From the Dipartimento di Farmacologia Sperimentale ed
Applicata, Facoltà di Farmacia, Università di Pavia,
Pavia 27100, Italy, § Dipartimento di Biologia e Patologia
Cellulare e Molecolare, Consiglio Nazionale delle Ricerche,
Napoli 80131, Italy, ¶ Dipartimento di Medicina
Sperimentale, Facoltà di Medicina di Catanzaro, Università
di Catanzaro, Catanzaro 88100, Italy, and
Laboratory of Cellular
and Molecular Neurophysiology, NICHD, National Institutes of Health,
Bethesda, Maryland 20892
Received for publication, August 21, 2000, and in revised form, December 29, 2000
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ABSTRACT |
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Activation of the cAMP-dependent
protein kinase A (PKA) pathway may induce cAMP-response element-binding
protein (CREB) phosphorylation either directly or via cross-talk
mechanisms with other signal transduction pathways. In this study, we
have investigated in striatal primary cultures the mechanism by which
activation of the cAMP/PKA-dependent pathway leads to CREB
phosphorylation via the extracellular signal-regulated kinase
(ERK)-dependent pathway. We have found that PKA-induced
CREB phosphorylation and CREB-dependent transcription are
mediated by calcium (Ca2+) release from intracellular
stores and are blocked by inhibitors of the protein kinase C and
ERK pathways. This mechanism appears to be mediated by the small
G-protein Rap1, whose activation appears to be primed by PKA-induced
Ca2+ release but not further induced by direct or indirect
PKA- or protein kinase C-dependent phosphorylation. These
results suggest that, in striatal neurons, intracellular
Ca2+ release, Rap1, and ERK pathway play a crucial
role in the PKA-induced CREB phosphorylation and
CREB-dependent transcription.
The dopaminergic striatal system is the main target of the
antipsychotic agents used in the treatment of schizophrenia (1) and of
the psychostimulant drugs cocaine and amphetamines (2). The prolonged
administration of these drugs increases the synaptic availability of
dopamine and induces many dopamine-dependent adaptive responses culminating in the transcription of striatal cAMP-response element
(CRE)1-dependent
genes, such as the immediate early gene c-fos and the neuropeptides dynorphin, substance P, and enkephalins.
CREB phosphorylation, initially thought to be mediated exclusively by
the cAMP/protein kinase A (PKA) pathway (3), is also induced by
Ca2+-dependent signal transduction pathways.
Two members of the Ca2+/calmodulin-dependent
kinase family (CaMK), CaMKII (4) and CaMKIV (5, 6), are activated by
Ca2+ entry through an L-type voltage-sensitive
Ca2+ channel or glutamate N-methyl-D-aspartic
acid (NMDA) receptors (7) and induce CREB phosphorylation. Moreover,
Ca2+ influx via L-type voltage-sensitive Ca2+
channel or The ERK pathway plays a pivotal role in stimulus-dependent
gene regulation in the central nervous system, because pharmacological manipulations of the ERK pathway functionality affect the synaptic plasticity mechanisms supposed to underlie learning and memory (13).
The cascade responsible of the ERK pathway activation requires the
stimulus-dependent recruitment of the small G protein Ras, which in turn activates the Raf and MEK kinases. Although Ca2+-dependent activation of Ras has been
demonstrated in several experimental models (14), in the central
nervous system an alternative route to ERK activation via the cAMP/PKA-
or Ca2+-dependent pathway has been recently
characterized. This route implies the participation of the small G
protein Ras-related Rap1 and the downstream kinase B-Raf (15, 16).
The importance of the Rap1/B-Raf system in the cross-talk between the
cAMP/PKA- and ERK-dependent pathways has received further attention by the recent identification of two novel families of Rap1GEF
activated by direct binding to Ca2+ and cAMP (17). However,
these results were obtained mainly in platelets and PC12 cells, rather
than in primary neural cells, which might display distinct signal
transduction pathways. Furthermore, the exact mechanism by which the
cAMP/PKA-dependent pathway induces ERK activation and the
role, if any, of Rap1 in CREB phosphorylation and
CRE-dependent transcription have not yet been investigated.
In an attempt to address these questions, in this study we have
investigated the mechanisms involved in cAMP/PKA-dependent ERK activation, CREB phosphorylation, and CREdependent
gene transcription in striatal neurons.
Materials--
The antibodies directed against phosphorylated
Ser133 CREB (P-CREB) and phosphorylation state-independent
CREB were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
The antibodies directed against dually phosphorylated ERK
(Thr202 and Tyr204) and phosphorylation
state-independent ERK were from New England Biolabs (Beverly, MA);
those against Rap1, B-Raf, and the protein-tyrosine kinase PYK2
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
The monoclonal antiphosphotyrosine PY20 antibody was purchased from
Transduction Laboratories (Lexington, KY). The Super signal kit was
from Pierce. Trizol, fetal bovine serum, horse serum, and all reagents
for cell cultures were from Life Technologies, Inc. The protein kinases
inhibitors
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89) and KT5720 were purchased from Biomol (Plymouth Meeting, PA).
8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate sodium salt
(8-CPT), chelerythrine chloride, bisindoleylmaleimide I, phorbol
12-myristate 13-acetate (PMA),
1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester (BAPTA-AM),
1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzo-furan-5-oxy]-2-(2'-amino-5'methylphenoxy)-ethane N,N,N',N'-tetraacetic acid
pentacetoxymethyl ester (fura-2/AM), A23187, 2'-amino-3'-methylflavone
(PD98059), EGTA,
[12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3, 4-c]carbazole]
(Gö6976), and thapsigargin were from Alexis Corp. (San Diego,
CA). Dopamine, sulpiride, SCH 23390, SKF-38393,
D-( Cell Culture--
Primary cultures were obtained as previously
described (18). Briefly, striata of 16-17-day-old Harlan
Sprague-Dawley rat embryos were dissected and incubated with papain.
Tissue fragments were mechanically dissociated, and the cells were
plated in 60-mm diameter poly-L-lysine-coated (10 µg/ml)
dishes and grown in 1:1 minimal essential medium/F-12 medium containing
2 mM glutamine, 2.5% fetal bovine serum, and 2.5% horse
serum. About 48 h after plating, 10 µM cytosine-C
arabinoside was added to the cultures to prevent the growth of
nonneuronal cells. The cells were grown for 6 days in vitro
for intracellular Ca2+ measurement experiments (see below)
or 12 days in vitro for all other experiments. The total
percentage of glial cells was below 5%, as assessed by glial
fibrillary acid protein staining and counterstaining with 1% cresyl
violet. Unless otherwise indicated, cells were rinsed twice with Krebs
buffer and then left for 20 min in Krebs buffer before stimulation with
the indicated agents.
In the experiments with BAPTA-AM, the cells were preincubated for the
indicated time with BAPTA-AM and then incubated with stimulating agents
in Krebs buffer. Experiments in which the role of excitatory amino
acids was studied were performed in the presence of 1 µM
tetrodoxin to block action potentials and excitatory amino acid release.
Intracellular Ca2+ Measurement--
The
intracellular Ca2+ concentration was measured with the
fluorescent Ca2+ indicator fura-2/AM. Striatal cells,
prepared as described above, were plated at low density into 24-well
multiwell plates containing 13-mm glass coverslips previously coated
with 10 µg/ml poly-L-lysine. After 6 days in
vitro, cells were washed twice with Krebs buffer and then
incubated in the same buffer containing 5 µM fura-2/AM at
37 °C in a humidified incubator with 5% CO2 for 30 min,
followed by a postincubation period in the same buffer for 30 min. The dye-loaded cells were epi-illuminated with light from a 75-watt xenon
lamp filtered through 340- and 380-nm interference filters, and the
fluorescence emitted at 510 nm was revealed by a photon-counting photomultiplier. The 340/380-nm fluorescence ratio, averaged over a
period of 2 s, was measured for 10 min in the Krebs buffer, and
this value was taken as basal reference value. The stimulating agents
were added as 10× concentrated solution, and then changes in the
340/380-nm ratio were monitored for the time indicated in Fig.
9. For each experimental condition, fluorescence responses were
taken in at least three fields on each coverslip.
Pull-down Assay for the Determination of Rap1
Activation--
The pull-down assay for the determination of Rap1
activation was carried out as previously reported (19). Briefly, the
GST fusion protein of the minimal Rap1-binding domain of ralGDS
(ral-RBD) was induced in Escherichia coli (strain BL21DE3)
by isopropyl-1-thio- Immunoblot Analysis--
Stimulated cells were rinsed twice with
ice-cold phosphate-buffered saline and then lysed in 400 µl of
ice-cold lysis buffer (10 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1% Nonidet P-40, 1 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 2 µg/ml pepstatin, and 2 µg/ml leupeptin). Cell lysate were
centrifuged at 16,000 × g for 5 min at 4 °C, and
the supernatant was incubated for 15 h at 4 °C with primary
antibodies and then with 15 µl of protein A-agarose for 1 h.
Immunoprecipitates were washed three times with lysis buffer and
resuspended either in SDS-sample buffer for immunoblot experiments or
in kinase buffer for immunocomplex protein kinase assay (see below).
Western blot analysis was carried out either on 20 µg of nuclear
extracts (for phosphorylated CREB, phosphorylation state-independent
CREB, and p90RSK), 20 µg of total cell lysate (for
phosphorylated ERK and phosphorylation state-independent ERK), or
immunoprecipitates for PYK2. Proteins resolved by 12.5% (PCREB, CREB,
ERK, and PERK) or 7% (PYK2 and p90RSK) SDS-polyacrylamide
gel electrophoresis were transferred to nitrocellulose membranes by
tank blotting overnight at 4 °C. The membranes were rinsed in
Tris-buffered saline (TBS) (composition 10 mM
Tris-HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween 20),
incubated for 1 h in TBS containing 4% nonfat dry milk (TBSM),
and then incubated overnight at 4 °C with primary antibodies in
TBSM. After three 15-min washes in TBS, the membranes were incubated in
TBSM for 1 h at room temperature with horseradish
peroxidase-conjugated goat polyclonal anti-rabbit IgG for rabbit
polyclonal primary antibodies, or with horseradish peroxidase-conjugated goat polyclonal anti-mouse IgG for mouse monoclonal primary antibodies. The chemiluminescent signals were detected using the Super signal kit, and the films were scanned using
an Agfa T1200 scanner with Photolook software. The band intensity was
quantified with NIH Image software. The same protocol was used for the
detection of tyrosine-phosphorylated PYK2 using the mouse monoclonal
antibody specific to phosphotyrosine PY20, except that 1% bovine serum
albumin was used instead of 4% nonfat dry milk.
Protein Kinase Assay--
Cells were preincubated with protein
kinase inhibitors as reported in the figure legends and then stimulated
with the indicated agents. In vitro PKA and PKC activities
were performed as previously reported (18, 20).
Northern Blot Analysis--
Striatal cultures were treated with
appropriate stimuli for the indicated time and rinsed twice with
phosphate-buffered saline. The RNA extraction reagent Trizol was added
directly to each culture dish. Total RNA was extracted with a 1:10
volume of chloroform, and the upper aqueous phase was precipitated
overnight at Statistical Analysis--
Results are expressed as mean ± S.E., and the significance of differences among treatments was
evaluated using Student's t test for paired data or
analysis of variance by Dunnett's test.
Activation of the cAMP/PKA Pathway Induces CREB Phosphorylation in
Striatal Neurons--
The striatum is the brain area with the most
abundant dopaminergic innervation, and striatal neurons in primary
cultures express all of the dopaminergic receptor subtypes. We
first sought to determine which receptor subtypes and signal
transduction pathways are involved in CREB phosphorylation induced by
the neurotransmitter dopamine in cultured striatal neurons.
Dopaminergic regulation of Ser133 CREB phosphorylation was
investigated by using selective agonists and antagonists specific for
the D1-like and D2-like dopaminergic receptor
subtypes. Dopamine-induced increase in Ser133 CREB
phosphorylation (2.1 ± 0.2-fold basal level, n = 4) was mimicked by the D1-like receptor agonist SKF38393,
not affected by the D2-like receptor antagonist sulpiride,
and blocked by the D1-like antagonist SCH 23390 (Fig.
1A). To confirm the
involvement of cAMP and to rule out any possible G
protein-dependent activation of cAMP-independent signal
transduction pathways, CREB phosphorylation was stimulated by agents
capable of increasing cAMP formation downstream of dopamine receptors.
The membrane-permeable cAMP analogue 8-CPT (200 µM) and
the direct activator of adenylylcyclase forskolin (20 µM)
induced a significant increase (3.2 ± 0.5-fold basal level,
n = 4) of CREB phosphorylation, similar to that induced by 100 µM dopamine.
The recent characterization of a family of cAMP-binding proteins that
directly activates the Ras-like Rap1 protein (21), raises the
possibility that the forskolin-induced CREB phosphorylation might be
mediated by a cAMP-dependent but PKA-independent
mechanism; this mechanism seems unlikely under our conditions, since
forskolin-induced CREB phosphorylation was blocked by the PKA
inhibitors KT5720 and H89 (Fig. 1B).
PKA-induced ERK Activation and CREB Phosphorylation Require PKC
Activity--
In different experimental models (16, 22), the
activation of the cAMP/PKA-dependent pathway leads to
activation of the ERK pathway. To assess whether this cross-talk occurs
in cultured striatal neurons, we performed a set of experiments using a
phosphospecific antibody directed against the dually phosphorylated ERK
isoforms ERK1 and ERK2. We found that DA and forskolin stimulated
predominantly the phosphorylation of the ERK p42 isoform ERK2 (FSK:
4.5 ± 0.8-fold over basal level, n = 4; DA:
3.2 ± 0.5-fold over basal level, n = 4), and this
effect was PKA-dependent, being reduced by the PKA
inhibitor H89 (Fig. 2). PKA-induced
activation of the ERK pathway was an intermediate step in PKA-induced
CREB phosphorylation, since the ERK kinase (MEK) inhibitor PD98059
strongly blocked CREB phosphorylation induced by forskolin and dopamine
(Fig. 3). In previous in vitro
kinase assays, we found that, under our experimental conditions, the
effect of PD98059 was specific for the ERK pathway and did not
interfere with the activity of other kinases such as PKA and CaMKs
(data not shown).
Since in the hippocampus the ERK pathway could be activated by PKC
(22), we investigated the role of this kinase in cAMP/PKA-induced CREB
phosphorylation. The PKC inhibitor bisindoleylmaleimide I strongly
reduced DA- and FSK-induced ERK2 and CREB phosphorylation (Fig.
4A); the same results were
obtained using two other specific PKC inhibitors, chelerythrine
chloride and Gö6976 (Fig. 4B). In vitro
kinase assays demonstrated that, under our experimental conditions, PKC
inhibitors blocked PKC activity but did not interfere with nuclear PKA
activity (Fig. 4C). To further confirm a causal relationship
between PKC activation and ERK2/CREB phosphorylation, we investigated
the functional consequence of short and long term treatment of striatal
cells with the direct PKC activator PMA on ERK and CREB
phosphorylation. When cells were stimulated for 10 min with 1 µM PMA to activate PKC, the MEK inhibitor PD98059 blocked
both PMA-induced ERK2 and CREB phosphorylation (Fig.
5A). Accordingly, when cells
were treated for 18 h with 10 nM PMA to desensitize
PKC, forskolin-induced ERK2 and CREB phosphorylation were significantly
reduced, as compared with untreated cells (Fig. 5B). Taken
together, these experiments strongly suggest that PKA causes CREB
phosphorylation via a PKC/ERK-dependent pathway.
PKA-induced CREB Phosphorylation and CRE-dependent Gene
Transcription Require Intracellular Ca2+--
Previous
studies have shown that neuronal Ca2+ represents a link
between the ERK- and PKA-dependent pathways (16). These
observations prompted us to investigate the Ca2+
requirement in PKA-induced CREB phosphorylation mediated by ERK in
striatal neurons; a set of experiments was performed using the
cell-permeable intracellular Ca2+ chelator BAPTA-AM. When
neurons were preincubated with BAPTA-AM (50 µM) for 30 min, both ERK2 (Fig. 6A) and
CREB phosphorylation (Fig. 6B) induced by forskolin were
significantly reduced.
Having established the requirement of Ca2+ in PKA-mediated
CREB phosphorylation, we then analyzed the possible involvement of L-type Ca2+ channels and NMDA receptors in this mechanism.
Both L-type Ca2+ channels and NMDA receptors have been
previously shown to be involved in CREB phosphorylation and
CRE-dependent gene expression (7). Preincubation of
striatal cells with the competitive NMDA antagonist APV or the
noncompetitive NMDA antagonist MK-801 did not interfere with
forskolin-induced CREB phosphorylation (Fig. 7A). This finding clearly
demonstrates that in striatal neurons NMDA receptors are not
responsible for the intracellular Ca2+ elevation required
in forskolin-induced CREB phosphorylation.
In a subset of striatal neurons, PKA-induced phosphorylation of the
L-type voltage-sensitive Ca2+ channel increases the peak
current through these channels, thereby augmenting the amount of
Ca2+ entering the cell (23). We therefore investigated
whether extracellular Ca2+ was required for PKA-induced
CREB phosphorylation in striatal neurons. To our surprise, we found
that neither the extracellular Ca2+ chelator EGTA nor the
specific L-type voltage-dependent Ca2+ channel
blocker nifedipine interfered with CREB phosphorylation induced by
forskolin (Fig. 7B). To test whether an increase in intracellular Ca2+ could induce CREB phosphorylation, we
analyzed the effect of the SERCA pump inhibitor thapsigargin. As
expected from the previous experiments, thapsigargin induced an
increase (2.8 ± 0.3-fold basal level, n = 4) of
CREB phosphorylation (Fig. 7B). Interestingly, we found that
neither nifedipine nor MK-801 interfered with forskolin-induced ERK2
phosphorylation (Fig. 7C), strongly suggesting, although without proving definitively, that ERK2 activation mediates
PKA-dependent CREB phosphorylation.
To investigate whether intracellular Ca2+ was also required
for the cAMP/PKA-induced gene transcription, we measured the mRNA levels of the immediate early gene c-fos, which contains in
its promoter a CRE sequence. Treatment of striatal neurons for 30 min
with 20 µM forskolin induced a 2.3 ± 0.3-fold
increase (n = 4) of c-fos mRNA over
basal levels; when striatal neurons were preincubated for 30 min with
BAPTA-AM (50 µM) to chelate intracellular Ca2+ or with the MEK inhibitor PD98059 to block the ERK
pathway, forskolin-induced increase of c-fos mRNA was
prevented (Fig. 8).
PKA Activation Releases Intracellular Ca2+--
Our
experiments with BAPTA-AM and thapsigargin indicate that PKA activation
might induce the release of intracellular Ca2+ from
Ca2+ store organelles in striatal neurons. To test this
hypothesis, intracellular Ca2+ measurements in striatal
neurons were performed using the fluorescent Ca2+ probe
fura-2/AM. The Ca2+ ionophore A23187 (5 µM),
which causes a large Ca2+ entry, was first tested as a
positive control (Fig. 9, open
squares, n = 4). Application of forskolin
(20 µM) to fura-2/AM-loaded neurons caused a significant
long lasting (about 30 s) and slowly decreasing rise in
intracellular Ca2+ levels (Fig. 9, full
circles, n = 4). The amplitude, but not the
shape, of the forskolin-induced Ca2+ response was greatly
reduced by preincubating the cells with the PKA inhibitor H89 (20 µM; Fig. 9, open triangles,
n = 4). Finally, to study the role of extracellular
Ca2+, fura-2/AM-loaded neurons were preincubated with the
chelator of extracellular Ca2+ EGTA (5 mM) and
then stimulated with forskolin. In the absence of extracellular
Ca2+, forskolin still elicited a significant sharp, very
short lasting (about 7 s) and fast decreasing rise in
intracellular Ca2+ levels (Fig. 9, open
circles). Taken together, these data indicate that, in
addition to playing a role in regulating voltage-dependent Ca2+ channel function, PKA may also increase intracellular
Ca2+ by releasing Ca2+ from intracellular
stores.
PKA Stimulation Induces Rap1 and PYK2 Activation--
In several
experimental models, the family of small G proteins plays a key role in
Ca2+-induced activation of ERK. Recently, it has been
reported that Rap1, a Ras-like small G protein that may activate the
ERK pathway by interacting with the kinase B-Raf, is activated by
membrane depolarization in a PKAdependent manner (16) and
by PKA via the ERK pathway (24). To test the possibility that Rap1
participates in the signaling pathway responsible for PKA-induced CREB
phosphorylation, we decided to investigate the activation of Rap1 by
means of a pull-down assay using the GST-ralRBD fusion protein.
Stimulation of striatal cells with 20 µM forskolin for 10 min induced a large increase in the amount of Rap1-GTP (Fig.
10A). We then investigated the effects of both kinase inhibitors and of agents capable of interfering with intracellular Ca2+ levels.
Forskolin-induced Rap1 activation was blocked by the PKA inhibitor H89,
but not by the specific PKC inhibitor bisindoleylmaleimide I. Intracellular Ca2+ release appears to be necessary and
sufficient for Rap1 activation, because Rap1 activation was not
observed in the presence of BAPTA-AM and was mimicked by elevation of
intracellular Ca2+ induced by thapsigargin (Fig.
10A). These data indicate that forskolin mainly stimulates
Rap1 activation in a PKA/Ca2+-dependent but
PKC-independent manner, suggesting that the role of PKC in
PKA-dependent CREB phosphorylation is likely to be
downstream of Rap1.
Finally, since in the central nervous system the concomitant elevation
of intracellular Ca2+ and activation of PKC leads to
tyrosine phosphorylation and activation of PYK2 (25), we investigated
whether PYK2 was activated by PKA under our experimental conditions. As
expected, PKA activation induced an increase of tyrosine
phosphorylation of PYK2 immunoprecipitated from total cell lysate of
striatal neurons (Fig. 10B).
This study describes a novel mechanism by which activation of the
cAMP/PKA-dependent pathway may stimulate CREB
phosphorylation in striatal neurons (Fig.
11). Activation of PKA, induced by
dopamine or forskolin, releases Ca2+ from the intracellular
stores, which in turn activates the Rap1·B-Raf complex whose
main target is the phosphorylation of ERK. Finally, the activation of
ERK induces CREB phosphorylation, probably via the translocation of the
ERK·RSK complex into the nucleus. The prevailing model dictates that
an increase of cAMP induces CREB phosphorylation via a mechanism in
which the PKA catalytic subunit translocates into the nucleus and
directly phosphorylates CREB; in this mechanism, the localization of
PKA may contribute to enhance cAMP-dependent CREB
phosphorylation and CRE-dependent gene expression (20).
However, several recent studies have demonstrated that, in addition to
this direct PKA-dependent pathway, other signaling pathways, such as the ERK pathway (10), can induce CREB phosphorylation in a PKA-dependent manner. The participation of the ERK
pathway appears to be very important for PKA-dependent
transcription, because several neurotransmitter receptor systems (22)
as well as depolarization-induced Ca2+ influx (16) have
been shown to activate the ERK pathway via a PKA-mediated mechanism.
This mechanism appears to have an important functional role, since a
previous study in Aplysia has shown that long term
facilitation involves PKA-dependent activation of ERKs (26). In agreement with these data, we have demonstrated that in
striatal neurons the novel mechanism involving PKA-induced intracellular Ca2+ release participates in ERK and CREB
phosphorylation and ultimately regulates the transcription of the
immediate early gene c-fos.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors and the release of Ca2+ from intracellular
stores, elicited by the stimulation of growth factors receptors,
activate the extracellular signal-related protein kinase
(ERK)/mitogen-activated protein kinase pathway and induce CREB
phosphorylation via the ribosomal S6 kinase 2 in PC12 cells (8, 9), in
primary neuronal cultures (10, 11), and in brain slices (12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-2amino-5-phosphonopentanoic acid (APV), and
(+)-MK-801 hydrogen maleate (dizolcipine) were from RBI (Natick, MA).
GST-Sepharose beads and Hybond nylon membranes were from Amersham
Pharmacia Biotech. The bacterial lysate containing the GST fusion
protein of the minimal Rap1-binding domain of ralGDS (GST-ral-RBD) was
a generous gift of Dr. M. Freissmuth (Institute of Pharmacology,
University of Vienna, Austria). N-Acetyl myelin basic
protein 4-14 was from Roche Molecular Biochemicals.
[
-32P]ATP was from PerkinElmer Life Sciences.
All the other reagents were purchased from Sigma.
-D-galactopyranoside, and bacterial
lysates were prepared as described (19). The GST fusion protein
was immobilized by incubating the bacterial lysate for 1 h at
4 °C with glutathione-Sepharose preequilibrated in pull-down buffer
(50 mM Tris buffer, pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 10% glycerol, 1% Nonidet P-40, 2 µg/ml aprotinin, 1 µg/ml leupeptin). Sepharose beads were washed three times to remove
excess GST fusion protein. After stimulation with agonists for 10 min,
the cells were rinsed twice with stimulation buffer and then chilled
immediately in pull-down buffer, and then cell lysate was cleared by
centrifugation at 10,000 × g for 10 min at 4 °C.
Approximately 5% of the total cell lysate was analyzed to normalize
for the total amount of Rap1. The remaining supernatant (about 500 µg
of proteins) was incubated with the GST-Sepharose beads (50 µl of a
1:1 slurry containing about 10 µg of immobilized GST fusion protein)
for 2 h to allow for the association of activated Rap1 with the
effector-GST fusion protein. Samples were washed twice with pull-down
buffer, resuspended in Laemmli sample buffer and applied to
SDS-polyacrylamide gels. The Rap1 band was then visualized using
specific antibodies in a 1:1000 dilution by immunoblot analysis (see below).
20 °C with a 1:1 volume of isopropyl alcohol.
Total RNA was pelleted by centrifugation at 20,000 × g
for 15 min at 4 °C, washed twice with ethanol, and quantified by UV
spectrophotometry at 260 nm. Total RNA (10 µg) was size-resolved on a
1.1% denaturing agarose gel containing 1 M
paraformaldehyde in 20 mM MOPS, pH 7.0, 5 mM sodium acetate, 1 mM EDTA and
capillary-blotted overnight in 10× SSC onto a positively charged
Hybond nylon membrane. The membranes were UV-autocross-linked and then
prehybridized for 1 h at 65 °C in 0.5 M
sodium-phosphate buffer, pH 7.3, 7% SDS, 1 mM EDTA. Hybridization was carried out for 16 h at 65 °C in 50 ml of
prehybridization solution to which about 1 × 106
cpm/ml of a full-length cDNA c-fos or 18 S ribosomal
gene (internal loading control) cDNA random primed
32P-labeled probes were added. Membranes were washed three
times for 2 min in 300 ml of prewarmed (65 °C) washing solution (50 mM
Na2HPO4-NaH2PO4, pH
7.4, 1% SDS) and once in 100 ml of the same solution on a shaking
platform for 30 min at 65 °C. The radioactivity associated with the
membranes was visualized and quantified with a Cyclone storage phosphor
system (Packard Instrument Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stimulation of D1-like receptors
and forskolin induce Ser133 CREB phosphorylation in primary
striatal cultures. A, cells were preincubated for 15 min with the D2-like antagonist sulpiride (SULP,
100 µM) or the D1-like antagonist SCH 23390 (SCH, 100 µM) and then left untreated ( ) or
stimulated for 10 min with dopamine (DA, 100 µM), with the D1-like agonist SKF 38393 (SKF, 100 µM), with dopamine in the presence
of sulpiride (DA + SULP), with dopamine in the
presence of SCH 23390 (DA + SCH), with the
membrane-permeable cAMP analogue 8-CPT (500 µM), and with
the adenylylcyclase activator forskolin (FSK, 20 µM). *, p < 0.05 compared with dopamine.
B, cells were preincubated with the PKA inhibitors KT5720
(10 µM) and H89 (20 µM) for 30 min and then
left untreated (
) or stimulated (+) for 10 min with forskolin
(FSK, 20 µM). *, p < 0.05 compared with forskolin. Nuclear protein extracts were analyzed by
Western blot with antibodies directed against CREB phosphorylated in
Ser133 (P-CREB; upper panel in
A and B) or against phosphorylation
state-independent CREB (CREB, lower panel in
A and B) to normalize for the total amount of
CREB. Data are reported as P-CREB/CREB immunoreactivity
(IR), and the average -fold increase (mean ± S.E.)
over basal level is reported in the bar graph
(n = 4).
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Fig. 2.
Forskolin- and dopamine-induced ERK2
phosphorylation is mediated by PKA. A, cells were
preincubated with the PKA inhibitor H89 (20 µM) for 30 min and then left untreated ( ) or stimulated (+) for 10 min with
forskolin (FSK, 20 µM) or with dopamine
(DA, 100 µM). Total cell lysate extracts were
then analyzed by Western blot with antibodies against dually
phosphorylated (Thr202 and Tyr204) ERKs
(P-ERK2, upper panel) or against phosphorylation
state-independent ERKs (ERK2, lower panel),
to normalize for the total amount of ERKs. Data are reported as dually
phosphorylated/phosphorylation state-independent ERK immunoreactivity
(IR), and the average -fold increase (mean ± S.E.)
over basal level is reported in the bar graph
(n = 4). *, p < 0.05 compared with the
correspondent stimulus without inhibitor.
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Fig. 3.
Forskolin- and dopamine-induced CREB
phosphorylation is mediated by MEK. Cells were preincubated for 30 min with the MEK inhibitor PD98059 (50 µM) and then left
untreated ( ) or stimulated (+) for 10 min with forskolin
(FSK, 20 µM) or with dopamine (DA,
100 µM). CREB phosphorylation in nuclear extracts was
analyzed as described in the legend of Fig. 1. Data are reported as
P-CREB/CREB immunoreactivity (IR), and the average -fold
increase (mean ± S.E.) over basal level is reported in the
bar graph (n = 4). *,
p < 0.05 compared with the correspondent stimulus
without inhibitor.
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Fig. 4.
Forskolin- and dopamine-induced CREB
phosphorylation is blocked by PKC inhibitors. A, cells
were preincubated for 30 min with the PKC inhibitor
bisindoleylmaleimide I (BIS, 10 µM) and then
left untreated ( ) or stimulated (+) for 10 min with forskolin
(FSK, 20 µM) or with dopamine (DA,
100 µM). P-CREB and P-ERK2 phosphorylation was analyzed
as described in the legends of Figs. 1 and 2, respectively. Data are
reported as P-ERK2/ERK2 immunoreactivity (IR) or P-CREB/CREB
immunoreactivity (IR), and the average -fold increase
(mean ± S.E.) over basal level is reported in the bar
graph (n = 4). *, p < 0.05 compared
with the correspondent stimulus without inhibitor. B, cells
were preincubated for 30 min with the PKC inhibitors chelerythrine
chloride (CLRT, 1 µM) or Gö6976 (5 µM) and then left untreated (
) or stimulated (+) for 10 min with forskolin
(FSK, 20 µM). CREB phosphorylation in nuclear
extracts was analyzed as described in the legend to Fig. 1. Data are
reported as P-CREB/CREB immunoreactivity (IR), and the
average -fold increase (mean ± S.E.) over basal level is reported
in the bar graph (n = 4). *, p < 0.05 compared with the correspondent stimulus without inhibitor.
C, cells were preincubated for 30 min with the PKC inhibitor
bisindoleylmaleimide I (BIS, 10 µM),
chelerythrine chloride (CLRT, 1 µM), or
Gö6976 (5 µM) and then left untreated (
) or
stimulated (+) for 10 min with forskolin (FSK, 20 µM). Cells were harvested, and PKA and PKC in
vitro activities were assayed as reported in the references cited
under "Experimental Procedures." Data are reported as
forskolin-induced kinase activity increase normalized to the
corresponding basal level in the presence of the indicated inhibitor.
The average -fold increase (mean ± S.E.) is reported in the
bar graph (n = 4). *,
p < 0.05 compared with forskolin without
inhibitor.
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Fig. 5.
Activation of PKC induces ERK2 and CREB
phosphorylation. A, cells were preincubated for 30 min
with the MEK inhibitor PD98059 (50 µM) and then left
untreated ( ) or stimulated (+) for 10 min with PMA (1 µM). P-CREB and P-ERK2 phosphorylation was analyzed as
described in the legends of Figs. 1 and Fig. 2, respectively. Data are
reported as P-ERK2/ERK2 or P-CREB/CREB immunoreactivity
(IR), and the average -fold increase (mean ± S.E.)
over basal is reported in the bar graph (n = 4). *,
p < 0.05 compared with the correspondent stimulus
without inhibitor. B, cells were left untreated (
) or
pretreated for 18 h with 10 nM PMA (des
PKC) to desensitize PKC. Then cells were left untreated (
) or
stimulated (+) for 10 min with forskolin (FSK, 20 µM). P-CREB and P-ERK2 phosphorylation was analyzed as
described in the legends of Figs. 1 and 2, respectively. Data are
reported as P-ERK2/ERK2 or P-CREB/CREB immunoreactivity
(IR), and the average -fold increase (mean ± S.E.)
over basal level is reported in the bar graph
(n = 4). *, p < 0.05 compared with
forskolin in PMA-untreated cells.
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Fig. 6.
Forskolin-induced ERK2 and CREB
phosphorylation requires intracellular Ca2+. Cells
were preincubated for 30 min with BAPTA-AM (50 µM) and
then left untreated ( ) or stimulated (+) for 10 min with forskolin
(FSK, 20 µM). P-CREB and P-ERK2
phosphorylation was analyzed as described in the legends of Figs. 1 and
2, respectively. Data are reported as P-ERK2/ERK2 or P-CREB/CREB
immunoreactivity (IR), and the average -fold increase
(mean ± S.E.) over basal level is reported in the bar
graph (n = 4). *, p < 0.05 compared with forskolin in BAPTA-untreated cells.
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Fig. 7.
Forskolin-induced CREB and ERK2
phosphorylation is not mediated by Ca2+ channels or NMDA
receptors. A, cells were preincubated for 15 min with
the competitive NMDA receptor antagonist APV (100 µM) or
with the noncompetitive NMDA receptor antagonist MK-801 (10 µM) and then left untreated ( ) or stimulated (+) for 10 min with forskolin (FSK, 20 µM). P-CREB
phosphorylation was analyzed as described in the legend to Fig. 1. Data
are reported as P-CREB/CREB immunoreactivity (IR), and the
average -fold increase (mean ± S.E.) over basal level is reported
in the bar graph (n = 4).
B, cells were preincubated for 15 min with the extracellular
Ca2+ chelator EGTA (5 mM) and the
voltage-sensitive Ca2+ channel blocker nifedipine
(NIF, 10 µM) and then left untreated (
) or
stimulated (+) for 10 min with forskolin (FSK, 20 µM) or with the SERCA pump inhibitor thapsigargin
(TPG, 200 nM). P-CREB phosphorylation was
analyzed as described in the legend of Fig. 1. Data are reported as
P-CREB/CREB immunoreactivity (IR), and the average -fold
increase (mean ± S.E.) over basal level is reported in the
bar graph (n = 4). C,
cells were preincubated for 15 min with the voltage-sensitive
Ca2+ channel blocker nifedipine (NIF, 10 µM) or with noncompetitive NMDA receptor antagonist
MK-801 (10 µM) and then left untreated (
) or stimulated
(+) for 10 min with forskolin (FSK, 20 µM).
P-ERK2 phosphorylation was analyzed as described in the legend to Fig.
2. Data are reported as P-ERK2/ERK2 immunoreactivity (IR),
and the average fold increase (mean ± S.E.) over basal level is
reported in the bar graph (n = 4).
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Fig. 8.
Forskolin-induced c-fos
transcription is mediated by Ca2+ and ERK signaling
pathways. Cells were preincubated for 30 min with the
intracellular Ca2+ chelator BAPTA-AM (50 µM)
or with the MEK inhibitor PD98059 (50 µM) and then left
untreated ( ) or stimulated (+) with forskolin (FSK, 20 µM) for 30 min. Total RNA was extracted from the cell
lysates, separated by 1% gel agarose electrophoresis, and blotted to
nylon membranes. The membrane was hybridized with a c-fos
(upper panel) and an 18 S ribosomal RNA (18 S,
lower panel) probe and then exposed to x-ray film
for autoradiography. Data are reported as c-fos mRNA
levels normalized to 18 S mRNA, and the average -fold increase
(mean ± S.E.) over basal level is reported in the bar
graph (n = 4). *, p < 0.05 compared with forskolin without inhibitor.
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Fig. 9.
Forskolin induces intracellular
Ca2+ release. Fura-2/AM-loaded striatal neurons were
preincubated for 30 min with EGTA (5 mM) or the PKA
inhibitor H89 (10 µM) and then stimulated at 21 °C at
the time indicated by the arrows with forskolin
(FSK, 20 µM) or the Ca2+ ionophore
A23187 (5 µM). Fura-2 fluorescence (340/380-nm ratio) was
measured every 2 s, and data were analyzed as described under
"Experimental Procedures." The figure shows averages obtained from
a total of 15 cells in one representative experiment; the S.E. (not
shown) was less than 15%. Within each optic field, the number of cells
that responded to the treatment was 5.2 ± 1.4 (n = 24 optic fields). A total of four independent experiments (four
different sets of cultures) were performed and gave similar results. *,
p < 0.05 compared with their respective basal
(unstimulated) values.
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Fig. 10.
Forskolin activates Rap1 and PYK2.
A, cells were preincubated for 30 min with BAPTA-AM
(BAPTA, 50 µM), the PKC inhibitor
bisindoleylmaleimide I (BIS, 10 µM), and the
PKA inhibitor H89 (20 µM) and then left untreated ( ) or
stimulated (+) for 10 min with forskolin (FSK, 20 µM) or thapsigargin (TPG, 200 nM).
Preincubation with these agents did not modify basal Rap1-GTP levels
(not shown). Total cell lysate (0.5 mg) was processed for the pull-down
assay of Rap1 using the GST-ral-RBD protein as described under
"Experimental Procedures." Western blot analysis was performed on
the pulled down Rap1-GTP (upper panel) and on
total Rap1 (lower panel) in a 5% (25-µg)
extract of total cell lysate as a loading control (bottom
panel). Data are reported as Rap1-GTP immunoreactivity, and
the average -fold increase (mean ± S.E.) over basal level is
reported in the bar graph (n = 4). *, p < 0.05 compared with forskolin. B,
cells were stimulated for 10 min with forskolin (FSK, 20 µM), and then PYK2 was immunoprecipitated from total cell
lysate (1 mg). The immunoprecipitate was analyzed by Western blot with
antiphosphotyrosine (WB P-Tyr, upper
panel) or anti-PYK2 (WB PYK2, lower
panel) antibodies as described under "Experimental
Procedures." Data are reported as Tyr(P) immunoreactivity
(IR), and the average -fold increase (mean ± S.E.)
over basal level is reported in the bar graph
(n = 4). *, p < 0.05 compared with
basal level.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 11.
Model of PKA-induced CREB phosphorylation
mediated by the PKC/ERK-dependent pathway. Activation
of the PKA pathway by stimulation of D1-like dopamine
receptors or by direct activation of adenylylcyclase (AC) by
forskolin (FSK) causes the release of calcium from
intracellular stores. Calcium then activates the ERK signaling pathway
via a mechanism that could be mediated by PKC, PYK2, and the
Rap1·B-Raf complex. Activated ERK translocates into the nucleus and
induces CREB phosphorylation and CRE-dependent gene
expression (in this case an increase of c-fos mRNA)
probably via an indirect mechanism involving the family of RSKs.
Previous studies in various experimental models have shown that the functionality of intracellular Ca2+ store organelles could be modulated by PKA-dependent phosphorylation in vivo and in vitro. The neuronal type-I IP3 receptor, immunopurified from rat cerebellar membranes, can be phosphorylated by PKA in vitro (27), and activation of PKA induces phosphorylation of the type-I IP3 receptor in cerebellar slices (27) and in cultured cells (28, 29). Notably, a recent paper has described that, in cardiac tissue, PKA-dependent phosphorylation of ryanodine receptor/Ca2+ release channel regulates the channel open probability (30). These studies suggest that PKA-dependent phosphorylation could modulate the functionality of intracellular Ca2+ store organelles by increasing their sensitivity to physiological agonists or toward external Ca2+. Given the importance of the cross-talk between PKA- and Ca2+-dependent pathways, the precise mechanism of PKA-mediated intracellular Ca2+ release will certainly become a focus of future studies in neuronal cell physiology.
In PC12 cells and primary cortical neurons, ERK activation induced by
Ca2+ entry through voltage-sensitive channels or by the
release of Ca2+ from intracellular stores is mediated by
the small G protein Ras (14). Several factors, such as the route and
magnitude of Ca2+ influx or the subcellular
compartmentalization of protein kinases in the cell type under
investigation, contribute to determine which signaling pathway upstream
of ERK is activated. Several recent studies performed in various cell
types have demonstrated that Ca2+, in addition to Ras, may
also activate ERK via another route involving the Ras homologue Rap1
and its effector Raf-like kinase B-Raf. The Rap1·B-Raf system
participates in PKA-mediated ERK activation induced by growth factors
in PC12 cells (14, 31, 32) and by 2 adrenergic receptor
stimulation in HEK 293 cells (24). In agreement with these reports, we
propose that in striatal neurons the Rap1·B-Raf system is an
intermediate step in the activation of the ERK pathway triggered by
PKA-induced intracellular Ca2+ release. This model is in
contrast with what was found by other authors in PC12 cells (16), in
which depolarization-induced Ca2+ entry activates the ERK
pathway via a two-step mechanism involving first PKA and then Rap1. In
a previous study (33) and in experiments not reported here, we found
that, in cultured striatal neurons, Ca2+ entry induced by a
Ca2+ ionophore or by depolarization inhibits cAMP
formation, probably interacting with the Ca2+-inhibitable
type V adenylyl cyclase isoform, which is abundantly expressed in these
neurons (18).
The complexity of Rap1 activation mechanisms in neuronal cells has been emphasized by the characterization of new families of Rap guanine nucleotide exchange factors activated by Ca2+ and diacylglycerol (34) or by cAMP and Ca2+ (17, 21). On the other hand, our findings demonstrate that, in agreement with another report (35), Rap1 activation is induced by PKA-dependent release of intracellular Ca2+ (35). The differences in the mechanism of Rap1 activation are probably due to the differential expression of adenylyl cyclase isoforms and small G protein guanine nucleotide exchange factors in the cell types under investigation. Moreover, since Rap1 activation is not triggered by a PKA- or PKC-dependent phosphorylation, two mechanisms may be considered. First, Rap1 could be directly activated by the novel Rap1GEFs, which in turn are directly activated by Ca2+. Second, Rap1 could be activated indirectly via a multiple step mechanism involving the Ca2+-dependent stimulation of PYK2, which then interacts with Rap1GEF C3G (36). In agreement with this latter hypothesis, in the present study we have found that activation of the cAMP/PKA-dependent pathway leads to activation of the tyrosine kinase PYK2 (25). This is consistent with the observation that, in striatal brain slices, stimulation of D2 receptors induces tyrosine phosphorylation of PYK2 via a phospholipase C-mediated intracellular Ca2+ release (37). So far, it is still unclear whether in striatum activation of PYK2 is implicated in CREB phosphorylation, because D2-dependent CREB phosphorylation was not blocked by the broad spectrum tyrosine kinase inhibitor genistein (37).
Although the experiments presented in this study show that in striatal neurons PKC inhibitors block PKA-induced CREB phosphorylation, no conclusion can be drawn about the mechanism by which the cAMP/PKA-dependent pathway activates PKC and at what level PKC modulates CREB phosphorylation. Having excluded a direct role of PKC in Rap1 activation in our model, it is reasonable to assume that PKC acts downstream of Rap1 and upstream of B-Raf. This hypothesis is supported by studies showing that PKC may play a facilitator role on the small G protein-dependent ERK pathway activation by enhancing Raf-1 or B-Raf functionality (38).
In conclusion, we have found that intracellular Ca2+
release induced by PKA leads to CREB phosphorylation via a PKC- and
ERK-dependent mechanism, most likely involving Rap1
activation. Given the complexity of cross-talk between the cAMP/PKA and
the ERK pathways, the plethora of signaling pathways converging on ERK,
and the wide array of transcription factors activated by these
signaling pathways, this and other studies (16, 23) raise fundamental
questions concerning the role of Rap1 and the functional effects of its
activation at the level of transcription factor activation and
CRE-dependent gene expression.
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FOOTNOTES |
---|
* 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.
** To whom correspondence should be addressed: Dipartimento di Farmacologia Sperimentale ed Applicata, Facoltà di Farmacia, Università di Pavia, 14 Viale Taramelli, Pavia 27100, Italy. Tel.: 39 382 507739; Fax: 39 382 507405; E-mail: schnll@unipv.it.
Published, JBC Papers in Press, January 3, 2001, DOI 10.1074/jbc.M007631200
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ABBREVIATIONS |
---|
The abbreviations used are:
CRE, cAMP-response element;
PKA, protein kinase A;
CREB, cAMP-response
element-binding protein;
ERK, extracellular signal-regulated kinase;
PKC, protein kinase C;
CaMK, Ca2+/calmodulindependent kinases;
NMDA, N-methyl-D-aspartic acid;
RSK, ribosomal S6
kinase;
H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide;
8-CPT, 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate
sodium salt;
PMA, phorbol 12-myristate 13-acetate;
BAPTA-AM, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid tetra-(acetoxymethyl)-ester;
fura-2/AM, 1-[2-(5-carboxyoxazol-2-yl)- 6-aminobenzo-furan-5-oxy]-2-(2'-amino-5'methylphenoxy)-ethane
N,N-N',N'-tetraacetic acid
pentacetoxymethyl ester;
PD98059, 2'-amino-3'-methylflavone;
Gö6976, [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole];
APV, D-()-2-amino-5-phosphonopentanoic acid;
GST-ral-RBD, GST fusion protein of the minimal Rap1-binding domain of ralGDS;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
TBS, Tris-buffered saline;
TBSM, TBS containing 4%
nonfat dry milk;
MOPS, 3-(N-morpholino)propanesulfonic acid;
P-CREB, phosphorylated Ser133 CREB;
P-ERK2, phosphorylated
ERK2;
GST, glutathione S-transferase.
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