From the UPR 9023 CNRS, CCIPE-141, Rue de la
Cardonille, 34094 Montpellier Cedex 05 and ** UMR 146 CNRS,
Institut Curie, Bat. 110, Centre Universitaire,
91405 Orsay Cedex, France
Received for publication, May 13, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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The small GTPases Ras or Rap1 were suggested to
mediate the stimulatory effect of some G protein-coupled receptors on
ERK activity in neuronal cells. Accordingly, we reported here that pituitary adenylate cyclase-activating polypeptide (PACAP), whose G
protein-coupled receptor triggers neuronal differentiation of the PC12
cell line via ERK1/2 activation, transiently activated Ras and induced
the sustained GTP loading of Rap1. Ras mediated peak stimulation of ERK
by PACAP, whereas Rap1 was necessary for the sustained activation
phase. However, PACAP-induced GTP-loading of Rap1 was not sufficient to
account for ERK activation by PACAP because 1) PACAP-elicited Rap1
GTP-loading depended only on phospholipase C, whereas maximal
stimulation of ERK by PACAP also required the activity of protein
kinase A (PKA), protein kinase C (PKC), and calcium-dependent signaling; and 2) constitutively active
mutants of Rap1, Rap1A-V12, and Rap1B-V12 only minimally stimulated the ERK pathway compared with Ras-V12. The effect of Rap1A-V12 was dramatically potentiated by the concurrent activation of PKC, the cAMP
pathway, and Ras, and this potentiation was blocked by dominant-negative mutants of Ras and Raf. Thus, this set of data indicated that GPCR-elicited GTP loading of Rap1 was not sufficient to
stimulate efficiently ERK in PC12 cells and required the permissive co-stimulation of PKA, PKC, or Ras.
Since its original establishment by Greene and Tischler (1), the
PC12 pheochromocytoma cell line is a widely used model of neuronal
differentiation. The first isolated neurotrophin, nerve growth factor
(NGF)1 (2), was shown to
induce a neuronal like phenotype characterized by neurite
outgrowth (1). The effect of NGF is dependent on a long lasting
activation of the Ras-Raf-MEK-ERK pathway (3). Activation of the cAMP
pathway was also reported to induce PC12 differentiation (4, 5). In
line with the demonstrated role of ERK activation in NGF-induced PC12
differentiation, cAMP analogues, and forskolin, a direct activator of
adenylate cyclase (AC) was shown to stimulate ERK activity in this cell
line (5, 6). Similarly, mobilization of calcium and/or stimulation of
the diacylglycerol (DAG) production results in ERK activation and
eventually neurite outgrowth (7, 8).
The mechanisms responsible for the control of the ERK pathway by
receptor tyrosine kinases, cAMP analogues, and calcium/DAG were
intensively investigated, and Ras-like small GTP-binding proteins
emerged as key elements in this pathway. The products of the
H-Ras gene was shown to stimulate the activity of the MEK kinase Raf-1 following activation of receptor tyrosine kinases (9).
Cyclic AMP and calcium were also suggested to control the activity of
Ras in some cell types (7, 10), resulting in ERK activation. More
recently, the Ras superfamily member Rap1 and the protein kinase B-Raf
were suggested to link PKA activation by cAMP analogues to MEK1
stimulation in neuronal cells (11). On the other hand, several
mechanisms have been proposed for calcium-induced ERK activation,
including activation of the calcium/calmodulin-dependent kinase (12-14), the Pyk2 tyrosine kinase (15, 16), and the cAMP/Rap1/B-Raf pathway (17).
Based on the above-mentioned data, one would predict that activation of
GPCRs positively coupled to AC or PLC- Materials--
PACAP and mouse 2.5 S NGF were purchased from
Neosystem (Strasbourg, France) and Promega, respectively. Forskolin and
phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma. The
PKA inhibitors H89 and (Rp)-cAMP, the PKC
inhibitor bisindolylmaleimide-1, the calcium chelator BAPTA-AM, and the
calmodulin inhibitor W13 were purchased from Alexis Corp. (San Diego,
CA). The PLC inhibitor U-73122 and its inactive analogue, U-73343, were
from Calbiochem. Monoclonal antibodies to Rap1 and Ras were from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA), and monoclonal antibody to
HA (12CA5) was from Roche Molecular Biochemicals.
Phosphorylation-specific and total ERK antibodies were purchased from
New England Biolabs (Beverly, MA).
Cell Culture--
Wild-type and PKA-deficient PC12 cells
(A126-1B2) were kindly provided by Prof. J. A Wagner (Harvard Medical
School, Boston) and were grown on tissue culture dishes (Falcon) coated
with poly-L-lysine in Opti-MEM/Glutamax medium supplemented
with 5% fetal calf serum and 5% horse serum (Invitrogen). Culture
medium was further supplemented with 10 units/ml penicillin and 10 µg/ml streptomycin. Cells were maintained at 37 °C in a saturating
humidified atmosphere of 95% air and 5% CO2.
Endogenous ERK1/2 Activities--
PC12 cells were
plated in 6-well plates in complete medium until they reached 80%
confluence. Cells were then washed and cultured for an additional
16-18 h in RPMI 1640/Glutamax medium supplemented with 1% fetal calf
serum (low serum medium) before treatment with PACAP (100 nM), forskolin (20 or 50 µM), PMA (0.3 or 1 nM), or NGF (40 ng/ml) for the indicated time. Before
stimulation, the cultures were exposed to different inhibitors as
mentioned in the figure legends. Cells were then rapidly rinsed in
ice-cold PBS and lysed in Laemmli's sample buffer. Cell lysates were
boiled for 5 min, and proteins were resolved by SDS-PAGE, blotted onto nitrocellulose membrane, and probed with phospho-specific and phosphorylation state-independent antibodies following the
manufacturer's instructions (New England Biolabs).
Endogenous Rap1 and Ras GTP Loading--
Rap1 and Ras GTP
loading were measured with a non-radioactive method as described
previously (37, 38). Briefly, after stimulation, cells were rinsed
rapidly in ice-cold phosphate-buffered saline at pH 7.4 and solubilized
at 4 °C for 10 min in 0.3 ml of lysis buffer (10% glycerol, 1%
Nonidet P-40, 50 mM Tris, pH 7.4, 200 mM NaCl,
2.5 mM MgCl2, 250 µM
phenylmethylsulfonyl fluoride, 1 µM leupeptin, 0.1 µM aprotinin, 10 mM NaF, and 1 mM
Na3VO4). Lysates were clarified by
centrifugation at 10,000 × g for 10 min, and
supernatants were incubated with glutathione-Sepharose beads (Amersham
Biosciences) freshly coupled to 10 µg of GST-RalGDS-RBD to isolate
Rap1GTP or GST-Raf1-RBD to isolate Ras-GTP. Protein complexes were
allowed to form for 1 h at 4 °C. Precipitates were washed 3 times with lysis buffer. Finally, precipitates were resuspended in
Laemmli's sample buffer, and denatured proteins were loaded on a 12%
SDS-PAGE. The proteins were transferred onto polyvinylidene difluoride
Immobilon-P transfer membrane filters (Millipore, Bedford, MA).
Immunodetection was performed using an anti-Rap1 or anti-Ras antibody
and an anti-mouse IgG coupled to horseradish peroxidase as a secondary
antibody. Blots were developed with an enhanced chemiluminescence
Western blot detection system (Pierce).
Transfections--
Cells were transfected with LipofectAMINE
2000 according to the manufacturer's instructions (Invitrogen) and
lysed 28 h after transfection.
Gal4-Elk1-dependent Luciferase Activity--
PC12
cells were grown to 70% confluence in a 24-well plate prior to
transfection as described above. In all experiments, 2 µg of
(Gal4)5-E1B-TATA-luciferase reporter plasmid and 2 µg of a plasmid encoding the Gal4-(Elk1 transactivation domain) fusion protein (kindly provided by R. Hipskind, Institut de Genetique Moleculaire; Montpellier, France) were co-transfected with various amounts of a test plasmid as indicated in the figure legends. Rap1A-V12, Rap1A-N17, and Ras-V12 cDNAs were provided by J. de Gunzburg (INSERM U248, Institut Curie, Paris, France). Ras-N17 and
Rap1B-V12 were obtained from F. Zwartkruis (Utrecht University, The
Netherlands) and P. J. Stork (The Vollum Institute, Portland), respectively. The N-Raf1 dominant-negative mutant of Raf-1, lacking the
C-terminal kinase domain, was described previously (39). The total
amount of transfected DNA was kept constant with addition of pRK5-CAT.
Eight hours after transfection, cells were placed for 18 h in low
serum medium before stimulation with PACAP, forskolin, PMA, or NGF.
Luciferase activity was assayed 5 h later as described previously
(31).
Infection with Adenoviruses--
Adenoviruses expressing Gap1m
(Ras-GAP), Rap1-GAP, or LacZ (as a negative control) were kindly
provided by Dr. S. Hattori (National Institute of Neurosciences, Tokyo)
(40). Cells were infected in 6-well plates at a multiplicity of
infection of 100 in complete medium. Forty eight hours post-infection,
endogenous ERK activities were determined as indicated above.
Neurite Outgrowth and Immunostaining--
PC12 cells were
transfected with HA-Ras-V12, HA-Rap1A-V12, or EGFP-N1 as a control.
Twenty eight hours after transfection, cells in low serum medium for
18 h were rinsed with PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were then rinsed three times with
PBS/glycine 0.1 M, permeabilized with PBS/Triton X-100
0.1% for 10 min at room temperature, and incubated for 30 min in
PBS/bovine serum albumin 1%. Fixed cells were stained with a
monoclonal antibody to HA (12CA5, Roche Molecular Biochemicals) for
1 h at room temperature, rinsed in PBS, incubated with Alexa fluor
488-conjugated goat anti-mouse antiserum (1:1000) (Molecular Probes),
and examined with a Zeiss Axiovert microscope.
Sustained Activation of ERK1/2 by PACAP--
Because
quantitative and qualitative differences were reported among PC12 cells
used in different laboratories, we verified ERK regulation by PACAP in
the PC12 cells used throughout this study. As expected, PACAP-38
induced a biphasic phosphorylation of both ERK1 and -2 at
Thr-202 and Tyr-204 (Fig.
1A). ERK phosphorylation was
maximally stimulated at 5 min, and then gradually diminished but
remained above resting level for several hours. This kinetic profile
was qualitatively similar to the one obtained with NGF (Fig.
1A). The effect of NGF was however more pronounced and
lasted longer, in agreement with the reported more robust effect of NGF on neurite outgrowth.
PACAP Stimulates Endogenous Rap1 and Ras--
We then evaluated
the enhancement of Rap1 and Ras GTP loading induced by PACAP-38 which
activates AC and PLC in PC12 cells (21). Both monomeric G proteins were
rapidly activated upon PACAP-38 addition (Fig. 1, B and
C). Rap1 activation was sustained, comparable in amplitude
to the one obtained with forskolin and superior to the one induced by
NGF (Fig. 1B). On the other hand, PACAP-induced Ras
stimulation was transient and weaker than that obtained with NGF (Fig.
1C).
Rap1A Is Critical for Long Term PACAP-induced Stimulation of
ERK--
To assess the role of Rap1 and Ras in PACAP-induced ERK
activation, we co-transfected dominant-negative mutants of both G proteins, Ras-N17 and Rap1A-N17, respectively, with a plasmid encoding
a Gal4-Elk1 fusion protein, and we measured the activity of a
luciferase reporter gene under the control of a minimal promoter incorporating five Gal4-responsive elements. In this system, ERK activation induces Gal4-Elk1 phosphorylation and the subsequent transcriptional regulation of the reporter gene, providing a read-out for long term stimulation of the ERK pathway. The induction of the
luciferase activity by PACAP was completely abolished upon preincubation with the MEK-specific antagonist U0126 (10 µM, 30 min), attesting for the specificity of the
reporter system (Fig. 2A).
PACAP-induced Elk1-transactivating activity was blocked more efficiently by Rap1A-N17 than by Ras-N17 (Fig. 2B), whereas
it was the reverse for NGF (Fig. 2C). Together with the data
presented in Fig. 1 which indicate that Rap1 stimulation is sustained
and ample whereas Ras stimulation is more transient and weaker, these results indicated that both Ras and Rap1 were involved in
PACAP-elicited ERK activation and that Rap1 played a prominent role in
long term ERK activation.
To confirm the relative role of Ras and Rap1 in
PACAP-induced ERK stimulation, we used adenoviruses encoding LacZ,
Gap1m (Ras-Gap), or Rap1GAP (40) (Fig.
3). As expected from the data obtained using the Elk1 reporter system, deactivation of Ras or Rap1 by the
GTPase-activating activity of the GAPs led to the attenuation of
PACAP-elicited endogenous ERK1/2 phosphorylation. Interestingly, Ras-GAP was more efficient than Rap-GAP at 5 min following PACAP addition, whereas it was the reverse at 15 min. These observations suggested that Ras was prominently involved in the peak stimulation of
ERK phosphorylation by PACAP, whereas the sustained stimulation of the
ERK pathway required Rap1 activation.
PACAP-elicited Rap1 Stimulation Is Dependent on PLC but Independent
of Calcium, PKC, and PKA--
To determine whether Rap1 GTP loading
elicited by PACAP was indeed sufficient to account for PACAP-induced
ERK stimulation, we compared the signaling pathways involved in
Pac1-elicited Rap1 and ERK stimulations. We first tested the
effects of pharmacological inhibitors of different signaling pathways
on Rap1 activation. The only effective molecule was U73122 (Fig.
4A), a specific blocker of
PLC. Significantly, the inactive analogue U73343 had no effect (Fig.
4A). PKC, calcium, and calmodulin were also found
dispensable because bisindolylmaleimide-1, EGTA, BAPTA-AM, and W13 were
ineffective (Fig. 4A). PKA was also not involved because H89
and (Rp)-cAMP did not alter Rap1 activation by
PACAP (Fig. 4A). This finding was further confirmed using
the A126-1B2 variant of PC12 cells, which displays only 10% of the
PKAII activity (41), and in which PACAP stimulated Rap1 GTP loading
(Fig. 4B). A role for a cAMP-GEF could also be excluded
because the stimulation of Rap1 by forskolin was abolished in A126-1B2
cells (Fig. 4B).
PLC, PKA, PKC, Calcium, and CaM Are Necessary for ERK Activation by
PACAP--
We then tested whether PLC-elicited Rap1 activation is
indeed sufficient for PACAP-induced ERK activation. As expected U73122 blocked ERK phosphorylation induced by PACAP (Fig. 4C)
confirming the involvement of the PLC pathway. Surprisingly, H89
efficiently attenuated PACAP-induced ERK phosphorylation (Fig.
4C). The involvement of PKA was further confirmed using
A126-1B2 cells in which PACAP-elicited ERK phosphorylation was notably
attenuated (Fig. 4D). Interestingly, in wild-type PC12
cells, forskolin, a direct activator of AC, was able to induce ERK
phosphorylation less efficiently than PACAP (Fig. 4D),
whereas it was the reverse for the stimulation of the cAMP production
(data not shown). As expected the effect of forskolin was completely
abolished in A126-1B2 cells, whereas the effect of NGF was not affected
(Fig. 4D). Altogether, these data indicate that PKA is
necessary but not sufficient for PACAP-induced ERK activation.
Bisindolylmaleimide-1, the most specific PKC blocker (42), strongly
diminished PACAP-induced ERK phosphorylation (Fig. 4C), showing that the activation of PKC is necessary for PACAP to stimulate efficiently ERK. Similarly, a rise in intracellular calcium is necessary as addition of EGTA to the incubation medium or of the cell-permeant BAPTA-AM attenuated PACAP-induced ERK phosphorylation (Fig. 4C). Moreover, the effect of calcium was at least
partially mediated by CaM because addition of W13, a specific
calmodulin blocker, strongly diminished PACAP-induced ERK1/2
phosphorylation (Fig. 4C).
Activation of Rap1 Is Not Sufficient for Efficient Stimulation of
ERK Activity--
Because there was an apparent discrepancy between
the pathway involved in Rap1 GTP loading and those necessary for potent ERK stimulation by PACAP, we tested whether activated Rap1 is sufficient to stimulate ERK efficiently.
A constitutively active mutant of Ras, Ras-V12, was exquisitely potent
in inducing Gal4-Elk1 phosphorylation-dependent luciferase activity (Fig. 5A; note the
bi-logarithmic scale). Surprisingly, the corresponding constitutively
active mutant of Rap1A, Rap1A-V12, only minimally stimulated Elk1
phosphorylation-induced luciferase activity (Fig. 5A). The
observed weak activation of the ERK pathway was not specific for the
Rap1A isoform because a constitutive Rap1B mutant was also ineffective
(Fig. 5A). Because the effect of Rap1-GTP depends on the
presence of B-Raf (11, 43), we monitored the PC12 cells used in the
present study for the expression of B-Raf by Western blotting and
evidenced the expected 95- and 68-kDa bands (data not shown).
To exclude that the difference in the potency of Ras-V12 and
Rap1A/B-V12 was because of a difference in the protein levels, we
performed similar experiments with HA-tagged constructs and monitored
the expression levels of the mutants by Western blotting. As shown in
Fig. 5B, transfection of various amount of the different constructs resulted in similar levels of Ras-V12, Rap1A-V12, and Rap1B-V12. In these conditions, Ras-V12 was considerably more effective
than the corresponding Rap1A/B mutants (Fig. 5B; note the
logarithmic scale).
To confirm further the results obtained with the Elk reporter system,
we examined ERK activation following transient transfection of
constitutively activated mutants of Rap1A, Rap1B, and Ras. As shown in
Fig. 5C, HA-Ras-V12 efficiently stimulated ERK
phosphorylation, whereas similar levels of HA-Rap1A-V12 and
HA-Rap1B-V12 were inefficient.
These data indicated that GTP loading of Rap1 was not sufficient to
induce effectively ERK activation. These results were further confirmed at a more physiological level. We monitored neurite
outgrowth in transfected PC12 cells and could demonstrate that Ras-V12
efficiently induced neurite outgrowth, in line with its potency in
stimulating the ERK pathway. In contrast, Rap1A-V12 was completely
inefficient (Fig. 5D).
Activation of Ras, the cAMP Pathway, and PKC Synergize with
Constitutively Active Rap1A in ERK Activation--
We reasoned that
because multiple signaling pathways are involved in ERK activation by
PACAP, these signals may be required to potentiate the effect of
GTP-loaded Rap1A. In line with this hypothesis, we found that
Elk1-transactivating activity is synergistically activated by Rap1A-V12
and minute amounts of Ras-V12 (Fig.
6A). Although the synergy of
the constitutively active Rap1A and Ras mutants was apparently weak
(Fig. 6A), one should take into account that it resulted
from a limited number of transfected cells, due to the small amount of
Ras-V12 used in this experiment.
Similarly, efficient Elk1 phosphorylation-dependent
luciferase activation was obtained upon incubation of
Rap1A-V12-transfected cells with doses of forskolin and PMA, which by
themselves did not produce a significant effect (Fig.
6B).
Dominant-negative Mutants of Ras and Raf Prevent the Potentiation
of Rap1-V12-elicited ERK Stimulation by the cAMP Pathway and
PKC--
To elucidate further the relative roles of Ras, PKC, and the
cAMP pathway, we used dominant-negative mutants of Ras and its downstream kinase, Raf-1. Ras-N17 moderately attenuated the
potentiation by PMA and forskolin of Rap1A-V12-elicited stimulation of
Gal4-Elk1-dependent luciferase activity (Fig.
7). On the other hand, the N-terminal fragment of Raf-1 (10), N-Raf-1, strongly attenuated the effect of
Rap1A-V12, PMA, and forskolin. This fragment of Raf-1 includes the Ras
binding domain of Raf-1 and hence behaves as a blocker of Ras
signaling. Consistently, similar results were obtained with the
N-terminal fragment of B-Raf (data not shown). The more pronounced
inhibition of the potentiation by PMA and forskolin of
Rap1A-V12-elicited stimulation of Elk1-dependent luciferase activity induced by N-Raf-1 compared with Ras-N17 suggested a Ras-independent activation of Raf by PMA and/or forskolin. In this
context, the phosphorylation of Raf-1 by PMA-activated PKC was a
plausible candidate pathway (44). Altogether, these data indicated that
PMA and forskolin potentiated the stimulatory effect of Rap1A-V12
through both Ras-dependent (blocked by Ras-N17 and N-Raf-1)
and -independent (blocked by N-Raf-1 only) pathways.
Collectively, our data indicate that PACAP activated the ERK
pathway through the pleiotropic stimulation of several signaling pathways acting synergistically. We showed that PACAP enhanced GTP
loading of two small GTP-binding proteins, Rap1 and Ras (Fig. 1). The
stimulation of Rap1 GTP loading was more pronounced and lasted longer
than that of Ras. Experiments using adenoviruses encoding Ras- or
Rap1-GAP indicated that Ras is primarily involved in the initial peak
stimulation of ERK, whereas Rap1 is involved in sustained ERK
activation (Fig. 3). This is reminiscent of reports by York and
co-workers (45) and Garcia and co-workers (39), suggesting that
NGF-induced and thrombopoietin-mediated sustained activation of ERK in
PC12 and UT7-Mpl cells, respectively, also involved the successive
stimulation of the ERK pathway by Ras and Rap1. Hence, consecutive
stimulation of Ras and Rap1 may represent a common theme in sustained,
biphasic activation of the ERK pathway, whatever the nature of the
triggering receptor.
Although Rap1 activation was shown to be essential for PACAP-induced
ERK stimulation (Figs. 2 and 3), we found that it is not sufficient.
First, although PACAP-induced enhancement of Rap1 GTP-loading is
blocked exclusively by a PLC blocker (Fig. 4A), PACAP-induced ERK activation is blocked or strongly attenuated by
inhibitors of other signaling pathways (Fig. 4C). Second,
whereas Ras-V12, a constitutively active mutant of Ras, induced a
robust stimulation of an Elk1 reporter plasmid, the same dose of
Rap1A-V12 minimally stimulated this construct (Fig. 5, A and
B). These two lines of evidence indicated that stimulation
of Rap1 GTP-loading was not sufficient to induce a potent ERK
activation in PC12 cells. The lack of effect of Rap1A-V12 was
unexpected given the reported effect of Rap1B-GTP Because PACAP-induced Rap1 GTP-loading was dependent on PLC activity
only, whereas ERK activation was dependent on multiple signaling
pathways, we sought to determine more precisely the role of Ras, AC,
and PKC in ERK activation by PACAP. One alternative was that Ras, AC,
or PKC activation worked in parallel to PLC-dependent Rap1
activation, and the other was that these signaling pathways synergized
with Rap1 activation. Data presented in Fig. 6 support the second
alternative, because Ras, cAMP, and PKC strongly enhanced Rap1A-V12-induced Gal4-Elk1-dependent luciferase activity.
This synergistic effect may take place at different levels. First, Rap1
may require, in addition to GTP loading, a post-translational modification to potently stimulate the B-Raf-MEK-ERK pathway. One
obvious candidate is phosphorylation by PKA because this kinase was
reported to phosphorylate and activate Rap1 (46, 47). Phosphorylation
by PKA may thus facilitate Rap1 GTP loading and/or provide an
additional signal required for a productive interaction with B-Raf.
Rap1 phosphorylation by PKA may also help to alleviate the inhibitory
effect of Rap1 on Raf-1 (48) and hence favor Raf-1 interaction with Ras
(see below). Second, the activation of Ras, the cAMP pathway, or PKC
may be required to sensitize the B-Raf-MEK-ERK pathway to GTP-loaded
Rap1. Qiu and co-workers (49) demonstrated that the Rap1-B-Raf-MEK-ERK
pathway is modulated by the amount of 14-3-3 scaffolding protein
associated with B-Raf. In cells where little 14-3-3 protein is
associated with B-Raf, cAMP inhibits ERK activity, whereas it
stimulates ERK in cells where 5-fold more 14-3-3 is found associated
with B-Raf. One may therefore suggest that the cAMP pathway or PKC may
regulate the association of 14-3-3 with B-Raf and hence modulate the
potency of GTP-loaded Rap1 to stimulate B-Raf activity.
Data presented in Fig. 7 suggested a direct role for PKC and/or the
cAMP pathway in Ras activation, because the potentiation of
Rap1A-V12-activated Gal4-Elk1-dependent luciferase activity by PMA and forskolin was partially blocked by a dominant-negative mutant of Ras. Furthermore, PMA and/or forskolin probably also have a
Ras-independent effect on Raf, because an N-terminal fragment of Raf-1
was more efficient than Ras-N17 in blocking the potentiating effect of
PMA and forskolin. Besides the well documented activation of Raf-1 by
GTP-loaded Ras, some PKC isoforms were also reported to phosphorylate
and activate Raf-1 (44), suggesting a possible mechanism for
Ras-independent activation of Raf-1 by PKC.
The requirement of the Ras-Raf-1 module for the efficient stimulation
of the ERK pathway by GTP-loaded Rap1 is surprising, considering the
reported inhibitory effect of activated Rap1 on Raf-1 activity (11).
One may argue that differentiation-permissive activation of the ERK
pathway by extracellular stimuli was consistently found to be biphasic
in PC12 cells (3) and mediated by the successive activation of Ras and
Rap. However, we showed that Rap1 GTP loading is an early event that is
concomitant of Ras activation (Fig. 1). Hence, a mechanism certainly
exists that delays ERK stimulation by activated Rap1. In this view, the
prerequisite for Raf-1 activation for efficient ERK stimulation by
GTP-loaded Rap1 would warrant that the Rap1-B-Raf module is activated
after the Ras-Raf-1 module. Furthermore, Ras is found at the plasma membrane, whereas Rap1 is found co-localized with a perinuclear compartment (50). A recent study (51) suggested that the recruitment of
Raf-1 to the plasma membrane by activated Ras leads to its phosphorylation on tyrosine 341 and serine 338. Interestingly, phosphorylated Raf-1 is activated by Rap1, in contrast to
dephosphorylated Raf-1 which is inhibited by Rap1 (51). This
observation, together with the present data, suggest a mechanism in
which Ras activation leads to the recruitment of Raf-1 to the plasma
membrane and to its subsequent phosphorylation. Phosphorylated Raf-1
would then interact with intracellular, GTP-loaded Rap1 and allow
subsequent ERK activation by activated Rap1. This mechanism would
result in a compartmentalized activation of the ERK pathway at the
plasma membrane before activation at intracellular membrane domains. Hence, the requirement for Raf-1 activation for GTP-loaded Rap1 to
stimulate efficiently the ERK pathway may be involved in the spatio-temporal control of ERK activation during differentiation, which
is reminiscent of the observed pattern of Ras and Rap1 activation in
NGF-stimulated PC12 cells (50).
We also presented data on the signaling pathways involved in
PACAP-elicited Rap1 GTP loading. Because Pac1 is coupled to
AC and PLC, the signaling pathways downstream of these effectors are
candidates for the control of Rap1 GTP loading. Although forskolin stimulates Rap1 GTP loading, in a strictly PKA-dependent
manner (Fig. 4B), PKA activity is dispensable for
PACAP-induced Rap1 activation (Fig. 4, A and B).
This observation is somehow unexpected considering the tight and
efficient coupling of Pac1 to AC and the reported effect of
forskolin or constitutively activated PKA on Rap1 activation in PC12
cells (11). One possible interpretation is that the physiological level
of cAMP elicited by Pac1 activation is not sufficient for a
direct activation of Rap1 in PC12 cells. This is in line with the
reports by Huang an co-workers (18) and Williams and co-workers (19)
indicating that the stimulation of cAMP production at physiological
levels by GPCRs is not sufficient to trigger neurite outgrowth in PC12
cells. Likewise, PKC or calmodulin are not directly involved in Rap1
activation because the specific blockers, bisindolylmaleimide-1 and
W13, respectively, were ineffective (Fig. 4A). Because
Pac1 is coupled to both AC and PLC, cAMP-GEFs and
CalDAG-GEFs are ideal candidates to mediate the PACAP-induced Rap1
activation. Our data exclude a role for the cAMP-GEFs. Rap1 stimulation
by forskolin is completely abolished in PKA-deficient cells (Fig.
4B) indicating that elevation of cAMP level is not sufficient per se to activate Rap1 independently of PKA in
this cell line. Only U73122, a specific PLC blocker, was effective in
preventing PACAP-induced Rap1 GTP loading, indicating that the PLC
activity is indispensable for Rap1 stimulation by PACAP (Fig.
4A). To explain these observations, one should postulate the
existence of either a DAG-GEF, which would not require calcium to be
activated, an IP3-GEF, or a PLC with a Rap1-GEF activity. No DAG-GEF was characterized so far. However, Johan de
Rooij2 (52) indicates that
CalDAG-GEFIII translocates to membranes upon addition of
12,13-tetradecanoyl phorbol acetate and is insensitive to increased
levels of calcium. Similarly, no IP3-GEF has been described
so far. However, an IP4-GAP (GAP1IP4BP) protein
was isolated (53), indicating that proteins controlling Rap1 activity
in response to inositol phosphates binding do exist. The existence of
PLC Altogether, the present data indicate that activation of the PACAP
receptor triggered a pleiotropic signaling. The proximal effectors of
Pac1 are AC and PLC whose activation resulted in the
stimulation of a set of kinases directly controlled by second messengers, i.e. PKA, PKC, and
calcium/calmodulin-dependent kinase. Activation of the PACAP
receptor also resulted in Ras activation through a mechanism that was
not studied in detail in this report. Ras activation was primarily
involved in the initial peak stimulation of ERK activity. In parallel,
PACAP-elicited PLC activation resulted in Rap1 GTP loading which was,
per se, not sufficient to induce an efficient ERK
stimulation. The activation of other signaling pathways such as those
controlled by Ras, cAMP, and PKC, including Raf-1, were required to
potentiate the effect of GTP-loaded Rap1. Hence, the present work
revealed an unforeseen difference between Ras and Rap1 in the
stimulation of the ERK pathway. Whereas GTP-loaded Ras was fully
competent to stimulate the ERK pathway, this was not the case for Rap1
which required additional signals to become effective. This finding
will hopefully help to solve some of the "apparent discrepancies"
(52) found in the literature on the mechanism of stimulation of the ERK
pathway by Rap1 in neuronal cells. These data also reconciled previous
reports with respect to the role of the cAMP pathway in PACAP-
versus forskolin-induced ERK activation in PC12 cells (5, 6,
36, 58). Finally, our data shed new light on the control of Rap1
activity by GPCRs, an important event in the context of neuronal differentiation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
would lead to PC12
differentiation. However, in contrast to the effect of cAMP analogues
or forskolin, stimulation of the endogenous A2A adenosine
receptor (18) or of the transfected
1 adrenergic receptor (19), two GPCRs positively coupled to AC, does not result in
neurite outgrowth. Similarly, the situation is more complex than
anticipated for PLC-coupled GPCRs as exemplified by the differential
effects of the three closely related, PLC-coupled,
1
adrenergic receptor subtypes (8). These contradictory data led us to
study in detail the mechanisms involved in the stimulation of the ERK
pathway by the GPCR for PACAP. This neuropeptide belongs to the
vasoactive intestinal peptide-secretin-glucagon family of peptides and
was originally isolated through its ability to stimulate the adenylate
cyclase activity of pituitary cells in vitro (20). PACAP
neurotrophic activity was first reported for PC12 cells in which the
38-amino acid form of PACAP was shown to promote neurite outgrowth
(21). Indeed, the receptor for PACAP is the only endogenous GPCR to
stimulate neurite outgrowth in PC12 cells. This neurotrophic activity
was extended to the protection of cerebellar granule (22, 23) and
dorsal root ganglion neurons (24) from apoptosis, and of cortical (25) and hippocampal (26) neurons from ischemia-induced cell death. PACAP
was also shown to control the proliferation and differentiation of
cortical (27-29) and cerebellar neuroblasts (30). PACAP neurotrophic activity is mediated by the activation of Pac1, the
PACAP-specific GPCR. Pac1 displays a complex pattern of
alternative splicing that modulates its ligand binding and signaling
properties (31-34) resulting in the modulation of PACAP physiological
effects (28, 35). We and others (21, 31) showed that Pac1
potently stimulated the AC and PLC activities as well as the ERK
pathway that was implicated in its neurotrophic activity (23, 36). In
this context, we selected the PC12 cell line to assess the involvement of the small GTP-binding proteins Rap1 and Ras in the signaling pathways responsible for PACAP-induced ERK activation in this system.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
PACAP-induced ERK phosphorylation and GTP
loading of endogenous Rap1 and Ras. A, PACAP-induced
ERK-1 and -2 phosphorylation. PC12 cells were treated with 100 nM PACAP or 40 ng/ml NGF for the indicated times. Cells
were harvested and lysed, and samples were analyzed by Western blotting
using a phosphospecific ERK1/2 antibody
(P-ERK1/2) (upper panels). Equal
loading was verified by probing the blots with an anti-ERK1/2 antibody
(ERK1/2) (lower panels). The data are
representative of three independent experiments. B, PACAP
induced Rap1 GTP loading. Following addition of PACAP as in
A, cells were harvested and lysed. GTP-loaded Rap1 was
affinity-precipitated using GST-RalGDS (Rap-binding domain) and
immunoblotted with an anti-Rap1 antibody (upper panels). The
total amount of Rap1 protein is shown in the lower panels.
To evaluate the amplitude of Rap1 stimulation by PACAP, the same
experiment was performed with 50 µM forskolin
(Forsk.) or 40 ng/ml NGF. C, PACAP-stimulated Ras
GTP loading. GTP-loaded Ras was affinity-precipitated using GST-Raf1
(Ras-binding domain) and immunodetected with a Ras antibody. As a
positive control, cells were stimulated with NGF (40 ng/ml) for 5 min.
Data are representative of four (B) and three (C)
experiments.
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Fig. 2.
Ras and Rap1 are involved in the stimulation
of Gal4-Elk1-dependent transcriptional activity by
PACAP. A, PACAP-induced Elk1-transactivating activity
was MEK-specific. PC12 cells were transfected with a plasmid encoding a
Gal4-Elk1 fusion protein and with a
(Gal4)5-E1BTATA-luciferase reporter gene. PACAP-induced
stimulation of luciferase activity was completely blocked upon
preincubation with U0126 (10 µM, 30 min), a specific
blocker of MEK1/2. B and C, effect of
dominant-negative Rap1A or Ras mutants on PACAP- and NGF-induced
Elk1-transactivating activity. Ras-N17 (5 µg) or Rap1A-N17 (5 µg)
were co-transfected with the Elk reporter system as in A. Cells were treated with PACAP (100 nM) or NGF (40 ng/ml),
and luciferase activity was assayed 5 h later. Data are mean ± S.E. of four independent experiments performed in triplicate.
View larger version (34K):
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Fig. 3.
Activation of Ras and Rap were required for
the biphasic stimulation of ERK phosphorylation. PC-12 cells were
infected with adenoviruses encoding -galactosidase
(LacZ), Gap1m (Ras-GAP), or Rap1-GAP
(Rap-GAP). Forty eight hours later, the cells were incubated
with PACAP (100 nM) for the indicated times. Cells were
harvested and lysed, and samples were analyzed by Western blotting
using a phosphospecific ERK1/2 antibody (P-ERK1/2) (upper
panels). The same Western blot was exposed for different times to
provide optimal sensitivity for each time point. Equal loading was
verified by probing the blot with an anti-ERK1/2 antibody
(ERK1/2) (lower panels).
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Fig. 4.
Characterization of the signaling pathways
involved in PACAP-induced Rap1 GTP loading and ERK activation.
A, characterization of the signaling pathways involved in
PACAP-induced Rap1 activation using pharmacological inhibitors. PC12
cells were treated with PACAP (100 nM, 5 min) alone or
following pretreatment with U73122 (20 µM, 20 min),
U73343 (20 µM, 20 min), H89 (20 µM, 20 min), (Rp)-cAMP (50 µM, 1 h),
EGTA (3 mM, 5 min), bisindolylmaleimide-1 (20 µM, 1 h), BAPTA-AM (50 µM, 30 min), or
W13 (70 µM, 1 h). GTP-loaded Rap1 was
affinity-precipitated with GST-RalGDS-RBD and immunoblotted with an
anti-Rap1 antibody (upper panel). The total amount of Rap1
protein is shown in the lower panel. B, GTP
loading of Rap1 induced by PACAP and forskolin in wild-type
(WT) versus PKA-deficient (A126-1B2) PC12 cells.
C, characterization of the signaling pathways involved in
PACAP-induced ERKs activation using pharmacological inhibitors. PC12
cells were treated with PACAP (100 nM, 5 min) alone or
following pretreatment with U73122 (20 µM, 20 min),
U73343 (20 µM, 20 min), H89 (20 µM, 20 min), EGTA (3 mM, 5 min), bisindolylmaleimide-1 (20 µM, 1 h), BAPTA-AM (50 µM, 30 min), or
W13 (70 µM, 1 h). Equal amounts of cell lysate were
assayed for Western blotting using a phosphospecific ERK1/2 antibody
(P-ERK1/2; upper panel) and an ERK1/2
antibody (lower panel). D, phosphorylation of
ERK1/2 induced by PACAP, forskolin, and NGF in wild-type
(WT) versus PKA-deficient (A126-1B2) PC12 cells.
A-D, data are representative of three independent
experiments.
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Fig. 5.
Constitutively active Rap1 weakly stimulated
ERK activity. A, PC12 cells were transfected with
plasmids encoding a Gal4-Elk1 fusion protein, increasing amounts of
Ras-V12, Rap1A-V12, or Rap1B-V12 and a (Gal4)5-E1BTATA-luciferase
reporter gene. Data are mean ± S.E. of at least three independent
experiments. Note the bi-logarithmic scale. B, plasmids
encoding HA-tagged Ras-V12, Rap1A-V12, or Rap1B-V12 were transfected as
in A with the indicated amount of each plasmid. Luciferase
activity was determined as in A. Expression levels of
constitutively active mutants were verified by Western blotting using
an anti-HA monoclonal antibody. C, PC12 cells were
transfected with the indicated amounts of plasmids encoding HA-tagged
Rap1A-V12, Rap1B-V12, or Ras-V12. Equal amounts of cell lysate were
assayed for Western blotting using a phosphospecific ERK1/2 antibody
(P-ERK1/2; upper panel) and an ERK1/2
antibody (middle panel). The amount of each transfected
protein was monitored by Western blotting using an anti-HA monoclonal
antibody. As expected from experiments performed in B,
Ras-V12 induced ERK phosphorylation, whereas constitutively activated
mutants of Rap1 proteins were ineffective. D, PC12 cells
were transfected with plasmids encoding green fluorescent protein
(GFP) as a negative control, HA-Ras-V12 and HA-Rap1A-V12.
Twenty eight hours later, transfected cells were visualized with a
fluorescent microscope, directly (green fluorescent protein) or
following immunohistochemistry with an anti-HA monoclonal antibody.
Ras-V12 (1 µg)-transfected cells displayed long neurites, whereas the
morphology of Rap1A-V12 (5 µg)-transfected cells did not differ from
control, green fluorescent protein (5 µg)-transfected, cells.
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Fig. 6.
ERK activation by Rap1 is potentiated by
activated Ras, the cAMP pathway, and PKC. A, PC12 cells
were transfected with plasmids encoding a Gal4-Elk1 fusion protein,
Ras-V12 (0.5 ng), and/or Rap1A-V12 (5 µg), and a
(Gal4)5-E1BTATA-luciferase reporter gene. B, cells were
transfected with Rap1A-V12 (5 µg) and a (Gal4)5-E1BTATA-luciferase
reporter gene. Where indicated, transfected cells were treated with PMA
(1 nM) and/or forskolin (20 µM) before the
luciferase activity was measured 5 h later. Data are mean ± S.E. of one representative experiment performed in triplicate.
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Fig. 7.
Potentiation of Rap1A-V12-elicited ERK
activation by cAMP and PMA is blocked by dominant-negative mutants of
Ras and Raf-1. PC12 cells were transfected with plasmids encoding
a Gal4-Elk1 fusion protein, a (Gal4)5-E1BTATA-luciferase reporter gene,
Rap1A-V12 (5 µg), Ras-N17 (5 µg), and N-Raf-1 (5 µg). Where
indicated, cells were treated with PMA (0.3 nM) and
forskolin (20 µM), and the luciferase activity was
measured 5 h later. Data are mean ± S.E. of one
representative experiment performed in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (43) on B-Raf
activity in vitro and of Rap1B-V12 on an Elk1 reporter
system (11). To test for a difference in the activity of Rap1A and
Rap1B, we transfected the Rap1B-V12 cDNA used by Vossler and
co-workers (11) and evidenced a stimulation of the Elk1 reporter system
similar to the one obtained with the Rap1A-V12 cDNA (Fig.
5A). We concluded that, in the PC12 cells used in the
present study, an additional signaling pathway should be activated in
order to potentiate the ERK stimulation by activated, GTP-loaded, Rap1.
Constitutive activation of this pathway at a permissive level in the
cell clone used by Vossler and co-workers (11) is a possible
explanation for the observed differences.
, a PLC with a Rap1-GEF activity, was recently reported (54-57).
PLC
is the founding member of a novel family of
polyphosphoinositide-specific phospholipases that integrate multiple
signaling pathways. Its PLC activity is controlled by G
12 and Ras, and PLC
bears a GEF activity toward Ras
and Rap1. Hence PLC
was a good candidate to mediate the observed
PACAP-induced Rap1 GTP loading. By using two different primer pairs for
rat PLC
, we performed RT-PCR and evidenced PLC
expression in rat kidney but not in PC12 cells (data not shown).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. A. Bellmann, B. Bilanges, S. Desagher, and A. Varrault for helpful discussions. We also thank Drs. J. L. Bos, J. de Gunzburg, R. Hipskind, and P. J. Stork for the generous gift of plasmids. We are grateful to Dr. F. Zwartkruis for advice on the Rap pull-down assay. We acknowledge the gift of A126- 1B2 and wild type PC12 cells by Dr. J. A. Wagner. Adex adenoviruses encoding LacZ, Gap1m, and Rap1GAP were generously provided by Dr. Seisuke Hattori.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Centre National de la Recherche Scientifique and Grant CT-1999-00602 from the European Commission (to L. J.).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.
§ Both authors contributed equally to this work.
¶ Recipient of a pre-doctoral fellowship from the Ministère de la Recherche, the Fondation pour la Recherche Médicale, and the Association pour la Recherche Contre le Cancer.
Recipient of post-doctoral fellowships from the Fondation pour
la Recherche Médicale, the Association pour la Recherche Contre le Cancer, and the Spanish Ministerio de Educacion, Cultura y Deporte.
To whom correspondence should be addressed. Tel.:
33-467-142932; Fax: 33-467-542432; E-mail:
journot@montp.inserm.fr.
Published, JBC Papers in Press, December 6, 2002, DOI 10.1074/jbc.M204652200
2 J. de Rooij, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
NGF, nerve growth
factor;
AC, adenylate cyclase;
BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetrakis(acetoxymethyl ester);
CaM, calmodulin;
DAG, diacylglycerol;
ERK, extracellular signal-regulated kinase;
GEF, guanine nucleotide exchange factor;
GAP, GTPase-activating protein;
GPCR, G protein-coupled receptor;
IP3, inositol
1,4,5-triphosphate;
MEK, mitogen-activated protein kinase/ERK kinase;
PACAP, pituitary adenylate cyclase-activating polypeptide;
PKA, protein
kinase A;
PKC, protein kinase C;
PLC, phospholipase C;
PBS, phosphate-buffered saline;
HA, hemagglutinin;
PMA, phorbol 12-myristate
13-acetate;
GTPS, guanosine
5'-3-O-(thio)triphosphate.
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