From the Grup de Neurobiologia Molecular, Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Universitat de Lleida, 25198 Lleida, Catalonia, Spain
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ABSTRACT |
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Evidence suggests that membrane depolarization is
able to promote neuronal survival through a sustained, although
moderate, increase in the intracellular calcium. We have used the PC12
cell line to study the possible intracellular pathways that can be activated by calcium influx. Previously, we observed that membrane depolarization-induced calcium influx was able to activate the extracellular-regulated kinase (ERK)/mitogen-activated protein kinase
pathway and most of this activation was
calmodulin-dependent. We demonstrated that a part of the
ERK activation is due to the phosphorylation of the epidermal growth
factor receptor. Here, we show that both the epidermal growth factor
receptor phosphorylation and the Shc-Grb2-Ras activation are not
calmodulin-modulated. Moreover, dominant negative mutant
Ha-ras (Asn-17) prevents the activation on ERKs by membrane
depolarization, suggesting that Ras and calmodulin are both necessaries
to activate ERKs by membrane depolarization. We failed to observe any
significant induction and/or modulation of the A-Raf, B-Raf or
c-Raf-1 kinase activities, thus suggesting the existence of a MEK
kinase different from the classical Raf kinases that directly or
indirectly can be modulated by Ca2+/calmodulin.
Several studies have reported that chronic depolarization of
plasma membrane prevent the cell death that occurs after deprivation of
neurotrophic factors in many populations of neurons in culture. This
effect is mediated by a sustained increase of the intracellular-free Ca2+ concentration
([Ca2+]i)1
that enters the cell through voltage-gated calcium channels (VGCC) (1-4). E. M. Johnson's laboratory conceptualized this phenomenon in the "Ca2+ set-point hypothesis," which postulates
that moderate increases in the
[Ca2+]i (less than 100 nM above the basal values) can promote the survival of
neurons in culture even in the absence of trophic support (for review,
see Ref. 5). However, the molecular basis relevant for the
depolarization-induced neuronal survival have been shown to be
controversial and remain to be elucidated (6-8). One possible
mechanism by which membrane depolarization could promote survival is by
activating signaling pathways similar to those activated by
neurotrophic factors. Among these, the Ras/MAP kinase and the
phosphatidylinositol 3-kinase (PI 3-kinase) pathways have been shown to
be the most relevant (9, 10).
Although membrane depolarization and subsequent increase in
[Ca2+]i is not able to promote
cell survival or differentiation in PC12, in low serum medium some
effects of increased [Ca2+]i have
been described. For example, membrane depolarization induced by adding
KCl (high-K+) to the culture medium is able to preserve
priming and preexisting neurites induced by NGF treatment (11). It can
also induce differentiation in cells sensitized with levels of NGF,
which alone are insufficient to induce morphological differentiation
(12). Finally, it has been reported that potassium combined with Bay K
induces long term survival and differentiation of PC12 cells (13).
Variations in the [Ca2+]i has been
shown to be determinant in the regulation of the Ras/MAP kinase pathway
through mechanisms not completely understood (14, 15). Rusanescu
et al. (13) demonstrated that an increase in
[Ca2+]i directly or indirectly
induces Shc tyrosine phosphorylation, which in turn associates with
Grb2 and Sos, resulting in the activation of Ras. Moreover, membrane
depolarization is able to induce tyrosine phosphorylation of the EGFR
to a sufficient extent to activate the ERK/MAP kinase pathway (16-18)
and this activation seems to be necessary to activate this signaling
pathway (18). Furthermore, PYK2, an intracellular tyrosine kinase
related to the focal adhesion kinase, is also activated by increases in
the [Ca2+]i that can in turn
activate Ras through a Src-dependent pathway (19).
As Ca2+ ions enter the cytosol, they encounter a number of
proteins that regulate their biochemical effects. Central among them is
calmodulin (CaM), a small Ca2+-binding protein, which can
bind up to four Ca2+ ions. After binding to
Ca2+, CaM changes its conformation and it is able to
regulate the activity of many different proteins. In several cell
types, CaM has been shown to modulate Ras either directly or
indirectly. Farnsworth et al. (20) have demonstrated
that Ca2+ stimulation of Ras can be mediated by CaM through
a Ras-GTP exchange factor which contains an IQ motif, referred to as
Ras-GRF (20). Moreover, some CaM-binding Ras-like GTPases have been
described (21, 22), even though its functional activity on MAP kinase activation has not yet been tested. These include RIT, which is widely
distributed in human tissues, and RIN, whose expression is unusually
confined to the nervous system. However, none of these proteins has
been shown to be present in the PC12 cell line. Additionally, CaM has
been shown to be able to regulate several CaM-dependent
protein kinases (CaM-K) being CaM K II and IV the best characterized.
CaM-K IV has been linked to the activation of several MAP kinases
(including JNK-1, p38, and to a lesser extent ERK2) (23). However,
CaM-K IV has been shown to be absent in PC12 cells and therefore the
involvement of this kinase in the activation of ERK MAP kinase is
controversial (23).
Recently, it has been suggested that PI 3-kinase can be involved in the
regulation of the ERK MAP kinase pathway although its implication seems
to be cell type- and ligand-specific (24). For example, in Swiss 3T3
fibroblasts, PI 3-kinase seems to regulate the prolonged activation of
the ERK MAP kinases (25). Additionally, it has been demonstrated that
integrin-dependent activation of the ERK MAP kinases is
reverted by wortmannin and LY294002, two selective PI 3-kinase
inhibitors (26). Moreover, a report from the laboratory of Sacks (27)
has shown that CaM is able to bind and modulate the activity of the PI
3-kinase. The relevance of these results in the PC12 cell system
remains to be proved.
Our laboratory has previously shown that membrane depolarization of
PC12 cells (17) and chicken motoneurons (8) is able to activate the ERK
MAP kinase pathway through a Ca2+/CaM-dependent
mechanism. In the present work, we have investigated more precisely the
level at which CaM is acting on the Ras/MAP kinase pathway. We show
here that this modulation is located lower to Ras but upstream of MEK.
Moreover, our results suggest that CaM action is independent of the
classical forms of Raf (c-Raf-1, A-Raf, or B-Raf) indicating the
existence of a MEK kinase, different from Raf and activated by a
Ras-dependent mechanism after membrane depolarization, that would be regulated directly or indirectly by
Ca2+/CaM.
Cell Culture, Cell Stimulation and Cell Lysates--
PC12 cells
were grown on 75-cm2 tissue culture dishes (Corning) in
Dulbecco's modified Eagle's medium supplemented with 6% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 6%
heat-inactivated horse serum (Life Technologies, Inc.). Medium was
further supplemented with 10 mM Hepes. The M-M17-26 PC12
subline was grown in RPMI 1640 medium supplemented with 5%
heat-inactivated fetal calf serum (Life Technologies, Inc.) and 10%
heat-inactivated horse serum (Life Technologies, Inc.). Culture medium
from two cell lines was further supplemented with 20 units/ml
penicillin and 20 µg/ml streptomycin. Cells were maintained at
37 °C in a saturating humidified atmosphere of 95% air and 5%
CO2.
For experiments, PC12 and M-M17-26 cells were allowed to proliferate
in polyornithine precoated tissue culture dishes (Corning) until they
reached 80% confluence. Before acute stimulation with NGF (100 ng/ml),
KCl (75 mM), or PMA (1.6 µM), cells were
washed three times and cultured for an additional 15-20 h in
serum-free medium. Before acute stimulations, the indicated cultures
were exposed to different protein inhibitors: the CaM inhibitors
calmidazolium chloride (Calbiochem-Novabiochem Corp., San Diego, CA),
trifluoperazine dimaleate (Calbiochem-Novabiochem Corp.), W5 and W7
(Sigma), and W12 and W13 (Sigma); the PKC inhibitor BIM I
(Calbiochem-Novabiochem Corp.); the MEK inhibitor PD98059
(Calbiochem-Novabiochem Corp.); or the PI 3-kinase inhibitor LY 294002 (Calbiochem-Novabiochem Corp.). After stimulation, cells were rinsed
rapidly in ice-cold phosphate-buffered saline at pH 7.2 and solubilized
at 4 °C in 0.4 ml of lysis buffer (see below). After 15 min of
incubation on ice, cells were scraped from the dishes and cell lysates
were orbitally rotated for 30 min at 4 °C. Nuclei and cellular
debris were removed by microcentrifuge centrifugation at 10,000 × g and 4 °C for 15 min. Protein concentration in the
supernatant was quantified by a modified Lowry assay as described by
the provider (Bio-Rad DC protein assay).
Western Blot--
Western blot assay was performed with
immunoprecipitates or cell lysates by resolving the proteins in
SDS-polyacrylamide gels. The proteins were transferred onto
polyvinylidene difluoride Immobilon-P transfer membrane filters
(Millipore, Bedford, MA) using a Pharmacia semidry Trans-Blot (Amersham
Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's
instructions. Antibodies against the phosphorylated forms of ERK1 and
ERK2, MEK1/2 and Akt (New England Biolabs, Inc., Beverly, MA), pan-ERK
and c-Raf-1 (Transduction Laboratories, Lexington, KY), pan-MEK (New
England Biolabs, Inc.), MEK1 (UBI, Lake Placid, NY), PLC Immunoprecipitation--
Immunoprecipitation of Shc, PLC MEK and Raf Kinase Activity Assay--
MEK in vitro
kinase assay was performed in MEK immunoprecipitates by using
recombinant GST-ERK2 (UBI) and [
Raf kinase activity was measured by using wild-type MEK1 (Santa Cruz
Biotechnology Inc.) and [ PI 3-Kinase Activity Assay--
After stimulation, cells were
solubilized in 1% Nonidet P-40 buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 mM
benzamidine, and 20 µg/ml leupeptin). Nuclei and debris were removed
by centrifugation, and approximately 750 µg of protein were subjected
to immunoprecipitation overnight at 4 °C with the anti-Tyr(P)
antibody, 4G10. Immunocomplexes were collected with protein A-Sepharose
preconjugated with a rabbit anti-mouse IgG antibody and sequentially
washed with lysis buffer, LiCl buffer (100 mM Tris, pH 7.5, 0.5 M LiCl, 1 mM EDTA, and 1 mM
sodium orthovanadate), and TNE buffer (25 mM Tris, pH 7.5, 100 mM NaCl, and 1 mM EDTA). Immunocomplexes
were incubated with a mixture of L- Ras Activity--
Ras activity was measured with a
non-radioactive method as described previously (28). Briefly, treated
cells were solubilized for 15 min in lysis buffer containing 25 mM Tris, pH 7.5, 5 mM EGTA, 15 mM
NaCl, 5 mM MgCl2, 1% Triton X-100, 1%
N-octyl- Materials--
The rest of biochemicals were obtained from
Sigma. Anti-Grb2 was a gift from Dr. J. Ureña (University of
Barcelona, Barcelona, Spain), anti-pan-Ras was from Dr. O. Bachs and
Dr. N. Agell (University of Barcelona, Barcelona, Spain), anti-EGFR was
from Dr. G. Capellà and Dr. C. García (Hospital de Sant
Pau, Barcelona, Spain), and anti-Tyr(P) (4G10) were from Dr. D. Martin-Zanca (CSIC-University of Salamanca, Salamanca, Spain). The
GST-RBD construct was obtained from Dr. F. McKenzie (State University
of New York, Stony Brook, NY) through Dr. O. Bachs and Dr. N. Agell.
The PC12 subline M-M17-26, kindly provided by Dr. G. M. Cooper
(Harvard Medical School, Boston, MA) through Dr. A. Aranda (CSIC,
Madrid, Spain), was obtained after transfection with the dominant
negative mutant Ha-ras (Asn-17). 7 S NGF was prepared in our
laboratory from salivary glands as described previously (29).
Functional Inhibitors of CaM Can Prevent the Depolarization-induced
Activation of the ERK MAP Kinase--
We have previously reported that
ERK MAP kinase activation induced by high-K+ in PC12 cells
is specifically blocked by the CaM antagonist W13, but not by its
structural analogue W12, which is about 5 times less potent (17, 30,
31) (Fig. 1A). In the present
report, this result has been extended to other CaM inhibitors that
include W7/W5 (30, 32) (Fig. 1B), calmidazolium (33, 34)
(Fig. 1C), and trifluoperazine (35, 36) (Fig.
1D). In all cases, these inhibitors showed a similar effect
to that elicited by W13, i.e. when PC12 cell were pretreated
with the inhibitors, they prevented the activation of the ERK MAP
kinase induced by high-K+ (Fig. 1). The blockade of
high-K+-induced ERK activation was
dose-dependent (data not shown) and specific, since
structural homologues (W5 and W12) of active inhibitors (W7 and W13,
respectively) were not able to block the ERK activation (Fig. 1,
A and B). Moreover, prevention of ERK activation
exerted by the CaM inhibitors was not mediated by an alteration of the calcium currents after membrane depolarization, i.e. these
drugs did not alter the kinetics of Ca2+ entry into the
cytoplasm (data not shown) (8, 17).
CaM Inhibitors Do Not Prevent Tyrosine Phosphorylation of the EGFR
Induced by Membrane Depolarization--
It has been previously
reported that transactivation of receptors with tyrosine kinase
activity such the EGFR in PC12 cells by membrane depolarization seems
to be necessary to reach a complete activation of the ERK MAP kinase
pathway (16-18). These data suggested us the possibility that CaM
inhibitors may exert its effects on the high-K+-induced
activation of ERKs indirectly through the blockade of the kinase
activity of the EGFR. To test this hypothesis, the EGFR was
immunoprecipitated from 2- and 5-min depolarized cells pretreated or
not with the CaM antagonists W12 and W13. To study the kinase activity
of the receptor, immunoprecipitates were analyzed on Western blot with
an anti-Tyr(P) antibody. As shown in Fig. 2, high-K+ was able to
activate the tyrosine kinase activity of the EGFR although to a much
lesser extent than EGF. However, in cell lysates where the ERK
phosphorylation due to membrane depolarization was completely prevented
by W13 (data not shown), neither W13 nor W12 pretreatment was able to
modify the level of tyrosine phosphorylation of the receptor (Fig. 2).
Therefore, the observed lack of ERK phosphorylation in the W13-treated
cultures after membrane depolarization could not be attributed to an
inhibition of the kinase activity of the EGFR.
CaM Inhibitors Modulate neither the Tyrosine Phosphorylation of Shc
nor the Association of Shc to Grb2--
The ERK MAP kinase cascade is
usually initiated by the interaction of trophic factors with their
corresponding tyrosine kinase receptors resulting in the
autophosphorylation of the receptor. Phosphorylated tyrosine kinase
receptors activate Ras by a mechanism that requires the tyrosine
phosphorylation of Shc (for review, see Ref. 9). The ability of Shc to
activate Ras is mediated by the association of Shc to Grb2 and Sos. It
has been previously reported that depolarization-induced ERK activation
can result from a direct depolarization-induced phosphorylation of
tyrosine kinase receptors such as the EGFR (18). Nevertheless, the
activation of EGFR was not sensitive to W13 (see above). Shc has also
been reported to play a central role in the Ca2+-induced
ERK activation after membrane depolarization (15). We have analyzed the
possibility that the W13 CaM antagonist can modulate the ERK activity
through the function of Shc. To test this possibility, we have studied
the effects of CaM antagonists over the tyrosine phosphorylation of Shc
and its association to Grb2 after membrane depolarization. For this,
PC12 cells were pretreated with W13 and then the cells were depolarized
for 2 and 5 min with 75 mM KCl. The structurally related
W12 homologue was included in the experiments to assess the specificity
of the W13 effects. Extracts were subjected to immunoprecipitation with an anti-Shc antibody that recognizes the 66-, 52-, and 46-kDa isoforms
of this protein. Immunoprecipitates were resolved in SDS-PAGE, blotted,
and probed with an anti-Tyr(P) antibody or, alternatively, with an
anti-Grb2 antibody. As shown in Fig.
3A, high-K+
induced the tyrosine phosphorylation of the 66-kDa isoform of the Shc
protein, although to a much lesser extent than NGF. The same pattern of
tyrosine phosphorylation was observed for the 52- and 48-kDa isoforms
of Shc protein (data not shown). W13 treatment did not affect the level
of tyrosine phosphorylation of the 66-kDa (Fig. 3A), 52- and
46-kDa isoforms (data not shown) of Shc protein at any time tested
after membrane depolarization. On the other hand, at 5 min,
high-K+ allowed the co-immunoprecipitation of Shc with
Grb2, although the amount of co-immunoprecipitated Grb2 was by far
lower to that obtained after NGF stimulation (Fig. 3B).
Nevertheless, as seen for the tyrosine phosphorylation of Shc, in
high-K+-stimulated W13-pretreated cultures, the amount of
Grb2 co-immunoprecipitated with Shc was not apparently decreased when
compared with cultures stimulated with 75 mM KCl alone
(Fig. 3B). When protein extracts from the different
experimental conditions described above were analyzed to assess the
state of ERK phosphorylation, it was found that membrane-depolarized,
W13-pretreated cultures, showed a significant reduction in the level of
ERK phosphorylation when compared with the extracts from depolarized
cultures without any drug pretreatment or pretreated with W12 (data not
shown). Therefore, it seems that W13 effects on ERK activity are not
attributable to an alteration in the function of the Shc proteins
because this drug modulates neither the tyrosine phosphorylation of Shc
proteins nor their association with Grb2 after membrane
depolarization.
Depolarization-induced PLC Modulation of the ERK MAP Kinase Pathway by CaM Inhibitors Occurs
Downstream of p21ras--
Rosen et al. (16)
have demonstrated that the Ca2+ influx induced by membrane
depolarization is able to activate ERK MAP kinases by a mechanism that
involves the small G protein p21ras in PC12
cells and in primary cultures of cortical neurons (14). Most of the
signaling pathways that have been proposed to be involved in the
activation of the ERK MAP kinases after increasing the [Ca2+]i converge on
p21ras, thus indicating a key role of this
protein in this process (15). In this context, we were interested into
know whether the W13 CaM inhibitor can explain its inhibitory effect on
ERK activity through the inhibition of the Ras activity. We approached
this issue using a method that has been previously reported by de Rooij and Bos (28). We found that a 2- or 5-min depolarizing stimulus was
able to moderately activate p21ras (Fig.
5A). However, W13 pretreatment
of cultures did not alter the level Ras activity induced by
high-K+ at any of the times tested. As an additional
control, the same extracts used in the Ras activity were analyzed by
Western blot with an anti-phospho-ERK antibody. Results shown in Fig.
5B demonstrate that the level of ERK phosphorylation in
high-K+-stimulated, W13-pretreated cultures was
significantly reduced than those stimulated with high-K+
without W13 pretreatment. These results indicate that the W13 modulation of the ERK activation does not depend on an effect of CaM
inhibitors on the classical form of Ras.
To determine whether endogenous Ras is necessary for
high-K+ to activate the ERK MAP kinase pathway we studied
the ability of membrane depolarization to activate ERKs in a PC12
subline (M-M17-26) that constitutively expresses the dominant
inhibitory Ha-ras (Asn-17) mutant (44). These cells respond
to membrane depolarization with a normal Ca2+ influx as
measured by using the fluorescent dye fura-2 (data not shown). When we
depolarized these cells, we observed that ERK phosphorylation was
strongly reduced in the M-M17-26 cells when compared with the level of
ERK phosphorylation observed in the wild type PC12 cells after membrane
depolarization (Fig. 5C). This result suggest that
high-K+ requires a functional p21ras
to activate the ERK MAP kinases.
The PI 3-Kinase/Akt Pathway Is Not Required for
Depolarization-dependent ERK Activation--
Another protein
that becomes tyrosine-phosphorylated and activated after trophic factor
receptor stimulation (i.e. Trk or EGFR) is the PI 3-kinase.
Recently, it has been suggested that PI 3-kinase can contribute to the
activation of ERK MAP kinases (24-26). Moreover, it has been suggested
that Ras may modulate the PI 3-kinase activity (45-49). Interestingly,
a recent study by Joyal et al. (27) has shown that CaM is
able to bind and modulate the activity of the PI 3-kinase. These
studies suggest that PI 3-kinase can be a key element in the mechanism
by which CaM antagonists exert its inhibitory effect over the
activation of the ERK after membrane depolarization. To study the
contribution of the PI 3-kinase on the activation of ERK MAP kinases
after high-K+ treatment, we have used several approaches.
We first studied the activation of this enzyme by assessing the state
of tyrosine phosphorylation of its p85 subunit that has been shown to
be a good criterion of its state of activation (6). Cells were
stimulated with high-K+ or NGF (used as a positive control)
for 1 or 5 min. Phosphoproteins from these cultures were
immunoprecipitated with an anti-Tyr(P) antibody and submitted to
Western blot analysis to detect the p85 subunit of PI 3-kinase using a
polyclonal antibody. This assay showed that membrane depolarization
failed to activate the PI 3-kinase (Fig.
6A). However, Western blot
from cells treated with NGF showed a clear band corresponding to p85
thus indicating the activation of this enzyme after NGF treatment (Fig.
6A). We have also measured the activity of PI 3-kinase in
different experimental conditions by immunoprecipitating
phosphoproteins and assaying the immunoprecipitates for their ability
to generate L-
Taken together, these results demonstrate that activation of ERK MAP
kinases after membrane depolarization does not depend on the activation
of PI 3-kinase/Akt pathway.
Depolarization-induced MEK Phosphorylation and MEK Activity Are
Inhibited by CaM Antagonists--
It has been reported that ERKs are
activated by phosphorylation on threonine and tyrosine residues by the
dual specificity MAPK/ERK kinases
MEK1 and MEK2 (54-56). In PC12 cells, the implication of MEK1 in the
activation of ERKs after membrane depolarization was first reported by
Rosen et al. (14). We have corroborated this result using
the selective MEK kinase inhibitor PD98059 (57, 58), which, when used
at 10-100 µM, blocked almost completely the
depolarization-induced ERK phosphorylation (Fig.
7A). The central role of MEK
in translating membrane depolarization into ERK activation, together
with the observation that CaM antagonists block ERK phosphorylation
(Fig. 1A), suggest that CaM inhibitors may exert its
inhibitory effect on ERK activity by blocking MEK activity. To examine
this possibility, we performed MEK kinase assays in which the ability
of the W13 CaM inhibitor to block MEK activity was tested. The increase
in MEK kinase activity observed in depolarized cultures (Fig.
7B) was almost completely abolished when cultures were
pretreated with W13 (Fig. 7B). These results seem to be
specific since W12 was found to be ineffective in preventing depolarization-induced MEK activity increase (data not shown). Thus,
these results suggest that the lack of phosphorylation on ERKs after
membrane depolarization in cultures pretreated with the W13 calmodulin
antagonist requires a functionally active MEK.
Although MEK is a dual specific serine/tyrosine kinase, it is itself
regulated by phosphorylation mainly on specific serines in response to
trophic factor stimulation by the Raf family of kinases (59, 60). On
the basis of our previous results, the question that arose was whether
the CaM antagonists block directly MEK activity or this inhibitory
effect occurs at some upstream element in the MAP kinase cascade. To
approach this issue, we have used an antibody that is able to detect
MEK1/2 only when phosphorylated at Ser-217/221, a situation that
reflects its functional activation. As shown in Fig. 7C, the
W13 calmodulin antagonist showed a dose-dependent
inhibition on depolarization-induced MEK phosphorylation. At 70 µM W13, the maximal inhibitory effect is achieved. At
this concentration, pretreatment with W12 did not have any significant
effect, supporting the specificity of the inhibitory effect observed
with W13 (Fig. 7D). Moreover, the kinetics of
phosphorylation blockade induced by W13 on MEK, i.e. when
measured after 2 or 5 min after depolarization, strongly correlated
with the kinetics of phosphorylation blockade of ERK (Fig.
7E), thus suggesting that MEK and ERK are in the same
CaM-dependent pathway.
A-Raf, B-Raf, and c-Raf-1 Activation after Membrane
Depolarization--
Our results show that W13 inhibits MEK activity
and MEK phosphorylation (Fig. 7, C and D). We
have also shown that Ras plays a critical role in the activation of
ERKs after membrane depolarization although W13 does not inhibits Ras
activity. These results argue for the existence of MEK kinases,
activated by Ras, that would be modulated directly or indirectly by
Ca2+-CaM. The best characterized kinases that are able to
activate MEK are the Raf family members, which include c-Raf-1, B-Raf, and A-Raf (55, 61). All of them have been reported to be expressed in
PC12 cells (54, 62). Moreover, the three Raf members are activated in
response to growth factors such as EGF or NGF, thus suggesting that
they can contribute to the activation of ERK MAP kinases after growth
factor stimulation (62). On the basis of our previous results, we
wanted to know whether W13 blockade of MEK phosphorylation after
membrane depolarization is due to an inhibitory effect of W13 over any
of the Raf isozymes. To approach this issue, we depolarized PC12 cells
for 5 min and we performed kinase assays in the immunoprecipitates
obtained with specific antibodies against each of the Raf isoforms.
A-Raf showed an undetectable kinase activity after NGF or
high-K+ treatments, suggesting that in our PC12 cell model,
none of these stimuli are able to activate this enzyme (Fig.
8A). On the other hand, B-Raf
showed a slight increase in its kinase activity after high-K+ or NGF treatment (Fig. 8B), whereas
c-Raf-1 activity was poorly stimulated after high-K+
treatment when compared with the activity obtained after NGF treatment
(Fig. 8C). The high-K+-induced kinase activity
in B-Raf and c-Raf-1 immunocomplexes was not blocked significantly by
the pretreatment of cultures with the W13 CaM antagonist (Fig. 8,
B and C). In all Raf activity studies, the state
of ERK phosphorylation was verified using specific anti-phospho-ERK
antibodies as described in Figs. 5B and 7E. In all cases, high-K+ induced a significant increment of ERK
phosphorylation that was specifically prevented by W13 pretreatment
(data not shown). Therefore, we conclude that the regulation of ERK
activity by the W13-sensitive pathway is not due to the inhibition of
any of the Raf activities.
We have previously reported that CaM is involved in the mediation
of ERK activation after membrane depolarization in chicken spinal cord
motoneurons (8) and PC12 cells (17). We observed this phenomenon by
using the W13 CaM antagonist. In the present work, we have broadened
this analysis in PC12 cells using other CaM inhibitors that include W7,
calmidazolium, and trifluoperazine. In all cases, the CaM antagonist
prevents the activation of ERK MAP kinases after membrane
depolarization. The aim of this work was to find the mechanism by which
W13 exerts its inhibitory effect.
Activation of the Ras-ERK MAP kinase pathway by membrane depolarization
was first reported by Rosen et al. (14). In that report, it
was demonstrated that p21ras has an important
role in mediating ERK MAP kinase activation after membrane
depolarization (14). The mechanism by which Ras is activated after
membrane depolarization has not been clearly defined. To date,
different Ca2+-dependent pathways have been
involved in this activation (15). Thus, it has been suggested that
membrane depolarization needs the activation of the EGFR, in a
ligand-independent manner, to activate ERKs (18). However, on the basis
of the results presented here (see also Ref. 17), the inhibition of ERK
activity by CaM inhibitors could not be explained by a modulation of
the EGFR phosphorylation by W13, since this inhibitor does not modify
the phosphorylation state of the receptor induced by membrane
depolarization (Fig. 2).
Shc plays a central role in most of the pathways proposed to activate
the Ras-ERK MAP kinase pathway after Ca2+ influx (15). As
proposed by Zwick et al. (18), the Shc tyrosine phosphorylation observed after membrane depolarization seems to be
dependent on the activation of the EGFR. Other authors have suggested
that activation of Shc after membrane depolarization is also dependent
on the activity of the intracellular tyrosine kinase Src (13).
Alternatively, the protein-tyrosine kinase PYK2 has been proposed to
play a key role in the activation of the Ras-ERK MAP kinase pathway
after Ca2+ influx through a Shc-dependent
mechanism (19). Our results demonstrate that CaM antagonists do not
modulate the Shc function since they do not modify the Shc tyrosine
phosphorylation or the association of Shc to Grb2 after membrane
depolarization, thus suggesting that CaM modulation occurs downstream
of Shc.
The activation of the PLC The most important point in the present analysis is to test the
relevance of the activation of Ras observed after membrane depolarization in the activation of ERK MAP kinases. For this, we used
the M-M17-26 PC12 cell line, which constitutively express a dominant
negative form of Ras. Moreover, we have used this cell line since these
cells responds with an normal entry of Ca2+ after membrane
depolarization, contrary to what it has been reported for other PC12
cell lines that express inducible forms of dominant negative Ras such
as GsrasDN6 PC12 cells (14). In M-M17-26 cells, depolarization is
unable to activate ERKs, suggesting that Ras activity is necessary for
membrane depolarization-induced ERK activation, as has been reported
for similar systems (13, 14). This result discards the possibility that
parallel pathways to Ras can be operative in depolarized PC12 cells to
activate ERKs. When we studied the level of Ras activity in depolarized
wild type PC12 cells, a significant increase in Ras activity was found, as measured by the method described by de Rooij and Bos using the
GST-RBD tool (28). However, pretreatment of cells with W13 does not
significantly modify the Ras activity. This is a relevant result since
it suggest that proteins with a GTP exchange activity that contains CaM
binding domains, such as Ras-GRF, are not probably involved in the
regulation of Ras activity after membrane depolarization in PC12 cells
(20).
Recent studies have shown that PI 3-kinase could be a good candidate to
explain the CaM-dependent effects on the ERK MAP kinase pathway activation after membrane depolarization. First, the kinase activity of this enzyme has been shown to be modulated by CaM in
vitro (27). Second, PI 3-kinase has been reported to become activated after Ca2+ influx in cerebellar granule neurons
(7). Third, it has been demonstrated that PI 3-kinase may be a
regulable molecule downstream of Ras in some cellular systems (45-49).
Forth, and more important, it has been reported that PI 3-kinase
activity is able to modulate the ERK MAP kinase pathway in some
cellular models (25, 26). However, our results clearly demonstrate
that, after membrane depolarization, neither PI 3-kinase activity nor
Akt phosphorylation, a downstream element of the PI 3-kinase, can be
detected. Moreover, the selective PI 3-kinase inhibitor LY 294002 was
not able no prevent the activation of the ERK MAP kinases after
membrane depolarization at doses that are effective to inhibit the Akt
phosphorylation after NGF stimulation. Therefore, these results suggest
that the PI 3-kinase does not mediate the activation of the ERK MAP
kinases after membrane depolarization in PC12 cells. These observations are in agreement with reports that depolarization-induced survival is
not dependent on the PI 3-kinase activity in some neuronal populations
(6, 8).2
Functionally active MEK has been reported to be necessary to reach a
complete activation of ERK MAP kinases after membrane depolarization
(14). Here we provide further evidence on this phenomenon by using the
selective MEK inhibitor, PD 98059. When used at the appropriate
concentrations, PD 98059 is able to prevent ERK phosphorylation after
membrane depolarization. Moreover, the CaM inhibitor W13 is able to
modulate both the MEK activity and its state of phosphorylation to a
sufficient extent to explain the absence of ERK phosphorylation and
activity in high-K+-stimulated PC12 cells. This observation
suggests that CaM regulation of ERK activity occurs upstream of MEK.
PC12 cells possess the three isoforms of the MEK kinase Raf,
i.e. A-, B-, and c-Raf-1 (54, 62). We addressed the study of
the involvement of these enzymes in the MEK activation after membrane
depolarization. Our results demonstrate that the activity of Raf
isozymes in the activation of MEKs after membrane depolarization is not
very relevant. First, we were unable to detect any significant A-Raf
activation after high-K+-stimulation in PC12 cell cultures.
Second, B-Raf activation after membrane depolarized was comparable to
that obtained after NGF-stimulation. However the total level of
activation is very low (~20-30% of increase respect to the
unstimulated cells). More important, the CaM inhibitor W13 is poorly
effective in preventing the activation of B-Raf after membrane
depolarization. Moreover, B-Raf activity has been involved in
sustained, rather than transient activation of the ERK MAP kinases (62,
65, 66). Therefore, membrane depolarization, which promotes a rapid and
transient activation of ERKs (17), will not probably use B-Raf as an
effector system. Finally, c-Raf-1 has been involved in the acute
stimulation of the ERK MAP kinases after NGF or EGF stimuli in PC12
cells (62). In our hands, c-Raf-1 is only moderately activated
(~60%) after membrane depolarization. In the parallel assays, we
have found 500-600% activation when PC12 cells were stimulated with
NGF. It is then clear that the rapid activation of ERKs observed after membrane depolarization could not be attributable the moderate activation of c-Raf-1. The controls established in order to assess the
amount of immunoprecipitated enzyme in each assay demonstrated that the
three Raf isozymes are present in our PC12 cells. Therefore, the poor
or undetectable Raf activity obtained in high-K+ treated
cultures can be attributable neither to a lack of the presence of these
enzymes in our cells nor to a inefficiency of the antibody to
immunoprecipitate them. Therefore, these results indicate the existence
of alternative forms of MEK kinases, different from the Raf family
members, that would be more relevant in the translation of the raising
in [Ca2+]i into the ERK MAP kinase
pathway activation.
Taken together, our results suggest that Ras and CaM are both necessary
to activate the ERK MAP kinases after membrane depolarization. This
activation seems to be independent of the PI 3-kinase/Akt pathway and
of the PKC activity. Moreover, our results point out that CaM
regulates, directly or indirectly, the activity of a MAP kinase kinase
kinase, activated by Ras and different from Raf isozymes, that would be
the main element involved in the activation of MEK and ERK MAP kinases
after membrane depolarization. Activation of MEK and ERK MAP kinases
independent of Raf has been previously reported in other cell systems.
For example in Swiss-3T3 and COS 7 cells, cAMP, which is able to block
the activation of c-Raf-1, is not able to prevent the activation of MEK
and ERK activities (67). On the other hand, dominant positive mutants
of the atypical PKC
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
EXPERIMENTAL PROCEDURES
(Transduction Laboratories), EGFR, A-Raf and B-Raf (Santa Cruz
Biotechnology Inc., Santa Cruz, CA), or pan-Shc (Transduction
Laboratories) were used according to the supplier instructions. After
incubation with specific peroxidase-conjugated secondary antibodies,
membranes were developed with an enhanced chemiluminescence Western
blotting detection system (Pierce)
, or
c-Raf-1 (Transduction Laboratories) and EGFR, A-Raf, or B-Raf (Santa
Cruz Biotechnology Inc.) was performed with specific antibodies
according to the supplier's instructions. Immunoprecipitated proteins
were electrophoresed, transferred, and detected essentially as
described above. To detect Grb2 association in Shc immunoprecipitates,
membranes were blocked with TBS-T20 (20 mM Tris-HCl, pH
7.4, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat
milk, probed with a polyclonal anti-Grb2 antibody diluted in TBS-T20
containing 0.2% nonfat milk and, finally, incubated with a specific
peroxidase-conjugated secondary antibody. To detect
tyrosine-phosphorylated PLC
and EGFR, membranes were blocked with
TBS-T20 containing 5% bovine serum albumin, probed with the 4G10
anti-phosphotyrosine monoclonal antibody (anti-Tyr(P)) and incubated
with a specific peroxidase-conjugated secondary antibody. To
immunoprecipitate the p85 subunit of the PI 3-kinase, extracts from
NGF-treated or depolarized PC12 cells were subjected to
immunoprecipitation overnight at 4 °C with the anti-Tyr(P) antibody
4G10 (1/100). Immunocomplexes were precipitated with protein
A-Sepharose coupled to rabbit anti-mouse polyclonal antibody. p85 was
detected using an specific anti-p85 antibody (UBI) as described by the supplier.
-32P]ATP (Amersham
Pharmacia Biotech) as substrates. After treatment, cells were lysed
with lysis buffer (1% Nonidet P-40, 0.25% deoxycholate, 50 mM Tris, pH 7.5, 1 mM EGTA, 50 mM
-glycerophosphate, 150 mM NaCl, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 mM
benzamidine, and 20 µg/ml leupeptin). After the removal of nuclei and
cellular debris, cell lysates were precleared for 1 h at 4 °C
with 20 µl (v/v) of protein A-Sepharose. Five hundred µg of protein
of the supernatant was transferred to a new tube, and the anti-MEK1
antibody was added (1/250) (UBI). After 2 h at 4 °C,
immunocomplexes were precipitated with 40 µl (v/v) of protein
A-Sepharose for an additional 1 h at 4 °C. Precipitates were
washed three times with lysis buffer and three more times with assay
kinase buffer (20 mM MOPS, pH 7.2, 1 mM
dithiothreitol, 5 mM EGTA, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate). Precipitates were resuspended in 50 µl (final volume) of assay kinase
buffer supplemented with 100 µM ATP, 15 mM
MgCl2, 6 µCi of [
-32P] ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech), and 400 ng of GST-ERK2 (UBI).
Kinase assay was allowed to proceed for 30 min at 30 °C. Reaction
was stopped with 5× SDS-PAGE sample buffer, and products were
separated by SDS-PAGE. After drying the gel, the phosphorylation signal
was quantified on a PhosphorImager (Boehringer Mannheim). Radioactive
spots were also detected by autoradiography by exposing the TLC plate
to Fuji medical x-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan)
overnight at -70 °C.
-32P] ATP (Amersham Pharmacia
Biotech) as substrates. After treatments, cells were lysed with lysis
buffer (1% Triton X-100, 20 mM Tris, pH 7.5, 2 mM EDTA, 50 mM
-glycerophosphate, 137 mM NaCl, 25 mM NaF, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 mM benzamidine, and 20 µg/ml
leupeptin). After the removal of nuclei and cellular debris, cell
lysates were precleared for 1 h at 4 °C with 20 µl (v/v) of
protein G-Sepharose. Seven hundred and fifty µg of protein of the
supernatant was transferred to a new tube, and 1 µg of anti-c-Raf-1
antibody (Transduction Laboratories), anti-B-Raf antibody, or
anti-A-Raf antibody (Santa Cruz Biotechnology Inc.) was added. After
2 h at 4 °C, immunocomplexes were precipitated with 40 µl
(v/v) of protein G-Sepharose for an additional 1 h at 4 °C.
Precipitates were washed three times with lysis buffer and three more
times with assay kinase buffer (25 mM Hepes, pH 7.5, 1 mM dithiothreitol, 1 mM EGTA, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate).
Precipitates were resuspended in 25 µl (final volume) of assay kinase
buffer supplemented with 100 µM ATP, 50 mM
MgCl2, 6 µCi of [
-32P] ATP (3000 Ci/mmol) (Amersham Pharmacia Biotech), and 120 ng of wild type MEK1
(Santa Cruz Biotechnology Inc.). Kinase assay was allowed to proceed
for 20 min at 30 °C. Reaction was stopped with 5× SDS-PAGE sample
buffer, and products were separated by SDS-PAGE. After drying the gel,
the phosphorylation signal was quantified and detected as described above.
-phosphatidylinositol
and L-
-phosphatidyl-L-serine (final
concentration 0.5 mg/ml each) and 10 µCi of [
-32P]
ATP. Incubation was allowed to proceed for 20 min at room temperature. Phosphorylated lipids were then extracted and resolved by TLC using
n-propanol:H2O:acetic acid (66:33:2, v:v:v) as
solvent. Radioactive spots were detected by autoradiography by exposing the TLC plate to Fuji medical x-ray film (Fuji Photo Film Co. Ltd.)
overnight at -70 °C.
-D-glucopyranoside, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 mM
benzamidine, and 20 µg/ml leupeptin. Nuclei and cellular debris were
removed, and 50 µg of the recombinant GST-RBD protein previously
coupled to glutathione-Sepharose (Amersham Pharmacia Biotech) were
added to approximately 750 µg of protein. Protein complexes were
allowed to form for 2 h at 4 °C. Precipitates were washed three
times with lysis buffer without
N-octyl-
-D-glucopyranoside and once with
phosphate-buffered saline. Finally precipitates were resuspended with
SDS-PAGE loading buffer and denatured proteins were loaded in a 12%
SDS-PAGE. Immunodetection was done using an anti-pan-Ras antibody
(Oncogene Research Products, Cambridge, MA) and a anti-mouse IgG
coupled to horseradish peroxidase as a secondary antibody. Blots were
developed with the enhanced chemiluminescence Western blotting
detection system described above.
RESULTS
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Fig. 1.
Inhibition of depolarization-induced ERK
phosphorylation after pretreatment with several CaM antagonists.
PC12 cells were serum-starved, and pretreated (+) or not ( ) for
1 h with different CaM antagonists: 70 µM W12 or W13
(A), 100 µM W5 or W7 (B), 25 µM calmidazolium (CMZ) (C), 50 µM trifluoperazine (TFP) (D), or
with the vehicle control (0.1% Me2SO) (B-D)
and then stimulated (+) or not (
) for 5 min with 75 mM
KCl. After treatment, cells were lysed and protein extracts were
analyzed on Western blot with an anti-phospho-ERK1/2 antibody
(upper panels) and stripped and reprobed with an
anti-pan-ERK antibody (lower panels) as a control
of the protein content per lane. Arrows labeled
P-ERK1 and P-ERK2 or ERK1 and
ERK2 indicate the position of the phosphorylated and
non-phosphorylated forms of ERK1 and ERK2 proteins, respectively.
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Fig. 2.
Induction of EGFR tyrosine phosphorylation
following membrane depolarization and after pretreatment with W13.
PC12 cells were serum-starved, and pretreated (+) or not ( ), for
1 h with 70 µM W12 or W13 and then stimulated (+) or
not (
), for 2 or 5 min with 75 mM KCl, for 5 min with 100 ng/ml NGF or left unstimulated (NS). After treatment, cells
were lysed and protein extracts were subjected to immunoprecipitation
with an anti-EGFR antibody and immunoprecipitates were analyzed on
Western blot with the 4G10 anti-Tyr(P) antibody (upper
panel) and stripped and reprobed with an anti-EGFR antibody
(lower panel) as a control of the protein content
per lane. EGFR-labeled arrows indicate the
position of the EGFR protein.
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Fig. 3.
Induction of Shc tyrosine phosphorylation and
Grb2-Shc association following membrane depolarization and after
pretreatment with W13. A, PC12 cells were
serum-starved, and pretreated (+) or not ( ), for 1 h with 70 µM W12 or W13 and then stimulated (+) or not (
), for 2 or 5 min with 75 mM KCl, for 5 min with 100 ng/ml NGF or
left unstimulated (NS). After treatment, cells were lysed
and protein extracts were subjected to immunoprecipitation with an
anti-Shc antibody and immunoprecipitates were analyzed on Western blot
with the 4G10 anti-Tyr(P) antibody (upper panel)
and stripped and reprobed with an anti-Shc antibody (lower
panel) as a control of the protein content per lane. Shc
labeled arrows indicate the position of the 66-kDa Shc protein.
B, PC12 cells were serum-starved, pretreated (+) or not
(
), for 1 h with 70 µM W12 or W13 and then
stimulated (+) or not (
), for 5 min with 75 mM KCl, with
100 ng/ml NGF or left unstimulated (NS). After treatment,
cells were lysed and protein extracts were subjected to
immunoprecipitation with an anti-Shc antibody and immunoprecipitates
were analyzed on Western blot with an anti-Grb2 antibody
(upper panel) and stripped and reprobed with an
anti-Shc antibody (lower panel) as a control of
the protein content per lane. Grb2- and
Shc-labeled arrows indicate the position of the Grb2 and
66-kDa Shc proteins, respectively. T.L., total cell
lysate.
Tyrosine Phosphorylation Is Not
Prevented by CaM Inhibitors: Involvement of PKC--
Besides the
tyrosine phosphorylation of Shc it has been suggested that activated
tyrosine kinase receptors were able to activate ERKs through a
PLC
-dependent mechanism (9, 37). PLC
activity generates diacylglycerol that, together with Ca2+ ions, act
as activators of PKC (38). PKC activity can be the mechanism by which
PLC
activates Ras-ERK MAP kinase pathway since phorbol esters, which
mimic diacylglycerol, are able to activate ERKs (39-42). Since
Shc-Grb2 association is not affected by W13, we have explored whether
or not membrane depolarization is able to activate PLC
. In order to
address this issue, protein extracts similar to those obtained for the
study of the Shc-Grb2 interaction were subjected to PLC
immunoprecipitation with a specific antibody and immunoprecipitates
were probed with an anti-Tyr(P) antibody. The results of this assay
showed that PLC
becomes tyrosine-phosphorylated after
high-K+ treatment (Fig.
4A), although to a lesser
extent to that obtained after NGF stimulation (Fig. 4A).
This result indicates that membrane depolarization can induce a
redundant ERK activation through a PLC
- and
Shc-dependent mechanism. We then further assayed whether or
not CaM inhibitor W13 was able to prevent depolarization-induced PLC
tyrosine phosphorylation. As shown in Fig. 4A, W13 is not able to significantly alter the degree of tyrosine phosphorylation of
PLC
when compared with that induced by high-K+ on
cultures treated with W12 or in non-pretreated cultures. Therefore, the
W13 effects on depolarization-induced ERK activity cannot be explained
by a blockade of the PLC
activation. The limited activation of
PLC
observed after membrane depolarization suggests that this enzyme
is not involved in the ERK activation found in depolarized cells.
However, to further analyze the relevance of this PLC
activity on
the activation of ERKs, we assessed the involvement of PKC. To approach
this issue, we used the specific PKC inhibitor BIM I (43) and we
studied whether or not this drug was able to prevent the activation of
ERKs after membrane depolarization. As shown in Fig. 4B,
concentrations of the inhibitor from 1 to 5 µM failed to
inhibit the depolarization-induced ERK phosphorylation. As a control,
we used the phorbol ester PMA (1.6 µM), which induces ERK
phosphorylation through a PKC-dependent mechanism. As shown
in Fig. 4C, PMA induced a strong ERK phosphorylation that
was totally prevented by BIM I. These results, together with the poor
activation of PLC
, suggest that the hypothetical contribution of the
PLC
-PKC pathway to the activation of ERKs after membrane depolarization seems not very relevant.
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Fig. 4.
Induction of PLC tyrosine
phosphorylation following membrane depolarization and after
pretreatment with W13: involvement of PKC in the induction of ERK
activity after membrane depolarization. A, PC12 cells
were serum-starved, pretreated (+) or not (
), for 1 h with 70 µM W12 or W13 and then stimulated (+) or not (
), for 5 min with 75 mM KCl or with 100 ng/ml NGF. After treatment,
cells were lysed and protein extracts were subjected to
immunoprecipitation with an anti-PLC
antibody and immunoprecipitates
were analyzed by Western blot with the 4G10 anti-Tyr(P) antibody
(upper panel) and reprobed with an anti-PLC
antibody (lower panel) as a control of the
protein content per lane. PLC
-labeled arrows
indicate the position of PLC
protein. B, PC12 cells were
serum-starved, pretreated (+) or not (
), for 1 h with the
indicated concentrations of the PKC inhibitor BIM I or with the vehicle
(0.17% Me2SO), and then stimulated (+) or not (
), for 5 min with 75 mM KCl. After treatment, cells were lysed and
protein extracts were analyzed on Western blot with an
anti-phospho-ERK1/2 antibody (upper panel) and
stripped and reprobed with an anti-pan-ERK antibody (lower
panel) as a control of the protein content per lane.
Arrows labeled P-ERK1 and P-ERK2 or
ERK1 and ERK2 indicate the position of the
phosphorylated and non-phosphorylated forms of ERK1 and ERK2 proteins,
respectively. C, PC12 cells were serum-starved, pretreated
(+) or not (
), for 1 h with the indicated concentrations of the
PKC inhibitor BIM I or with the vehicle (0.17% Me2SO), and
then stimulated (+) or not (
), for 5 min with 1.6 µM
PMA. After treatment, cells were lysed and protein extracts were
analyzed on Western blot with an anti-phospho-ERK1/2 antibody
(upper panel) and stripped and reprobed with an
anti-pan-ERK antibody (lower panel) as a control
of the protein content per lane. Arrows labeled
P-ERK1 and P-ERK2 or ERK1 and
ERK2 indicate the position of the phosphorylated and
non-phosphorylated forms of ERK1 and ERK2 proteins, respectively.
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Fig. 5.
Profile of p21ras activation
following membrane depolarization after pretreatment with W13.
PC12 cells were serum-starved, pretreated (+) or not ( ), for 1 h
with 70 µM W13 or W12, and then stimulated (+) or not
(
), for 2 or 5 min with 75 mM KCl or for 5 min with 100 ng/ml NGF. After treatment, cells were lysed and protein extracts were
obtained. A, protein extracts were subjected to
precipitation with 50 µg of the recombinant GST-RBD protein
precoupled to protein G-Sepharose (see "Experimental Procedures").
Precipitates were analyzed by Western blot with an anti-pan-Ras
antibody. TL, total cell lysate. GST-RBD- and
Ras-labeled arrows indicate the position of the
recombinant GST-RBD protein and the p21ras
protein, respectively. B, protein extracts were analyzed on
Western blot with an anti-phospho-ERK1/2 antibody (upper
panel) and stripped and reprobed with an anti-pan-ERK
antibody (lower panel) as a control of the
protein content per lane. Arrows labeled P-ERK1
and P-ERK2 or ERK1 and ERK2 indicate
the position of the phosphorylated and non-phosphorylated forms of ERK1
and ERK2 proteins, respectively. C, wild type PC12 cells
(WT) and M-M17-26 cells (MM), a PC12 subline
that constitutively expresses the dominant inhibitory Ha-ras
(Asn-17) mutant, were serum-starved and then stimulated (+) or not
(
), for 5 min with 75 mM KCl or with 100 ng/ml NGF. After
treatment, cells were lysed and protein extracts were analyzed on
Western blot with an anti-phospho-ERK1/2 antibody (upper
panel) and stripped and reprobed with an anti-pan-ERK
antibody (lower panel) as a control of the
protein content per lane. Arrows labeled P-ERK1
and P-ERK2 or ERK1 and ERK2 indicate
the position of the phosphorylated and non-phosphorylated forms of ERK1
and ERK2 proteins, respectively.
-phosphatidylinositol 3-phosphate from
L-
-phosphatidylinositol (PI). The results obtained with
the different experimental conditions were essentially the same as
those described for the p85 precipitation, i.e. membrane depolarization was unable to activate the PI 3-kinase whereas NGF
induces a strong activation (Fig. 6B). Consistent with this result, when the state of phosphorylation of Akt, a well known downstream element of the PI 3-kinase pathway (50-52), was tested with
an anti-phospho-Akt-specific antibody, we found that NGF was able to
phosphorylate Akt whereas membrane depolarization was not (Fig.
6C). Finally, LY295002, a specific inhibitor of the PI
3-kinase activity (53), was not able to prevent the
high-K+-induced ERK phosphorylation in PC12 cultures (Fig.
6D). Nevertheless, at the dose of 25 µM,
LY295002 was able to prevent the Akt phosphorylation induced by NGF
thus demonstrating that the drug was effective to inhibit PI 3-kinase
activity (Fig. 6E).
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Fig. 6.
Effect of membrane depolarization on PI
3-kinase/Akt pathway. A, PC12 cells were serum-starved
and then stimulated for 1 or 5 min with 75 mM KCl, 100 ng/ml NGF, or left unstimulated (NS). After treatment, cells
were lysed and protein extracts were subjected to immunoprecipitation
with the 4G10 anti-phosphotyrosine antibody. Immunoprecipitates were
analyzed on Western blot with an anti-p85 antibody.
p85-labeled arrow indicates position of the p85
protein. B, PC12 cells were serum-starved and then
stimulated for 1 min with 75 mM KCl, 100 ng/ml NGF, or left
unstimulated (NS). After treatment, cells were lysed and
protein extracts were subjected to immunoprecipitation with the 4G10
anti-phosphotyrosine antibody. PI 3-kinase activity was assayed in the
immunoprecipitates as described under "Experimental Procedures"
with L- -phosphatidylinositol as substrate.
PI-3P-labeled arrow indicates de position of the
L-
-phosphatidylinositol 3-phosphate.
Origin-labeled arrow indicates the position of
the point of sample application. C, PC12 cells were
serum-starved and then stimulated for 5 min with 75 mM KCl,
100 ng/ml NGF, or left unstimulated (NS). After treatment,
cells were lysed and protein extracts were analyzed on Western blot
using an anti-phospho-Akt antibody (upper panel)
and stripped and reprobed with an anti-pan-ERK antibody
(lower panel) as a control of the protein content
per lane. P-Akt-1- and ERK2-labeled
arrows indicate the position of the phosphorylated form of
Akt-1 and ERK2 proteins, respectively. D, PC12 cells were
serum-starved, pretreated or not (
), for 30 min with the indicated
concentrations of LY 294002 or with the vehicle (0.2%
Me2SO) and then stimulated (+), or not (
), for 5 min with
75 mM KCl. After treatment, cells were lysed and protein
extracts were analyzed on Western blot with an anti-phospho-ERK1/2
antibody (upper panel) and stripped and reprobed
with an anti-pan-ERK antibody (lower panel) as a
control of the protein content per lane. Arrows labeled
P-ERK1 and P-ERK2 or ERK1 and
ERK2 indicate the position of the phosphorylated and
non-phosphorylated forms of ERK1 and ERK2 proteins, respectively.
E, PC12 cells were serum-starved, pretreated (+) or not
(
), for 30 min with 25 µM LY 294002 or with the vehicle
(0.05% Me2SO), and then stimulated (+) or not (
), for 5 min with 100 ng/ml NGF. After treatment, cells were lysed and protein
extracts were analyzed on Western blot with an anti-phospho-Akt
antibody (upper panel) and stripped and reprobed
with an anti-pan-ERK antibody (lower panel) as a
control of the protein content per lane. P-Akt-1- and
ERK2-labeled arrows indicate the position of the
phosphorylated form of Akt-1 and ERK2 proteins, respectively.
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Fig. 7.
Inhibition of depolarization-induced MEK
activity and MEK phosphorylation after pretreatment with W13.
A, PC12 cells were serum-starved, pretreated or not ( ),
for 30 min with the indicated concentrations of the MEK inhibitor PD
98059 or with the vehicle (0.2% Me2SO), and then
stimulated (+) or not (
), for 5 min with 75 mM KCl. After
treatment, cells were lysed and protein extracts were analyzed on
Western blot with an anti-phospho-ERK1/2 antibody (upper
panel) and stripped and reprobed with an anti-pan-ERK
antibody (lower panel) as a control of the
protein content per lane. Arrows labeled P-ERK1
and P-ERK2 or ERK1 and ERK2 indicate
the position of the phosphorylated and non-phosphorylated forms of ERK1
and ERK2 proteins, respectively. B, PC12 cells were
serum-starved, pretreated (+) or not (
), for 1 h with 70 µM W13 or W12, and then stimulated for 2 or 5 min with 75 mM KCl, for 5 min with 100 ng/ml NGF, or left unstimulated
(NS). After treatment, cells were lysed and protein
extracts were subjected to immunoprecipitation with an anti-MEK
antibody. Immunoprecipitates were used to determine the kinase activity
of MEK (see "Experimental Procedures") using recombinant GST-ERK2
as substrate (upper panel) and were analyzed on
Western blot with the same antibody used in the immunoprecipitation
step (lower panel) as a control of the enzyme
content in the immunoprecipitates. MEK activation was quantitated with
a Phosphor Imager and is expressed as the relative kinase activity
where the activity of untreated cells was taken as equal to unity.
C, PC12 cells were serum-starved, pretreated or not (
),
for 1 h at the indicated concentrations of W13, and then
stimulated (+) or not (
), for 5 min with 75 mM KCl. After
treatment, cells were lysed and protein extracts were analyzed on
Western blot with an anti-phospho-MEK1/2 antibody (upper
panel) and stripped and reprobed with an anti-pan-MEK1/2
antibody (lower panel) as a control of the
protein content per lane. D, PC12 cells were serum-starved,
pretreated (+) or not (
), for 1 h with 70 µM W13
or W12, and then stimulated (+) or not (
), for 5 min with 75 mM KCl. After treatment, cells were lysed and protein
extracts were analyzed on Western blot with an anti-phospho-MEK1/2
antibody (upper panel) and stripped and reprobed
with an anti-pan-MEK1/2 antibody (lower panel) as
a control of the protein content per lane. E, PC12 cells
were serum-starved, pretreated (+) or not (
), for 1 h with 70 µM W13, and then stimulated for 2 or 5 min with 75 mM KCl, for 5 min with 100 ng/ml NGF or left unstimulated
(NS). After treatment, cells were lysed and protein extracts
were analyzed on Western blot sequentially with an anti-phospho-MEK1/2
antibody (1; P-MEK1/2), stripped and reprobed
with an anti-pan-MEK1/2 antibody (2; MEK1/2),
stripped and reprobed with an anti-phospho-ERK1/2 (3;
P-ERK1/2), and stripped and reprobed with an anti-pan-ERK
antibody (4; ERK). The Western blots with the
anti-pan-MEK1/2 antibody (2; MEK1/2) and the
anti-pan-ERK antibody (4; pan-ERK) were used as a
control of the protein content per lane. In C-E,
P-MEK1/2- and MEK1/2-labeled arrows
indicate the position of phosphorylated and non-phosphorylated forms of
MEK1/2 proteins, respectively. In E, arrows
labeled P-ERK1 and P-ERK2 or ERK1 and
ERK2 indicate the position of the phosphorylated and
non-phosphorylated forms of ERK1 and ERK2 proteins, respectively.
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Fig. 8.
Activation of A-Raf, B-Raf, and c-Raf-1
following membrane depolarization. PC12 cells were serum-starved,
pretreated (+) or not ( ), for 1 h with 70 µM W12
or W13, and then stimulated (+), or not (
), for 5 min with 75 mM KCl or with 100 ng/ml NGF. After treatment, cells were
lysed and protein extracts were subjected to immunoprecipitation with
an anti-A-Raf (A), an anti-B-Raf (B), or an
anti-c-Raf-1 antibody (C). Immunoprecipitates were used to
determine the kinase activity of the different Raf proteins (see
"Experimental Procedures") using wild type MEK1 as substrate
(upper panels) and were analyzed on Western blot
with the same antibody used in the immunoprecipitation step
(lower panels) as a control of the enzyme content
in the immunoprecipitates. A-Raf, B-Raf, and c-Raf-1 activation was
quantitated with a Phosphor Imager and is expressed as the relative
kinase activity where the activity of untreated cells was taken as
equal to unity. UD, unquantifiable activity.
DISCUSSION
by tyrosine kinase receptors has been
proposed to be a relevant mechanism by which ERK MAP kinases can be
activated (37). Such mechanism seems to involve, probably, some
specific isoforms of PKC (39). Moreover, in PC12 cells activated PKC is
able to activate Ras and thereafter the ERK MAP kinase pathway (42).
Our results demonstrate that membrane depolarization is able to
activate the tyrosine phosphorylation of PLC
, being a possible
pathway that connects Ca2+ influx to Ras activation. The
activation of PLC
as a consequence of the EGFR activation has been
previously reported (63, 64), and this could be the mechanism that will
operate in our system. However, the PKC-specific antagonist BIM I, at
concentrations that completely prevent the activation of ERKs after PMA
treatment, was not able to prevent the activation of ERKs after
membrane depolarization. This result suggests that the hypothetical
PLC
-PKC pathway does not make an important contribution to the
activation of ERK MAP kinases by depolarizing stimuli.
seem to activate ERKs in a
MEK-dependent manner without increasing c-Raf-1 activity
(68, 69). Finally, other MAP kinase kinase kinases have been
identified, different from Raf isozymes that potentially could activate
ERKs (see Refs. 55 and 61). The mechanisms by which these kinases are
regulated are unclear, which opens the possibility that some of them
could explain the phenomena reported here.
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ACKNOWLEDGEMENTS |
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We thank colleagues from our laboratory for criticisms and technical support, in particular M. Encinas, X. Dolcet, V. J. Palomar, and V. J. Yuste. The assistance of Dionisio Martin-Zanca in many aspects of our work is specially acknowledged. We are grateful for the generous gifts of the following antibodies: anti-Grb2 (Dr. J. Ureña), anti-pan-Ras (Drs. O. Bachs and N. Agell), anti-EGFR (Drs. G. Capellà and C. García), and 4G10 anti-Tyr(P) (Dr. D. Martin-Zanca). We also thank Drs. F. McKenzie, O. Bachs, and N. Agell for the generous gift of the prokaryotic expression vector containing the GST-RBD construct and Drs. G. M. Cooper and Ana Aranda for the generous gift of the M-M17-26 cells. We are grateful to Dr. J. Fibla for purification of NGF. We thank Isabel Sánchez for expert technical assistance and Dr. A. Porras for helpful technical comments in the PI 3-kinase kinase assay. The comments of Drs. O. Bachs, N. Agell, N. Rocamora, and the members of the Molecular Neurobiology Group are particularly acknowledged.
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FOOTNOTES |
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* This work was supported in part by Comisión Interministerial de Ciencia y Tecnología Grant PN-SAF 97-0094, Biotech Grant BIO4-CT96-0433, and a grant from the Fundación Francisca de Roviralta.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.
The authors dedicate this paper to Julia.
Predoctoral fellow of Generalitat de Catalunya.
§ To whom correspondence should be addressed: Grup de Neurobiologia Molecular, Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Rovira Roure 44, E-25198 Lleida, Catalonia, Spain. Tel.: 34-973-70-24-14; Fax: 34-973-70-24-26; E-mail: Joan.Comella{at}cmb.UdL.es.
2 J. X. Comella, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
[Ca2+]i, intracellular free
Ca2+ concentration;
VGCC, voltage-gated Ca2+
channel;
MAP, mitogen-activated protein;
PI 3-kinase, phosphatidylinositol 3-kinase;
high-K+, high level of
K+;
NGF, nerve growth factor;
EGF, epidermal growth factor;
EGFR, EGF receptor;
BIM I, bisindolylmaleimide I;
PMA, phorbol
12-myristate 13-acetate;
ERK, extracellular-regulated kinase;
MEK, MAPK/ERK kinase;
CaM, calmodulin;
CaM-K, CaM-dependent
protein kinase;
PAGE, polyacrylamide gel electrophoresis;
TBS, Tris-buffered saline;
anti-Tyr(P), anti-phosphotyrosine;
GST, glutathione S-transferase;
PI, L--phosphatidylinositol;
Trk, tropomyosin receptor
kinase;
PKC, protein kinase C;
PLC, phospholipase C;
MOPS, 4-morpholinepropanesulfonic acid..
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REFERENCES |
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