-p38, ERK, or p38. Essentially identical results were obtained
in another three experiments.
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The Inhibition of the MEK-ERK Pathway Induces Apoptosis by a
Caspase-dependent Mechanism--
Consistent with the
notion that ERK may provide survival signals are the results of Fig.
2A, demonstrating that the
incubation of HeLa cells with the MEK inhibitor PD98059 induces
apoptosis in cell cultures maintained in the absence of serum. The
presence of serum or the incubation with the caspase inhibitor z-VAD,
completely inhibited that effect. This suggests that the mechanism
whereby the inhibition of the MEK-ERK cascade induces apoptosis
involves the caspase system and that the presence of serum not only
inhibits the activation of p38 but also the induction of apoptosis.
Immunoblot analysis of parallel extracts demonstrate that the MEK
inhibitor actually blocked the ERK pathway and activated CPP32 in a
serum-inhibitable and z-VAD-dependent manner (Fig.
2B). The loss of CPP32 represents cleavage and activation of
the caspase.

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Fig. 2.
The inhibition of the ERK pathway induces
apoptosis. HeLa cells incubated either in the absence or in the
presence of serum were treated for 12 h with 50 µM
PD98059 with or without 100 µM caspase inhibitor z-VAD.
The induction of apoptosis was determined by TUNEL analysis
(A). Results are the means ± S.D. of three independent
experiments with incubations in duplicate. B, parallel
extracts were immunoblotted with antibodies to phospho-ERK, ERK, or
CPP32. Essentially identical results were obtained in another two
experiments.
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Caspase Inhibition Blocks the Activation of p38 in PD98059-treated
Cells--
In the next series of experiments we determined whether the
stimulation of p38 is mediated by the caspase system. Thus, HeLa cells
maintained in serum-free medium for 24 h were incubated or not
with the MEK inhibitor either in the absence or in the presence of
z-VAD. Results of Fig. 3A
demonstrate that the activation of p38 is completely blocked by the
inhibition of caspases.

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Fig. 3.
PD98059-activated p38 is inhibited by z-VAD
but not by actinomycin D. HeLa cells incubated in the absence of
serum were treated with 50 µM PD98059 for 6 h with
or without 100 µM z-VAD (A) or 5 µg/ml
actinomycin D (Act. D) (B), after which cell
lysates from each experiment were analyzed in parallel gels by
immunoblotting with antibodies to phospho-ERK, phospho-p38, or p38.
Essentially identical results were obtained in another three
experiments.
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The Activation of p38 Is Independent of FADD--
Recent evidence
indicates that the stimulation of apoptosis by UV irradiation or myc
expression involves the activation of the Fas signaling cascade
(22-24). To determine whether the activation of p38 by the inhibition
of Erk may depend on the expression of Fas ligand, HeLa cells were
incubated with PD98059 either in the absence or in the presence of
actinomycin D. Results of Fig. 3B demonstrate that
inhibition of transcription has no effect on p38 activation. In
addition, reverse transcription-polymerase chain reaction and
immunoblot analysis demonstrate that Fas ligand levels were unaffected
upon the stimulation of HeLa cells with PD98059 (not shown). It is
possible that the ability of PD98059 to activate p38 is due to the
stimulation of the Fas system with no increase in the level of Fas
ligand (22, 23). Fas binds to FADD, which connects the receptor to the
caspase pathway through FADD-like interleukin-1
converting enzyme
(FLICE) (25, 26). A dominant negative mutant of FADD, which contains
just the death domain, not only inhibits the ability of Fas to induce
apoptosis but also that of tumor necrosis factor
(25, 27).
Therefore, we next determined whether the activation of p38 by the
inhibition of the MEK-ERK pathway is mediated by the FADD signaling
cascade. Thus, Flag-p38 was transfected along with an expression vector for dominant negative FADD in HeLa cells that were subsequently incubated in the absence of serum and activated with PD98059. Of note,
expression of dominant negative FADD has no effect on PD98059-induced
p38 (Fig. 4), indicating that the
activation of p38 by ERK inhibition is independent of the FADD-mediated
Fas and tumor necrosis factor
-signaling pathways.

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Fig. 4.
HeLa cells were transfected with a
Flag-tagged version of wild type p38 (10 µg) along with an empty
plasmid or an expression vector for a dominant negative FADD mutant (10 µg). Sixteen h post-transfection, cells were shifted to
serum-free medium, after which they were incubated with 50 µM PD98059 for 6 h. Afterward, Flag-tagged p38 was
immunoprecipitated, and its activity was determined by using
recombinant GST-Elk-1 as the substrate. Essentially identical results
were obtained in another three experiments.
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The PI 3-Kinase-Akt Pathway Regulates the Activation of
p38--
We next explored whether the blockade by serum of p38
activation depends on PI 3-kinase. HeLa cells were maintained either in
the absence or in the presence of serum and were incubated with the MEK
inhibitor either with or without wortmannin (an inhibitor of PI
3-kinase). Results of Fig. 5A
show that the ability of serum to block the activation of p38 by
PD98059 is completely abrogated by wortmannin, which has little or no
effect on cells incubated in the absence of serum. This suggests that
PI 3-kinase is required for the effects of serum on p38 stimulation.
The following experiments confirm these findings. Transfection of an
active PI 3-kinase mutant inhibited the activation by the MEK inhibitor
of a co-transfected Flag-tagged p38 construct (Fig. 5B).
Transfection of an active mutant of Akt also inhibits the activation of
p38, suggesting that Akt activation accounts for the inhibitory effects
of serum and PI 3-kinase on p38 activation. Consistently, transfection of the kinase-inactive mutant of Akt abrogates the inhibitory effect of
serum on the activation of p38 by the MEK inhibitor. Likewise,
expression of a dominant negative mutant of PI 3-kinase produces the
same effect (Fig. 5B).

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Fig. 5.
Role of PI 3-kinase in the ability of serum
to block PD98059-induced p38. A, HeLa cells incubated either
in the absence or in the presence of serum were treated with 50 µM PD98059 for 6 h with or without 100 nM wortmannin (WM), after which cell lysates
from each experiment were analyzed in parallel gels by immunoblotting
with antibodies to phospho-ERK, phospho-p38, or p38. Essentially
identical results were obtained in another three experiments.
B, HeLa cells were transfected with a Flag-tagged version of
wild type p38 (10 µg) along with an empty plasmid or expression
vectors for PI 3-kinase and Akt active mutants (10 µg) or dominant
negative Akt or a dominant negative p85 deletion mutant. Sixteen h
post-transfection, cells were either shifted to serum-free medium (in
the case of cells transfected with the active mutants) or kept in
serum-containing medium (in the case of the dominant negative mutants),
after which they were incubated with 50 µM PD98059 for
6 h. Afterward, Flag-tagged p38 was immunoprecipitated, and its
activity was determined by using recombinant GST-Elk-1 as the
substrate. The expression levels of the different constructs were
determined by using the corresponding anti-tag antibodies. Essentially
identical results were obtained in another three experiments.
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The PI 3-Kinase Pathway Interferes the p38 Cascade at the Level of
Caspase Activation--
To position the PI 3-kinase negative effects
on the PD98059-triggered p38 pathway, we initially determined whether
the inhibition of the ERK cascade actually activated the caspase
pathway. To this aim, the cleavage of CPP32 was determined in HeLa cell
cultures triggered with PD98059 either in the absence or in the
presence of serum. This treatment promoted the cleavage of CPP32 in a
time-dependent manner that was completely inhibited by the
presence of serum (Fig. 6A).
The CPP32 cleavage was reproducibly detectable at 10-30 min after ERK
inhibition and was maximal at 2 h (Fig. 6A). Of note,
wortmannin abrogated the inhibitory effects of serum (Fig. 6B), indicating that PI 3-kinase acts upstream in a pathway
leading to activation of the caspases, which are required for p38
stimulation by PD98059.

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Fig. 6.
Inhibition of the ERK pathway activates the
processing of CPP32. A, HeLa cells were incubated for
24 h either in the absence or in the presence of serum, after
which they were treated with 50 µM MEK inhibitor PD98059
for different times. Cell lysates were analyzed by immunoblotting with
an antibody to CPP32. B, HeLa cells incubated either in the
absence or in the presence of serum were treated with 50 µM of PD98059 for 1 h with or without 100 nM wortmannin, after which cell lysates were analyzed by
immunoblotting with an antibody to CPP32. Essentially identical results
were obtained in another three experiments. kD,
kilodalton.
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The PI 3-Kinase Pathway Inhibits PD98059-induced
Apoptosis--
Consistent with the above results are the data of Table
I showing that the reduction in cell
viability induced by the inhibition of the MEK-ERK pathway is not only
blocked by transfection of dominant negative p38 but also by expression
of active mutants of PI 3-kinase and Akt. This again demonstrates the
existence of a functionally relevant cross-talk between the MEK-ERK and PI 3-kinase pathways to control p38 activation and apoptosis.
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Table I
Effect of different kinase mutants on cell death induced by the
inhibition of the MEK-ERK pathway
HeLa cells were transfected with pCMV- gal (2.5 µg) and 5 µg of
either plasmid control pCDNA3 (control) or expression vectors for
active PI 3-kinase or Akt or a dominant negative p38 construct
(p38AGF). Sixteen-h post-transfection cells were changed to
serum-free medium for 24 h, after which they were treated or not
with PD98059 (50 µM) for 24 h, fixed, and stained
with 5-bromo-4-chloro-3-indolyl -D-galactopyranoside
(X-gal). Results (number of blue cells/35-mm dish) are the mean ± S.D. of three independent experiments with incubations in duplicate.
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DISCUSSION |
The role played by different signal transduction molecules in the
control of cell survival is the subject of intense discussion. The
stress-activated kinase cascades, which include the JNK/SAPK and the
p38 pathways, are activated in response to different apoptotic stimuli
and seem to play a decisive role in this process (3, 4, 6-9). Thus,
the simple activation of p38 by its physiological stimulant, MKK3/6, is
sufficient to induce apoptosis in transfected cells (7).
Conversely, expression of MKK3/6 or p38 dominant negative mutants
blocked apoptosis induced by nerve growth factor withdrawal (7) and UV
irradiation (8). On the other hand, there are two major kinase pathways
involved in the induction of cell survival: the ERK and the PI 3-kinase
cascades. Inhibition of ERK activity correlates with the activation of
JNK/SAPK and p38 as well as with the induction of apoptosis in nerve
growth factor-deprived PC-12 cells and UV-irradiated mouse fibroblasts (7, 8). Also, the inhibition of ERK below a basal threshold level
triggers apoptosis, suggesting that, in addition to its well
established role in cell cycle progression, ERK also controls survival.
The balance between the activity of the stress kinases to that of ERK
has been proposed to determine the cell fate (11). This is consistent
with results demonstrating the antagonistic effects of ceramide
versus sphingosine 1-phosphate. The former potently
activates the stress kinases, whereas the latter inhibits them and
activates ERK (28). This, again, suggests a link between both types of
mitogen-activated protein kinase cascades. Our own data shows that UV
irradiation or the inhibition of the atypical PKCs leads to an increase
in p38 activity and a decrease in ERK activity (8).
We show here that the simple inhibition of the ERK basal activity is
sufficient to trigger p38 activation in a caspase-dependent manner. This effect is blocked by the presence of serum through the PI
3-kinase-Akt signaling pathway that acts upstream of the caspase
system. Recent evidence suggests that the mechanism whereby certain
stimuli induce apoptosis may involve the activation of the Fas pathway
(22-24). In this case, however, we demonstrate that the blockade of
this pathway has no effect on the ability of the inhibition of ERK to
activate p38. Because the strategy employed here (the use of a FADD
dominant negative mutant) not only blocks Fas but also tumor necrosis
factor
signaling to cell death (25, 27), we conclude that neither
of these systems is involved in the activation of p38 by PD98059.
We have recently shown that the inhibition of the atypical isoforms of
PKC (
PKC and
/
PKC) is required for the induction of apoptosis
and the activation of p38 in mouse fibroblasts (8, 21). This involves
the interaction of
PKC and
/
PKC with the protein Par-4, whose
expression is sufficient to induce apoptosis in fibroblasts incubated
under low serum conditions (8, 21, 29). Interestingly, the ability of
Par-4 to induce apoptosis is a caspase-dependent process
that correlates with a decrease in ERK activity concomitant with an
increase in p38 activity (8). What we show here is that there is a link
between the inhibition of ERK, the activation of the caspase system,
and the stimulation of p38. These results are consistent with the model
depicted in Fig. 7, according to which an
apoptotic stress signal such as UV irradiation produces at least two
actions, one that by inducing Par-4, inactivates the atypical PKCs,
which leads to the inhibition of ERK, triggering the caspase system
that stimulates p38. Another action blocks PI 3-kinase, which allows
the caspase system to be operative. The fact that the atypical PKCs are
activated by PI 3-kinase products (30-33) indicates that these PKCs
are subjected to a double control mechanism in stress-activated cells:
the interaction with Par-4 and the inactivation of PI 3-kinase. From
all these results, at least three important issues arise for future
studies. One is the mechanism whereby Akt inhibits p38. Because the
caspase activation is required for p38 stimulation by PD98059, we are tempted to speculate that Akt may be affecting either directly or
indirectly the caspase system. The recent finding that Akt phosphorylates and inactivates Bcl-XL/Bcl-2-associated death promoter (34, 35) coupled with the existence of a link between Bcl-2 and the
caspases (36, 37) may explain why PI 3-kinase has to be inhibited for
p38 to be activated. The second issue, concerning the mechanism whereby
PI 3-kinase may be inhibited by UV irradiation, is less clear and will
need further work to be solved. However, the recent discovery that PI
3-kinase is blocked by c-Abl in the genotoxic stress response (38)
opens new possibilities for investigation. Finally, the mechanism
whereby p38 is activated in response to ERK inhibition remains to be
determined. We show here that the caspase system is essential, and
preliminary results demonstrate that it is mediated by
MKK3/6.2 Because the upstream
activator of MKK3/6 has not been established yet, it is very difficult
to address the link existing between this kinase and the caspases.
However, other evidences support a connection between the caspases and
the stress-activated kinases. Thus, Frisch and co-workers have recently
demonstrated that MEK kinase 1 activation during anoikis requires the
cleavage of the kinase by caspases, which seems critical for cell death
(39).
We are indebted to Esther Garcia, Carmen
Ibañez, and Beatriz Ranera for technical assistance. We want to
thank Gonzalo Paris and Isabel Perez for help and enthusiasm.