 |
INTRODUCTION |
The myristoylated alanine-rich protein kinase C substrate
(MARCKS)1 with an apparent
molecular mass of 80-87 kDa is a prototype of family members of
prominent cellular substrates for protein kinase C (PKC). Comparison of
known MARCKS sequences revealed three highly conserved regions: the N
terminus, which contains a myristoylation consensus sequence, the MH2
domain, and a basic effector domain, which contains the PKC
phosphorylation sites and calmodulin- and actin-binding sites (1-3).
Within the basic internal domain of 25 residues, three or four serine
residues are phosphorylated by PKC (4, 5). The potential to bind
calcium/calmodulin and cross-links filamentous (F)-actin is regulated
by PKC-dependent phosphorylation (4, 6). In addition, the
PKC-dependent phosphorylation introduces negative charges
into the basic cluster, reducing its electrostatic interaction with
acidic lipids and results in translocation of MARCKS from membrane to
cytoplasm (7-10). Genomic analysis with Southern blots and polymerase
chain reactions revealed the presence of only the 87-kDa MARCKS gene in
bovine and human genomes and only the 80-kDa MARCKS gene in the mouse
and rat genomes. These are equivalent genes in the different species,
and there is about 70% amino acid similarity between the 87-kDa and
80-kDa MARCKS (11). MacMARCKS, with a molecular mass of 48-60 kDa, is
another member of the MARCKS family, which has been cloned from mouse
macrophage (12) and mouse brain (13). MacMARCKS also has a
myristoylated N terminus, a highly conserved MH2 domain, and a basic
effector domain that contains PKC phosphorylation sites.
Protein and mRNA of MARCKS are widely distributed and are most
abundant in brain, spinal cord, spleen, and lung (14). In the brain,
MARCKS is widespread throughout the brain and is enriched in certain
regions, including the piriform and entorhinal cortices, portions of
the amygdaloid complex, the intralaminar thalamic nuclei, the
hypothalamus, the nucleus of the solitary tract, nucleus ambiguus, and
many catecholaminergic and serotonergic nuclei (15). In situ
hybridization also revealed a high expression of mRNA in the
hippocampal CA1 and dentate gyrus (16). Electron microscopic analysis
revealed immunoreactivity in axons, axon terminals, small dendritic
branches, and occasionally in dendritic spines. No immunoreactive product was observed in large dendrites, somata, or nuclei (15). Furthermore, disruption of the MARCKS gene in mice leads to abnormal brain development and perinatal death, with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (17). The observation suggests that expression of MARCKS during embryonic and fetal life in
the mouse is necessary for normal brain development of the central
nervous system.
The properties of MARCKS, including cross-linking F-actin and binding
to plasma membrane, suggest that MARCKS regulates actin-membrane interaction and in turn maintains cell shape and motility. Consistent with this hypothesis, MARCKS is phosphorylated during chemotaxis, secretion, and phagocytosis in neutrophils and macrophages (18, 19),
during neurosecretion (20, 21), and during mitogenesis (22, 23).
PKC-dependent phosphorylation is possibly involved in
functional roles of MARCKS. However, accumulating evidence has
suggested that MARCKS is also an in vivo and in
vitro substrate of proline-directed protein kinases, such as
mitogen-activated protein (MAP) kinase and
cycline-dependent protein kinase (cdk) 5. A mass
spectroscopic analysis of intact MARCKS purified from bovine brain
revealed at least 6 phosphorylation sites in the Ser-Pro motif in the
N-terminal domain and upstream of the phosphorylation sites for PKC
(24). Furthermore, the proline-directed protein kinases, such as MAP
kinase (25) and cdc2 kinase or cdk5 (26), can phosphorylate recombinant
mouse MARCKS and purified rat MARCKS, respectively.
MAP kinase, especially ERK 2, was found to be widely expressed in whole
rat brain and enriched in the hippocampal formation. In addition,
neurotransmitters and neurotrophic factors have been seen to activate
MAP kinase in neurons (27, 28). For example, activation of glutamate
receptors, especially the NMDA receptor, causes a large increase in MAP
kinase in the hippocampal neurons (29, 30), and stimulation of
AMPA/kainate receptors elevated MAP kinase activity in cultured
cortical neurons (31). Similarly, basic fibroblast growth factor,
epidermal growth factor, and brain-derived neurotrophic factor
stimulate MAP kinase activity to the same extent as seen with glutamate
(30, 32-34). Taken together, MAP kinase is a possible candidate for
phosphorylation of MARCKS in vivo in the central nervous
system. We now report that a long lasting increase in MARCKS
phosphorylation in rat hippocampal neurons following stimulation of the
glutamate receptor was mainly though the NMDA receptor activation and
is due to activation of MAP kinase rather than PKC. Furthermore, a
transient increase in the PKC-dependent phosphorylation of
MARCKS was observed following stimulation of the glutamate receptors.
 |
EXPERIMENTAL PROCEDURES |
Materials--
The following chemicals and reagents were
obtained from the indicated sources: Nu serum, Collaborative Research;
[
-32P]ATP, 125I-protein A, and
125I-calmodulin, NEN Life Science Products;
[32P]orthophosphate, ICN Biochemicals;
L-glutamate, AP3, and phorbol 12-myristate 13-acetate
(PMA), Sigma; D-AP5, CNQX, NMDA, ACPD, and AMPA, Tocris
Neuramin; calphostin C, Kyowa Medex Co. Ltd.; PD098059, Research
Biochemicals International; lysyl endopeptidase (Pseudomonas
aeruginosa), Seikagaku Co.; actin (rabbit skeletal muscle), Sigma;
monoclonal antibody against microtubule associated protein 2, Amersham
Pharmacia Biotech; and purified MAP kinase (sea star
Pseudomonas ochraceus oocytes), Upstate
Biotechnology (its specific activity is 0.5 µmol of phosphate/min/mg
using myelin basic protein as substrate). The polyclonal antibody
against CaM kinase II, which recognized both
and
subunits, was
prepared as described (35). PKC was partially purified based on the
procedures of Wooten et al. (36). The procedures contained
sequential chromatography on DEAE-Sephacel, Sephacryl S-100, and
phenyl-Sepharose CL-4B columns. The specific activity was 3.0 µmol of
phosphate/min/mg using myelin basic protein as substrate.
Preparation of MARCKS Antibodies--
MARCKS was purified from
rat brains according to the method by Patel and Kligman (37), except
that a DEAE-cellulose column was used instead of a Mono Q column.
MARCKS purified by this procedure was subjected to SDS-PAGE, and the
MARCKS protein band was excised from the gel. The gels were emulsified
in complete Freund's adjuvant and used to raise antisera in rabbits.
The IgG fraction from the antisera was prepared and used in the present
study. To prepare a phospho-specific antibody against PKC
phosphorylation sites in MARCKS, the phosphopeptide
KRFS(P)FKKS(P)FKLSG, which contains two PKC phosphorylation sites,
Ser-152 and Ser-156, was synthesized and used to raise antisera in
rabbits. The phospho-specific antibody was affinity-purified from the
serum by sequential chromatography on the nonphosphorylated
peptide-conjugated and the phosphopeptide-conjugated columns. Further
characterization of the phospho-specific antibody will be described
elsewhere by Yamamoto et
al.2 (manuscript in
preparation). The specificity of both antibodies is shown in Figs. 1
and 7.
Cell Culture--
Neonatal rat hippocampal cell cultures were
prepared as described (38). Briefly, hippocampi were removed from
Wistar rats on postnatal day 1 and placed in growth medium consisting
of Eagle's minimum essential medium (Life Technologies, Inc.)
containing 10% fetal calf serum, 10% horse serum, 2% Nu serum, 12 ng/ml nerve growth factor, and 30 mg/liter kanamycin. Cells were
mechanically dissociated by trituration with fire-polished Pasteur
pipettes and seeded at a density of 3.5 × 105
cells/35-mm dish pretreated with calf skin collagen (Sigma type III).
One day after plating the neurons, cultures were treated with 5 µM cytosine-
-arabinofuranoside to prevent the
replication of nonneuronal cells. The culture medium was replaced by
growth medium lacking 10% fetal calf serum at 2 and 6 days of culture. The cells were maintained in humidified 95% air and 5%
CO2 at 37 °C for 8-10 days before use.
Immunofluorescence Analysis--
Immunofluorescence analysis of
cultured hippocampal cells was carried out as described (38). In short,
cells in a 35-mm dish were fixed for 10 min at -20 °C with cold
methanol. After air drying the dishes, cells were washed in
phosphate-buffered saline (PBS) and permeabilized in 0.05% Triton
X-100 in PBS for 10 min. Nonspecific antibody binding was blocked by
preincubation in 5% goat serum in PBS (blocking solution) for 20 min.
Anti-MARCKS polyclonal (1 mg of IgG/ml) and anti-MAP2 monoclonal
(Amersham Pharmacia Biotech) antibodies were diluted 1:100 and 1:50,
respectively, in the blocking solution. Cells were incubated with the
primary antibodies overnight at 4 °C. The cells were then washed in
PBS and incubated in fluorescein-conjugated goat anti-rabbit IgG
(Cappel) and rhodamine-conjugated goat anti-mouse IgG (Tago, Inc.).
Negative controls were immunostained with the MARCKS antibody
preabsorbed with an excess of purified MARCKS. Following treatment with
secondary antibodies, the cultures were washed with PBS and covered
with a 22-mm coverslip.
Labeling of Cells--
Eight- to 10-day cultured hippocampal
cells were washed once with phosphate-free and serum-free minimum
essential medium and labeled in 1.0 ml of this medium containing
carrier-free [32P]orthophosphate (0.25 mCi/ml), as
described (39). After labeling for 5 h, the cells were incubated
with Krebs-Ringer HEPES (KRH) solution, which contained 128 mM NaCl, 5 mM KCl, 1 mM
NaHPO4, 2.7 mM CaCl2, 1.2 mM MgSO4, 10 mM glucose, and 20 mM HEPES (pH 7.4). After incubation for 30 min in KRH,
cells were incubated at 37 °C for the specified time with KRH ± MgCl2 without (controls) or with the specified test
agent. After incubation for the indicated time, the medium was quickly
aspirated, and the cells were frozen in liquid N2.
Immunoprecipitation and Quantitation of 32P-MARCKS
and 32P-CaM kinase II--
Cells were harvested and
homogenized in 0.4 ml of the solubilization solution containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.5% Triton
X-100, 10 mM EDTA, 1 mM
Na3VO4, 30 mM sodium pyrophosphate, 50 mM NaF, 100 nM calyculin A, 0.1% SDS, 0.1 mM leupeptin, 75 µM pepstatin A, and 0.1 mg/ml aprotinin. Sonication and centrifugation were performed as
described (38). Aliquots (20 µl) of the supernatant were used to
determine protein content and 32P incorporation into total
proteins by trichloroacetic acid precipitation. To determine the
32P incorporation, Whatman 3MM filter papers on which the
aliquots (20 µl) were spotted were washed with trichloroacetic acid,
acetone, and ethanol. After drying, the radioactivity was measured by a liquid scintillation counter. The radioactivities of the
32P incorporation into the total proteins were not
different between the control and the stimulated cells (data not
shown). The supernatant fraction containing the same amount of
radioactivity was incubated at 4 °C for 4 h with antibodies to
MARCKS (10 µg of IgG protein) and/or CaM kinase II (10 µg of IgG
protein) and 75 µl of protein A-Sepharose CL-4B suspension (50%
v/v). The immunocomplex immobilized on protein A was washed three times
with solubilization solution. After immunoprecipitation of
32P-MARCKS and 32P-CaM kinase II, the
immunoprecipitates were eluted from protein A-Sepharose CL-4B by
treatment with the SDS sample buffer (40) and boiled for 4 min.
Supernatants were subjected to SDS-PAGE (40), followed by
autoradiography. A Bio-Imaging Analyzer (BA100, Fuji Film) was used to
quantify the amount of 32P incorporation into MARCKS and
CaM kinase II subunits.
Analysis of Phosphopeptide Mapping by HPLC--
The
phosphorylation sites of MARCKS were determined as described (24) using
limited proteolysis of 32P-labeled MARCKS in gel pieces
with 1 or 2 µg of lysyl endopeptidase (P. aeruginosa). In
control experiments, purified rat brain MARCKS was phosphorylated by
MAP kinase or PKC in appropriate conditions for each kinase. MARCKS (2 µg) was incubated for 30 min with 20 nM MAP kinase in the
presence of 50 mM HEPES buffer, pH 7.5, 10 mM
MgCl2, 1 mM EGTA, 0.1 mM
[
-32P]ATP, and 1 mM dithiothreitol or with
20 nM PKC in the presence of 50 mM HEPES
buffer, pH 7.5, 10 mM MgCl2, 1 mM
CaCl2, 0.1 mM [
-32P]ATP, 1 mM dithiothreitol, 50 µg/ml phosphatidylserine, and 5 µg/ml 1,3-diolein. An additional control was prepared by
phosphorylation of MARCKS with both MAP kinase and PKC under the
conditions used in PKC-dependent phosphorylation. The
in vitro phosphorylated MARCKS and in situ
phosphorylated MARCKS immunoprecipitated with the antibody were
separated by SDS-PAGE and cut from the gel. After incubation in gel
pieces for 10 h at 35 °C with lysyl endopeptidase, the reaction
was terminated by addition of 0.1% of trifluoroacetic acid, at a final
concentration. After the gel pieces were removed by centrifugation, the
supernatant was applied to a C18 column (4 × 150 mm) in an HPLC
apparatus (Hitachi L-6000). The MARCKS peptides were eluted
with a linear gradient of H2O-acetonitrile in the presence
of 0.1% trifluoroacetic acid at a flow rate 1 ml/min. The eluate was
collected by a fraction collector, and the radioactivity of each
fraction was counted by liquid scintillation spectrometry.
Cross-linking of 125I-CaM to MARCKS--
Purified
MARCKS was cross-linked to 125I-CaM (65 µCi/µg) by the
method of Graff et al. (4) as described (26). MARCKS (1.5 µg) was incubated for 30 min at 30 °C without or with PKC or MAP kinase in the presence of 0.5 mM ATP. The MARCKS protein
(1.5 µg) were incubated for 1 h at 25 °C with
125I-CaM (0.2 µCi/3 ng) in 38 mM HEPES (pH
7.5) and 2.5 mM CaCl2 in a final volume of 50 µl. After addition of disuccinimidyl suberate in a final
concentration of 0.25 mM, the sample was further incubated for 20 min at 25 °C. The reaction was terminated by addition of Tris-HCl (pH 7.5) to a final concentration of 5 mM. The
sample was treated with the SDS-sample buffer and boiled for 2 min. The sample was subjected to SDS-PAGE, followed by autoradiography. The
cross-linking of 125I-CaM to MARCKS was totally abolished
by inclusion of an excess amount of nonlabeled CaM in the medium (data
not shown).
Interaction of MARCKS with F-actin--
The interaction of
MARCKS with F-actin was determined by a co-sedimentation assay as
described (42). After preparation of F-actin by polymerization of
G-actin, the MARCKS protein (1.5 µg) was incubated for 30 min with
F-actin (10 µg) in 60 µl of Buffer C (0.1 M KCl, 1 mM MgCl2, 0.1 mM EGTA, 0.2 mM ATP in 10 mM Tris-HCl, pH 7.8). When
indicated, the MARCKS protein was phosphorylated by PKC or MAP kinase
in appropriate conditions for each kinase before the co-sedimentation
assay. The samples were then centrifuged for 15 min at 100,000 × g. The pellet containing F-actin and co-sedimented MARCKS
was resuspended with SDS-sample buffer and subjected to SDS-PAGE. After
electrophoresis, gels were stained with Coomassie Brilliant Blue or
subjected to immunoblotting analysis using anti-MARCKS antibody to
determine amount of MARCKS bound to F-actin.
Other Methods--
Protein was determined by the method of
Bradford (43) using bovine serum albumin as standard.
Statistical Evaluation--
Values are means ± S.E.
Comparison between two experimental groups were made by the unpaired
Student's t test. For multiple comparisons, one-way
analysis of variance with Sheffe's correction was used, and
p values of < 0.05 were considered to have statistical significance.
 |
RESULTS |
Specificity of Anti-MARCKS Antibody and Immunofluorescent
Localization of MARCKS in Cultured Neurons--
By Western blot
analysis, the anti-MARCKS antibody recognized MARCKS with the molecular
mass of 80 kDa in the purified rat MARCKS and in the hippocampal
homogenate from the rat brain (Fig. 1).
In addition, the 48-kDa protein was immunoreactive in the homogenate.
Preabsorption of the antibody with an excess of the purified MARCKS
abolished the immunoreactivity against both the 80-kDa protein and the
48-kDa protein. The 48-kDa protein may be a degraded product, as
reported (44). Although MacMARCKS, a MARCKS-related protein with a
molecular mass with 48-52 kDa, was seen to be present in the rat brain
(45) and was even cloned from mouse brain (13), the 48-kDa protein does
not seem to be MacMARCKS, because the antibody did not recognize the
MacMARCKS protein in rat peritoneal macrophages (data not shown).

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 1.
Immunospecificity of antibodies against rat
brain MARCKS. The purified rat brain MARCKS (2 µg) (lane
1) and the homogenate (50 µg) of rat hippocampus (lane
2) were separated by SDS-PAGE containing 10% acrylamide and
electrophoretically transferred to a nitrocellulose membrane. The
membranes were incubated with 10 µg of IgG/ml of anti-MARCKS
antibodies. Bound antibodies were visualized with
125I-labeled protein A. A, protein stained with
Coomassie Brilliant Blue. B, immunoblots of the protein
(lanes 1 and 2) and the MARCKS antibody was
preabsorbed with an excess of the purified MARCKS before immunoblotting
(lanes 3 and 4). Lanes 1 and
3 and lanes 2 and 4 correspond to the
purified MARCKS and brain homogenate, respectively. Two proteins with
molecular masses of 80 and 50 kDa in the homogenate were recognized by
the anti-MARCKS antibody, and both bands disappeared by the absorption
of the antibody with MARCKS. C, in the
32P-labeled experiments, the anti-MARCKS antibody could
specifically immunoprecipitate the 80-kDa MARCKS from cell extracts of
unstimulated or stimulated hippocampal cells with glutamate.
|
|
Using 32P-labeled hippocampal neurons, the antibody could
specifically immunoprecipitate the phosphorylated 80-kDa MARCKS from cell extracts of cultured hippocampal neurons incubated in the presence
or absence of glutamate (Fig. 1). Although MacMARCKS was reported to be
phosphorylated by stimulation by depolarization of rat brain
synaptosomes (45), there was no apparent phosphorylation of the 48-kDa
protein as determined by immunoblot analysis (Fig. 1C). The
finding also suggested that the antibody was not cross-reactive with
MacMARCKS. After 10 days in culture, hippocampal neurons were
investigated immunohistochemically for expression of MARCKS. The
cultured neurons displayed strong immunofluorescence with anti-MARCKS
antibody in somata and neurites of stellate and pyramidal-like neurons
that were strongly stained with anti-MAP2 antibody (Fig. 2, B and C). The
weak staining in somata was observed in the surrounding astrocytes
(Fig. 2B). In the neurites, the immunostaining showed in
punctate structures along thin neurites. This may represent localization of MARCKS in synaptic varicosities or nerve terminals on
the dendrites. Thus, MARCKS was primarily expressed in neuronal cells
of these cultures.

View larger version (133K):
[in this window]
[in a new window]
|
Fig. 2.
Immunofluorescence with the anti-MARCKS
antibody in cultured hippocampal cells. Hippocampal cells were
examined by phase contrast (A and D) and
immunofluorescence (B, C, and E) microscopy after
being stained with the anti-MARCKS antibody (B) and
anti-MAP2 antibody (C). The immunofluorescence was strong in
the cell bodies and neurites in stellate and pyramidal-like neurons. In
the neurite, punctate-staining was observed, whereas weak
immunofluorescence was observed in glial cells. The immunofluorescence
in glial cells, as well as neurons, disappeared by preabsorption of
antibodies with purified MARCKS (D and E).
|
|
Increased Phosphorylation of MARCKS by Stimulation of Glutamate
Receptors--
In initial experiments, we examined effects of
glutamate and glutamate receptor agonists on in situ MARCKS
phosphorylation in cultured hippocampal neurons. Treatment with 10 µM glutamate led to a large increase up to 170% in the
MARCKS phosphorylation within 3 min (Fig.
3). This increase in phosphorylation
gradually decreased but was still significantly elevated for at least
30 min with the continued presence of glutamate in the medium.
Similarly, treatment with 50 µM NMDA caused the long
lasting increase in the phosphorylation but to a smaller extent than
that seen with glutamate. In contrast, treatment with 50 µM AMPA or 500 µM ACPD caused a small and
transient increase in MARCKS phosphorylation. In particular, in the
case of ACPD, a significant increase was observed only at the 3-min
incubation. In separate experiments with cultured cortical astrocytes,
the concentration of glutamate required to potentiate MARCKS
phosphorylation was much higher, and 50 ± 10% increase in the
phosphorylation was observed with 500 µM glutamate. Ten
µM glutamate had no effect on phosphorylation in the
cultured astrocytes (data not shown). Therefore, the effect of
glutamate observed in the present study was considered to occur in the
neurons. Exposure of cultured neurons to over 100 µM
glutamate or 100 µM NMDA seemed to be toxic in the
cultured hippocampal neurons, as we reported earlier (46), and the
maximum effect on the MARCKS phosphorylation was obtained with 10 µM glutamate in these cultures. In addition, the 10-min
exposure to glutamate did not cause a significant neuronal toxicity,
such as cell death within the next 24 h (data no shown). We
therefore examined MARCKS phosphorylation using 10 µM
glutamate to clarify its physiological roles in the cultured
hippocampal neurons. However, because the persistent phosphorylation of
MARCKS was observed in the continued presence of glutamate or NMDA in
the medium, it may also be involved in pathological events such as the
glutamate-induced neuronal cell death.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
Increased phosphorylation of MARCKS by
stimulation of glutamate receptors. The time course of
agonist-induced MARCKS phosphorylation. The cultured hippocampal cells
(8-10 days in culture) were prelabeled with
[32P]orthophosphate for 5 h and washed once in KRH
solution. After incubation at 37 °C for 30 min in KRH, cells were
incubated without the agonist (control) in KRH or with the indicated
agonists: 10 µM glutamate, 50 µM NMDA in
Mg2+-free KRH with 1 µM glycine, 50 µM AMPA, or 500 µM ACPD in KRH. After
incubation for the indicated times, the cells were frozen on liquid
N2 and homogenized. The 32P-labeled MARCKS was
immunoprecipitated. The 32P-incorporation into MARCKS was
analyzed using a Bio-Imaging analyzer (BA100, Fuji Film). In control,
cells were incubated under the same conditions without an agonist, and
32P-incorporation into MARCKS was determined at each time
point. Values are expressed as a percentage when the
32P-incorporation into MARCKS from control cells was taken
as 100% at each time point. Values are means ± S.E.
(n = 4-6). The changes in MARCKS phosphorylation were
statistically significant versus control; *,
p < 0.05; **, p < 0.01.
|
|
To evaluate the glutamate receptor types associated with MARCKS
phosphorylation, several glutamate receptor antagonists were tested.
When hippocampal neurons cultured for 8-10 days were used, the basal
MARCKS phosphorylation did not change by treatments with AP-3, CNQX, or
MK801 alone, or a combination of CNQX and MK801. However, the basal
MARCKS phosphorylation was inhibited by 20% by treatment with MK801
but not with CNQX or AP3 in neurons cultured for 14-21 days, due to
the inhibition of MARCKS phosphorylation stimulated by spontaneous
synaptic activity observed in mature neurons in culture. The increase
in MARCKS phosphorylation following a 10-min incubation with glutamate
was weakly but significantly inhibited by CNQX, a non-NMDA receptor
antagonist, and strongly by MK801, a specific inhibitor of the NMDA
receptor, whereas AP3, a metabotropic receptor inhibitor, had little
inhibitory effect (Fig. 4B).
The combination of CNQX and MK801 abolished the glutamate-induced MARCKS phosphorylation as well as the increased autophosphorylation of
CaM kinase II (Fig. 4, A and B). This finding is
consistent with the observation that treatments with NMDA or AMPA
significantly potentiated MARCKS phosphorylation at the 10-min
incubation, as shown in Fig. 3. These observations suggest that the
long lasting increase in MARCKS phosphorylation by exposure to
glutamate was mainly through the NMDA receptor and more weakly through
the kainate or AMPA type receptor. The metabotropic glutamate receptor
did not contribute to the persistent MARCKS phosphorylation in
hippocampal neurons.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 4.
Effects of glutamate receptor antagonists on
glutamate-induced MARCKS phosphorylation. A,
autoradiograph showing in situ phosphorylation of MARCKS and
autophosphorylation of CaM kinase II after stimulation with glutamate
and their inhibition with MK801 and CNQX. Hippocampal cells (8-10 days
in culture) were prelabeled and preincubated as in Fig. 3. The cells
were incubated for 10 min in KRH without (control) or 10 µM glutamate (Glu) in Mg2+-free
KRH in the presence or absence of 20 µM MK801 and 20 µM CNQX. After homogenization, the cell extracts were
incubated with 6 µg of the anti-MARCKS antibody and the anti-CaM
kinase II antibody. After immunoprecipitation, the
32P-incorporation into MARCKS and the and subunits
of CaM kinase II were analyzed by SDS-PAGE followed by autoradiography.
B, effects of glutamate receptor antagonists on the
glutamate-induced MARCKS phosphorylation. The hippocampal cells were
prelabeled and preincubated as in Fig. 3. The cells were incubated for
10 min in KRH without (control) or with 10 µM
glutamate in Mg2+-free KRH in the presence or absence of
the indicated glutamate receptor antagonists (1 mM AP3, 20 µM CNQX, 20 µM MK801, or the combination of
20 µM CNQX and 20 µM MK801). The
antagonists were added to the medium in the last 10-min preincubation
and during the stimulation. The 32P-incorporation into
MARCKS was determined as in Fig. 3. The MARCKS phosphorylation in
control cells was taken as 100%, and from this value, the
phosphorylation in each condition was calculated as a percentage.
Values are means ± S.E. (n = 6). The changes in
MARCKS phosphorylation were statistically significant versus
control (a) and versus treatment with glutamate
(b); p < 0.05.
|
|
Inhibition of MARCKS Phosphorylation by PD098059--
To study the
involvement of PKC in glutamate-induced MARCKS phosphorylation, we
stimulated cells by glutamate in the presence of 200 nM
calphostin C, a relatively specific and potent inhibitor for PKC, or
after down-regulation of PKC by pretreatment with PMA for 16 h.
Unexpectatively, treatment with calphostin C did not abolish the
glutamate-induced phosphorylation after 10-min stimulation (Fig.
5, A and B).
Similarly, the down-regulation of PKC did not inhibit the
glutamate-induced phosphorylation. Calphostin C and down-regulation of
PKC were working to inhibit PKC in these conditions, because the
PMA-induced MARCKS phosphorylation was largely prevented by treatment
with 200 nM calphostin C as well as by the down-regulation
of PKC (Fig. 5B). Interestingly, treatment with 50 µM PD098059, a specific inhibitor for MAP kinase kinase
inhibitor (47, 48) largely inhibited glutamate-induced MARCKS
phosphorylation (Fig. 5, A and B). When the MAP
kinase activity was measured by in-gel kinase assay using
SDS-polyacrylamide gel containing myelin basic protein, as reported
(30), the MAP kinase activity increased to 336 ± 10% by 10-min
stimulation with glutamate. Inclusion of 50 µM PD098059
totally inhibited glutamate-induced MAP kinase activation to near
control levels (108 ± 6% as compared with the control). In
contrast, the increased phosphorylation by 2-min stimulation with
glutamate was partly abolished by PD098059 and calphostin C and totally
abolished by a combination of PD098059 and calphostin C (Fig.
5C). The down-regulation of PKC also significantly inhibited
the MARCKS phosphorylation. Thus, the glutamate-induced MARCKS
phosphorylation for a longer period was mainly due to activation of MAP
kinase in the cultured hippocampal neurons.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of PD098059 and calphostin C on the
glutamate-induced MARCKS phosphorylation. Cultured hippocampal
neurons were prelabeled and preincubated as in Fig. 3. When indicated,
50 µM PD098059 or 200 nM calphostin C was
added during the 30-min preincubation. In case of down-regulation of
PKC, cells were preincubated for 16 h with 100 nM PMA
in cultured medium. The cells were then incubated for 2 or 10 min with
10 µM glutamate plus 1 µM glycine in KRH
(C, control) or Mg2+-free KRH in the presence or
absence of 50 µM PD098059 or 200 nM
calphostin C. In case of PMA treatment, cells were incubated for 10 min
in the presence or absence of calphostin C. The 32P-MARCKS
was immunoprecipitated and analyzed by autoradiography. A,
autoradiographs showing effects of PD098059 and calphostin C on
glutamate-induced MARCKS phosphorylation. B, the cells were
stimulated for 10 min with glutamate or PMA in various conditions as
described above. C, the cells were stimulated for 2 min with
glutamate. The 32P-incorporation into MARCKS was analyzed
by a Bio-Imaging Analyzer and calculated as a percentage of the control
in each condition. The changes in MARCKS phosphorylation were
statistically significant versus control (a) and
versus treatment with glutamate (b) or PMA
(c); p < 0.05.
|
|
Phosphopeptide Mapping Analysis of Phosphorylated MARCKS--
To
further confirm the involvement of MAP kinase in the glutamate-induced
MARCKS phosphorylation by 10-min incubation, phosphopeptide mapping
analysis was carried out after limited proteolysis with lysyl
endopeptidase, according to the method of Taniguchi et al. (24). To clarify the in situ phosphorylation sites of
MARCKS, the purified rat brain MARCKS was in vitro
phosphorylated by purified PKC and MAP kinase in initial experiments
and was separated by SDS-PAGE. After cutting out gel bands
corresponding to MARCKS, MARCKS was digested in gel pieces with 1 or 2 µg of lysyl endopeptidase. After the digestion, the MARCKS peptides
were separated from the gel pieces and analyzed using a conventional
high performance liquid chromatography apparatus, as described under
"Experimental Procedures." When MARCKS peptides was eluted with a
linear gradient of acetonitrile-H2O in the presence of
0.1% trifluoroacetic acid, three major 32P-labeled
radioactive peaks were detected by PKC phosphorylation (Fig.
6A). Each peak was eluted in
fractions of 26, 39, and 41 (Fig. 6A). In contrast, one
major peak in the fraction of 32, a small shoulder in the fraction of
30, and several minor peaks were detected in MARCKS phosphorylated by
MAP kinase (Fig. 6B). As an additional control, the MARCKS
phosphorylated by both MAP kinase and PKC was analyzed (Fig.
6C). Three major peaks originated from PKC- and MAP
kinase-dependent phosphorylation sites were separated, but
the first peak of PKC peptides eluted in fraction 26 was shifted to
fraction 28 after the phosphorylation with both kinases. Although the
reason for the shifting of the first peak of PKC peptides is unclear,
dually phosphorylated peptides may be produced under the
phosphorylation conditions. Next, MARCKS that was in situ
phosphorylated by 2- or 10-min stimulation with glutamate was analyzed
using the same procedures. The elution patterns of in situ
phosphopeptides were apparently similar to that of in vitro
phosphorylation as shown in Fig. 6C. At 2 min, phosphopeptides of MARCKS of the control cells showed major peaks in
fractions of 29, 31, and 36 and several minor peaks (Fig.
6D). Stimulation with glutamate for 2 min mainly produced
phosphopeptides in fractions 29 and 31, which corresponded to PKC- and
MAP kinase-dependent phosphorylation sites, respectively,
as shown in Fig. 6C. In contrast, following 10-min
stimulation with glutamate, a peak of fraction 32 with a shoulder in
fraction 30 largely increased with minor peaks around the fractions
37-41 (Fig. 6E). However, changes in other minor peaks were
not consistent in repeated experiments. The increased phosphorylation
of PKC- and MAP kinase-dependent sites were also evident in
the PMA-stimulated MARCKS phosphorylation, because PMA is known to be a
strong activator for MAP kinase as well as PKC in the hippocampal
neurons (30). These results suggest that the PKC-dependent
phosphorylation in MARCKS is transient and that the persistent
glutamate-induced phosphorylation in MARCKS is made through MAP kinase
rather than PKC. Furthermore, as shown in Fig. 6, D-F, the
phosphorylation by MAP kinase and PKC already occurred in the basal
conditions in cultured hippocampal neurons.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 6.
Analysis of in vitro and in
situ phosphorylation sites of MARCKS by HPLC.
A-C, the purified MARCKS (2 µg) was in
vitro phosphorylated by PKC (A), MAP kinase
(B), and both kinases (C) in the presence of
[ -32P]ATP. After digestion with 1 µg of lysyl
endopeptidase at 35 °C for 10 h, the phosphopeptides were
analyzed by HPLC, as described under "Experimental
Procedures." The phosphopeptide profiles obtained from MARCKS
were similar after digestion with 1 or 2 µg of lysyl endopeptidase.
When MARCKS phosphorylated by both PKC and MAP kinase was analyzed, the
first peak of PKC phosphopeptides was slightly shifted to
fraction 28 (C). D-F, in case of in
situ phosphorylation of MARCKS, the hippocampal neurons
prelabeled with [32P]orthophosphate were incubated for 2 or 10 min with or without glutamate or PMA, as in Fig. 3. MARCKS from
the control and the stimulated cells was immunoprecipitated and
digested with 1 µg of lysyl endopeptidase. The phosphopeptides were
then analyzed using the same procedures. The experiments were repeated
more than three times in the control and the stimulated cells, and one
representative set of data is shown.
|
|
Phosphorylation of MARCKS by PKC--
MARCKS is originally known
to be a substrate for PKC. We further confirmed the transient increase
in phosphorylation of MARCKS by PKC following stimulation with
glutamate. We developed a method to detect sites phosphorylated by PKC.
A specific antibody that recognizes the phosphorylation sites Ser-152
and Ser-156 in MARCKS was produced by immunization of the
phosphopeptide of MARCKS.2 In control experiments, the
purified MARCKS was phosphorylated by PKC in the presence of
nonradioactive ATP and was subjected to immunoblot analysis. The
antibody to the phosphopeptide of MARCKS could detect only the
phosphorylated form by PKC, as shown in the last two lanes of Fig.
7A. In addition, PMA could
stimulate the phosphorylation of PKC-dependent sites (Fig.
7A), and its effect was abolished by addition of 200 nM calphostin C. Consistent with the results in Fig. 6, the
increased phosphorylation of PKC-dependent sites by
stimulation with glutamate was transient, reaching a maximum between 1 and 3 min, followed by a decline to the basal levels within 10 min
(Fig. 7B). The amount of MARCKS protein detected with
nonselective antibody did not change during the incubation with
glutamate (Fig. 7B) These results confirm that activation of
PKC following glutamate stimulation is transient and PKC can primarily
phosphorylate MARCKS during the early period by stimulation with
glutamate. In addition, basal phosphorylation of PKC sites was observed
in all these preparations. The basal phosphorylation by PKC is possibly
due to endogenous release of glutamate and/or other stimulants that
stimulate PKC in cultured hippocampal neurons.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Increased phosphorylation of MARCKS by PKC
following stimulation with glutamate. The nonlabeled hippocampal
neurons were preincubated for 1 h in Ca2+-free KRH and
then incubated in KRH (control) or in Mg2+-free
KRH with 10 µM glutamate for the indicated time, or in
KRH with 100 nM PMA for 10 min. After homogenization, the
cell extracts were treated with the SDS-sample solution (40) and
subjected to SDS-PAGE. In control experiments, purified MARCKS was
incubated with 20 nM PKC in the presence or absence of 0.1 mM ATP. After SDS-PAGE, the proteins were
electrophoretically transferred to Durapore membranes (Millipore) and
immunoblotted with the anti-phosphospecific antibody that recognized
PKC-phosphorylation sites. Immunoreactive bands were visualized by
incubation with 125I-protein A, followed by
autoradiography. A, autoradiographs showing specificity of
the antibody and immunoblots with the cell extracts. The
phosphospecific antibody recognized only phosphorylated MARCKS by PKC
in the presence of ATP, as shown in the right two lanes.
Immunoreactivities to the anti-phospho-specific antibody increased
after a 10-min incubation with PMA. B, inset,
autoradiographs showing a transient increase in phosphorylation by PKC
following treatment with glutamate. The immunoreactivity against the
anti-phospho-specific antibody transiently increased without changes in
the protein levels shown by the conventional anti-MARCKS antibody.
Immunoreactivity was measured using a Bio-Imaging Analyzer and is
expressed as a percentage versus control, in each condition.
Values represent means ± S.E. The changes in MARCKS
phosphorylation were statistically significant versus 0 min;
*, p < 0.05; **, p < 0.01.
|
|
Regulation of Functional Properties of MARCKS by MAP
Kinase--
Finally, we addressed question whether the functional
properties of MARCKS is regulated by phosphorylation by MAP kinase. We
then investigated effects of MARCKS phosphorylation by MAP kinase on
its CaM-binding ability assessed by cross-linking with 125I-CaM and interaction with F-actin determined by a
co-sedimentation assay. The effects were compared with the changes by
PKC-dependent phosphorylation. The purified rat brain
MARCKS was incubated without or with protein kinases in the presence of
0.5 mM ATP (Fig. 8). Under
these conditions, the total phosphate incorporated into MARCKS were 0.9 and 2.6 mol of phosphate/mol of MARCKS by MAP kinase and PKC,
respectively. There was no incorporation of phosphate without each
protein kinase. As reported previously (4), the CaM-binding ability of
MARCKS was totally abolished by PKC-dependent phosphorylation, as shown in Fig. 8. The phosphorylation by MAP kinase
slightly but significantly reduced its CaM-binding ability to 75% of
control. In contrast, the interaction between MARCKS and F-actin was
largely affected by the MAP kinase-dependent
phosphorylation to the same extent as seen for the
PKC-dependent phosphorylation (Fig.
9). These results suggest that MAP kinase
can functionally regulate the properties of MARCKS, especially in its
interaction with F-actin.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 8.
Effects on the calmodulin binding of MARCKS
phosphorylation by PKC and MAP kinase. The purified MARCKS (1.5 µg) was incubated in the absence (None) or presence of PKC
or MAP kinase (MAPK) at 20 nM each with 0.5 mM ATP for 30 min. The phosphorylated MARCKS was treated at
60 °C for 15 min and cross-linked to 125I-CaM (0.2 µCi) in the presence of 2.5 mM CaCl2.
A, the cross-linked MARCKS was subjected to SDS-PAGE in 9%
acrylamide, followed by autoradiography. The positions of MARCKS
cross-linked to 125I-CaM and free 125I-CaM are
indicated by arrowheads. B, the results from six
independent experiments were examined statistically. Values represent
means ± S.E. The changes were statistically significant
versus without protein kinase (None); **,
p < 0.01.
|
|

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 9.
Effect on the binding to F-actin of MARCKS
phosphorylation by PKC and MAP kinase. F-actin (10 µg) was
incubated with MARCKS that had been preincubated without
(None) or with PKC or MAP kinase (MAPK) as in
Fig. 8. After centrifugation, the pellet with F-actin and co-sedimented
MARCKS was separated by SDS-PAGE and stained with Coomassie Blue
(A). In order to detect the co-sedimented MARCKS, the gel
was subjected to immunoblotting analysis with the anti-MARCKS antibody,
followed by autoradiography (B). The position of MARCKS is
indicated by an arrow head. The results from six independent
experiments were examined statistically (C). Values
represent means ± S.E. The changes were statistically significant
versus without protein kinase (None); **,
p < 0.01.
|
|
 |
DISCUSSION |
In neutrophils and macrophages, MARCKS is phosphorylated during
chemotaxis, secretion, and phagocytosis (18, 19); during neurosecretion
(20, 21, 49, 50); and during mitogenesis (22, 23). Because this
phosphorylation seems to be closely associated with activation of PKC
and its abilities to bind calcium/calmodulin and cross-link actin
filaments are directly regulated by the PKC-dependent phosphorylation (4, 6), the physiological functions of MARCKS would
appear to be mainly regulated by PKC in vivo. However, mass spectrometrical analysis using purified bovine MARCKS demonstrated six
novel phosphorylation sites in addition to the known PKC
phosphorylation sites (24). The endogenous phosphorylation by PKC was a
minor portion and all the novel sites were serine residues, followed immediately by proline residues, which means that MARCKS is also a good
substrate for proline-directed protein kinase, including MAP kinase and
cdk5, in vivo.
We reported activation of PKC and MAP kinase, as well as CaM kinase II,
in cultured rat hippocampal neurons (30, 38). We focused on activation
of the protein kinase cascades following the activation of NMDA
receptors in the hippocampal neurons, because NMDA receptor activity
was exclusively associated with synaptic plasticity in the developing
brain as well as in the adult brain. For example, activation of CaM
kinase II in cultured hippocampal neurons was predominantly regulated
by the activity of the NMDA receptor, because glutamate-induced
activation of CaM kinase II was only inhibited by addition of the NMDA
receptor antagonist but not by antagonists of AMPA/kainate and the
metabotropic receptors (38). The NMDA receptor-dependent
activation of CaM kinase II was also evident in the long term
potentiation (LTP) in the hippocampal CA1 regions (51). The
potentiation of CaM kinase II was closely associated with the induction
of LTP, in an NMDA receptor-dependent manner (51, 52).
Similarly, activation of MAP kinase by stimulation with glutamate was
primarily due to activation of the NMDA receptor (30). The activation
of MAP kinase may be related to the induction of LTP in the CA1 region,
because PD098059 attenuated the induction of LTP (53). Thus, MAP kinase
became an attractive candidate related to the underlying the molecular
basis for expressing a stable LTP in CA1 regions.
In the present study, we demonstrated that MARCKS is one in
vivo substrate for MAP kinase following stimulation of glutamate receptors. MARCKS phosphorylation was sustained during more than 30 min
after glutamate stimulation, a long lasting increase predominantly due
to activation of the NMDA receptor. In addition, the long lasting
glutamate-induced MARCKS phosphorylation was largely prevented by
PD098059, a MAP kinase kinase inhibitor but not by a PKC inhibitor. When examining our previous results in terms of MAP kinase activation by stimulation of the glutamate receptors, the time course of glutamate-induced MARCKS phosphorylation was closely related to that of
MAP kinase activation, in which the NMDA receptor activation was mainly
involved (30). Treatment with PD098059 totally inhibited the
glutamate-induced MAP kinase activation, as shown in the present study.
Consistent with these observations, the major site for MAP kinase
detected by HPLC analysis was mainly potentiated after a 10-min
exposure to glutamate. The phosphorylation of the major site for MAP
kinase was elevated even after a 30-min incubation (data not shown).
The finding of one major peak in MARCKS phosphorylated in
vitro by MAP kinase is consistent with findings that Ser-113 in
mouse MARCKS and Ser-116 in bovine MARCKS were mainly phosphorylated by
the purified MAP kinase in vitro (24, 25). Furthermore, Schönwaßer et al. (25) demonstrated that the mutation
of Ser-113 to alanine in MARCKS largely abolished the phosphorylation
by MAP kinase. However, Ser-113 was not phosphorylated in permeabilized Swiss 3T3 cells after stimulation with platelet-derived growth factor
as well as PMA (25). Because platelet-derived growth factor activates
MAP kinase in a variety of cells, the phosphorylation of MARCKS by MAP
kinase may not generally occur. In hippocampal neurons, MARCKS may be
an important substrate for MAP kinase, because stimulation with
brain-derived neurotrophic factor in hippocampal neurons could also
elicit an increase in MARCKS phosphorylation by MAP
kinase.3
The present study demonstrated that stimulation of hippocampal neurons
with glutamate elicits biphasic increases in phosphorylation of MARCKS:
that is, phosphorylation with PKC through the metabotropic glutamate
receptor in a short period and with MAP kinase through activation of
the NMDA receptor over a long period. This was confirmed by using a
specific antibody that recognizes PKC-phosphorylation sites. These
results are consistent with previous observations that
glutamate-induced translocation of PKC from the cytosol to the membrane
fraction was transient and were reverted to basal levels in 5 min (38).
The translocation of PKC was only inhibited by addition of an inhibitor
for the metabotropic glutamate receptor (38). Transient increase in
phosphorylation of MARCKS was noted in hippocampal neurons (54),
cerebellar granule cells (55), and hippocampal slices from adult rats
(56). It is important to determine which kinase is involved in the
phosphorylation described in previous works (54-56).
The possibility remains that other proline-directed protein kinases,
such as cdc2 kinase and cdk5, are involved in glutamate-induced MARCKS
phosphorylation. The phosphopeptides eluted in fractions 38-42 were
not identified. It has not been elucidated how cdc2 kinase and cdk5 are
activated by external stimuli for receptors, although these kinases are
expressed in neurons (57-60) and have the potential to
stoichiometrically phosphorylate MARCKS in vitro (26,
61).
The present study is apparently the first finding to demonstrate the
in situ phosphorylation of MARCKS through MAP kinase following extracellular stimuli. MAP kinase-induced phosphorylation of
synapsin I was noted in cultured cortical neurons following stimulation
with brain-derived neurotrophic factor (41). Synapsin I is
predominantly located in presynaptic regions and regulates the binding
between synaptic vesicles and actin filaments. In vitro
phosphorylation of synapsin I by MAP kinase reduced the ability of
cross-linking of actin filaments but had no evident effect on the
binding ability to synaptic vesicles. The regulation of bundling of
actin filaments by synapsin I phosphorylation through MAP kinase
suggests that MAP kinase is implicated in neurite extension rather than
exocytosis in the phosphorylation of synapsin I. On the other hand,
MARCKS, which is also enriched in nerve terminals, regulates
interactions between actin filaments and plasma membranes. In this
context, we investigated the physiological significance of MARCKS
phosphorylation by MAP kinase. The phosphorylation by MAP kinase
significantly regulated the interaction between MARCKS and F-actin
rather than its CaM-binding ability. The results suggest a potential
role of MAP kinase in the regulation of F-actin-membrane interaction.
The site for MAP kinase, Ser-113, is located in the near upstream site
for the central PKC phosphorylation domain that includes Ser-152,
Ser-156, and Ser-163 and is conserved in all members of the MARCKS
family and MacMARCKS. The inhibitory effect of phosphorylation of
Ser-113 by MAP kinase on binding to calmodulin may be due to
conformational changes in the MARCKS structure. In cerebellar granule
cells, involvement of MARCKS phosphorylation in NMDA-stimulated neurite
outgrowth was suggested as MARCKS was present in neurites and growth
cones (55). The present study demonstrated that MARCKS is also present
in neurites and the varicosity-like structure, as based on light
microscopic observations of cultured hippocampal neurons (Fig. 2).
Further investigation of localization of MARCKS phosphorylated by MAP kinase is needed to clarify the roles of MARCKS in the central nervous
system. MARCKS may serve as a good substrate for MAP kinase during the
LTP expression, because MARCKS can provide a reversible cross-bridge
between the actin cytoskeleton and the plasma membrane, which may
contribute to reorganization or morphological changes in synapses in
the hippocampus during LTP expression.