Protein Kinase C Activation Modulates
-Calmodulin
Kinase II Binding to NR2A Subunit of N-Methyl-D-Aspartate Receptor
Complex*
Fabrizio
Gardoni
,
Camilla
Bellone,
Flaminio
Cattabeni, and
Monica
Di Luca
From the Institute of Pharmacological Sciences, University of
Milano, via Balzaretti 9, 20133 Milano, Italy
Received for publication, October 31, 2000, and in revised form, November 30, 2000
 |
ABSTRACT |
The N-methyl-D-aspartate
(NMDA) receptor subunits NR2 possess extended intracellular
C-terminal domains by which they can directly interact with a large
number of postsynaptic density (PSD) proteins involved in synaptic
clustering and signaling. We have previously shown that
PSD-associated
-calmodulin kinase II (
CaMKII) binds with
high affinity to the C-terminal domain of the NR2A subunit.
Here, we show that residues 1412-1419 of the cytosolic tail of NR2A
are critical for
CaMKII binding, and we identify, by site directed
mutagenesis, PKC-dependent phosphorylation of
NR2A(Ser1416) as a key mechanism in inhibiting
CaMKII-binding and promoting dissociation of
CaMKII·NR2A
complex. In addition, we show that stimulation of PKC activity in
hippocampal slices either with phorbol esters or with the mGluRs
specific agonist
trans-1-amino-1,3- cyclopentanedicarboxylic acid
(t-ACPD) decreases
CaMKII binding to NMDA receptor complex. Thus,
our data provide clues on understanding the molecular basis of a direct
cross-talk between
CaMKII and PKC pathways in the postsynaptic compartment.
 |
INTRODUCTION |
The excitatory glutamatergic synapse is thought to be controlled
by a complex net of signaling proteins that regulate the postsynaptic
ion channel activity. Electron microscopy and biochemical studies have
identified, in the postsynaptic compartment of the glutamatergic
synapse, the postsynaptic density
(PSD)1 fraction as a complex
structure containing signaling proteins, cytoskeletal components, and
ion channels (1, 2). Among ion channels, NMDA and
1-
-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)
glutamate receptors are particularly enriched in PSD (3-5). In
particular, NMDA receptors are critical players in synaptogenesis,
synaptic plasticity, as well as in the molecular pathogenesis of
neurological disorders (6, 7). NMDA receptors are oligomeric complexes
formed by the coassembly of members of three receptor subunit families:
NR1, NR2 (NR2A-D; Ref. 6), and NR3A (8). The NR2 NMDA receptor
subunits possess extended intracellular C-terminal domains by which
they can interact with a large number of PSD proteins (9-13). Due to
its anatomical localization and expression onset (14), NR2A is likely
to play a major role in synaptic plasticity modulating long term
potentiation and long term depression.
Although different groups identified NMDA receptor subunits NR2A/B as a
target for
CaMKII in PSD (13, 15-18), the exact nature of these
complex interactions requires further elucidation. In particular, the
molecular mechanism(s) responsible for CaMKII dissociation from NMDA
receptor complex is far from being clear. In the last 10 years,
the hypothesis of a specific interaction between CaMKII and PKC
pathways in the postsynaptic compartment has been put forward (19). The
intracellular domains of NR2 subunits contain serine residues that can
be phosphorylated by either CaMKII or protein kinase C (20-22). In
addition and very recently, PKC has been shown to potentiate NMDA
receptor activity even if the exact role of PKC phosphorylation on NMDA
receptor complex function is still not completely understood (23, 24). Here we show that PKC-mediated phosphorylation on Ser1416
residue of NR2A C-terminal tail decreases its binding affinity for
CaMKII. Moreover we provide evidence that PKC stimulation in
hippocampal slices either through phorbol esters or mGluR activation decreases the interaction between
CaMKII and NMDA receptor complex.
 |
EXPERIMENTAL PROCEDURES |
PSD Preparation--
To isolate PSD from rat hippocampus, a
modification of the method by Carlin et al. (25) was used,
as described previously (16, 17).
Cloning, Expression, and Purification of GST Fusion
Proteins--
NR2A fragments were subcloned downstream of GST in the
BamHI and HindIII sites of the expression plasmid
pGEX-KG by polymerase chain reaction using the Pfu
polymerase (Stratagene, La Jolla, CA) on a cDNA containing plasmid
for NR2A (kind gift from S. Nakanishi). GST-NR2A fusion constructs were
expressed in Escherichia coli and purified on
glutathione-agarose beads as described previously (17). Mutated
GST-NR2A fusion proteins were produced by using the
QuikChangeTM Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA) or the ExSiteTM PCR-Based
Site-Directed Mutagenesis Kit (Stratagene). The inserts were
fully sequenced with ABI Prism 310 Genetic analyzer (ABI Prisma).
"Pull-out" Assay--
Aliquots of 5 µg of hippocampal PSD
were diluted with phosphate-buffered saline, 1% SDS and incubated (1 h, 37 °C) with glutathione-agarose beads saturated with GST fusion
proteins or GST alone. The beads were then extensively washed with
phosphate-buffered saline, 0.2% Triton X-100. Bound proteins were
resolved by SDS-PAGE and subjected to immunoblot analysis with a
monoclonal anti-
CaMKII antibody.
Overlay Technique--
An overlay procedure has been used to
detect the binding of eluted GST-NR2A fusion proteins to either
PSD-associated or purified
CaMKII. PSD proteins or purified
CaMKII were separated on a 6% SDS-PAGE gel and transferred
overnight to nitrocellulose. The membrane was blocked for 1 h in
50 mM Tris-HCl, 200 mM NaCl (TBS), pH 8.0, containing 1% Tween 20 (v/v) and nonfat powdered milk (5% w/v). The
membrane was washed in TBS containing 0.1% v/v Tween 20 and nonfat
powdered milk (5% w/v) and then incubated for 2 h at room
temperature with constant shaking in overlay buffer TBS, 5% (w/v)
nonfat powdered milk, 0.1% Tween 20 containing the fusion protein.
After extensive washes, the membrane was subjected to immunoblot
analysis using a polyclonal anti-GST antibody.
GST-NR2A Fusion Protein Phosphorylation--
For
PKC-dependent phosphorylation of NR2A, GST-NR2A-purified
fusion proteins were incubated with 50 units of purified PKC (Sigma-Aldrich, Milan, Italy) for 30 min at 37 °C, in presence of 10 mM Tris-HCl, pH 7.4, 10 mM
MgCl2, 0.5 mM CaCl2, and 10 µM ATP ([
-32P]ATP, 2 µCi/tube; 3000 Ci/mmol; Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).
Preparation of Hippocampal Slices--
Hippocampal slices were
obtained as described previously (26). Briefly, brains were removed and
placed into chilled (4 oC) oxygenated Krebs buffer.
After removal of meninges, hippocampal slices were prepared quickly
with a McIlwain tissue chopper and placed in custom-made chambers
equilibrated continuously with 95% O2/5% CO2
(v/v). Slices were then equilibrated at 37 °C (95% O2/5% CO2) for 30 min. After the equilibration
period, slices were incubated for 15 min in the absence or presence of
either t-ACPD (10 µm) or phorbol 12,13-dibutyrate (PDBu, 0.1 µM) or H-7 (10 µM). After incubation,
slices were homogenized in cold 0.32 M sucrose containing 1 mM Hepes, 1 mM MgCl2, 1 mM NaHCO3, 0.1 mM
phenylmethylsulfonyl fluoride, pH 7.4, in presence of a complete set of protease inhibitors (CompleteTM, Roche Molecular
Biochemicals, Mannheim, Germany) and phosphatase inhibitors. The
homogenized tissue was centrifuged at 1,000 × g for 10 min. The resulting supernatant was centrifuged at 3,000 × g for 15 min to obtain a fraction of mitochondria and
synaptosomes. The pellet was resuspended in hypotonic buffer (in
presence of proteases inhibitors) in a glass-glass potter and
centrifuged at 100,000 × g for 1 h. The resultant
pellet was resuspended in 1 ml of buffer containing 75 mM
KCl and 1% Triton X-100 and centrifuged at 100,000 × g for 1 h. The final pellet was homogenized by 10 strokes in a glass-glass potter in 20 mM Hepes. An equal
volume of glycerol was added and stored at
80 °C. The protein
composition of this preparation was carefully tested for the absence of
presynaptic markers and for the enrichment in the PSD proteins
(
CaMKII, PSD-95, NMDA receptor subunits; Ref. 27). This fraction is
referred to as "Triton-insoluble fraction."
Immunoprecipitation--
Triton-insoluble fraction proteins (50 µg) were incubated overnight at 4 °C in buffer A containing: 200 mM NaCl, 10 mM EDTA, 10 mM
Na2HPO4, 0.5% Nonidet P-40, 0.1% SDS in a
final volume of 200 µl with antibodies against NR2A/B as indicated in
the text (dilution 1:50). Protein A-agarose beads (5 mg/tube), or
Staphylococcus aureus Cowan I cells (0.5%) washed in the
same buffer, were added and incubation was continued for 2 h. The
beads were collected by centrifugation and washed three times with
buffer A. Sample buffer for SDS-PAGE was added, and the mixture was
boiled for 3 min. Beads were pelletted by centrifugation, and
supernatants were applied to 6% SDS-PAGE.
Antibodies--
Monoclonal
CaMKII antibody was purchased from
Roche Molecular Biochemicals; polyclonal antibody against
anti-active p286-
CaMKII was purchased from Promega (Madison,
WI); polyclonal antibody against NR2A/B was purchased from Chemicon
International, Inc., (Temecula, CA); a polyclonal antibody against GST
was produced in rabbits using recombinant GST (17).
 |
RESULTS |
We first investigated whether PKC was capable to phosphorylate in
an in vitro assay the NR2A C-terminal tail. To this
end, GST fusion proteins with different NR2A C-terminal domains were incubated with purified PKC in presence of [
-32P]ATP
as a phosphate donor. Fig. 1 shows a
representative autoradiograph of in vitro
PKC-dependent phosphorylation of NR2A-(1244-1464) and NR2A-(1349-1464) GST fusion proteins; a radioactive band
corresponding to both NR2A-(1349-1464) and NR2A-(1244-1464) GST
fusion protein phosphorylation is clearly visible (Fig. 1), indicating
the presence of several PKC phosphosites distributed in the NR2A
C-terminal domain; the phosphorylation signal shown by the NR2A
fragment is specific, since GST alone did not show any phospho-band
(leftmost lane).

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Fig. 1.
In vitro phosphorylation of
GST-NR2A fusion proteins. GST, GST-NR2A-(1244-1464), and
GST-NR2A-(1349-1464) purified fusion proteins were incubated with
purified protein kinase C in the presence of [ -32P]ATP
as phosphate donor (2 µCi/tube; 3000 Ci/mmol). Proteins were
separated by SDS-PAGE (running gel: 11% acrylamide) and
phosphoproteins revealed by autoradiograph.
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|
Previous studies of our laboratory (17) identified NR2A domain
1349-1464 as the binding region for both recombinant and PSD-associated
CaMKII; using the pull-out procedure we asked whether the PKC-dependent phosphorylation of this region
could influence the NR2A-
CaMKII binding affinity.
GST-NR2A-(1349-1464) fusion proteins, in vitro
phosphorylated by purified PKC in presence or absence of ATP as
phosphate donor, were incubated in a pull-out assay with solubilized
hippocampal PSD (Fig. 2). PKC
phosphorylation of GST-NR2A-(1349-1464) is able to decrease (
75.6% ± 9.3%, mean ± S.D.; n = 6, p < 0.01) the binding of PSD-associated
CaMKII to NR2
C-terminal tail (Fig. 2). Inspection of this NR2A region revealed the presence of at least two domains
(YLR-S1403-S1404-LRS and
SDR-S1416-RGH) containing serine residues
(Ser1403-Ser1404, Ser1416)
that might represent a putative phosphate acceptor site for PKC and
that might be of relevance in
CaMKII binding to NR2A.

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Fig. 2.
Effect of PKC-dependent phosphorylation on
NR2A- CaMKII association. GST-NR2A-(1349-1464) fusion
protein was phosphorylated in a PKC-dependent manner in
absence of presence of ATP and then incubated in a pull-out assay with
purified hippocampal PSD. Bound proteins were eluted from the
beads with SDS sample buffer, separated by SDS-PAGE, and analyzed on
Western blotting with anti- CaMKII monoclonal antibody.
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|
A point mutation strategy was used to determine the effects of PKC
site-specific phosphorylation of NR2A:
GST-NR2A-(1349-1464)S1416A, GST-NR2A-(1349-1464)S1403A/S1416A, and
GST-NR2A-(1349-1464)S1403A/S1404A/S1416A fusion proteins were
generated and in vitro phosphorylated by PKC in presence of
[
-32P]ATP as phosphate donor. As shown in Fig.
3, the single-mutated S1416A product
displays per se a decrease of 63.7% ± 5.3% (mean ± S.D.; n = 6, p < 0.01) of the
32P incorporation as compared with the nonmutated protein,
thus indicating that this site represents a major phosphate consensus site for PKC in the NR2A C-terminal tail. The concomitant
S1403A/S1404A/S1416A mutations lead to the complete inability of NR2A
fusion protein to be in vitro PKC-phosphorylated.

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Fig. 3.
In Vitro PKC-dependent phosphorylation of
GST- NRA2-(1349-1464) fusion proteins. GST,
GST-NR2A-(1349-1464), GST-NR2A-(1349-1464)S1416A,
GST-NR2A-(1349-1464)S1403A/S1416A, and
GST-NR2A-(1349-1464)S1403A/S1404A/S1416A were phosphorylated by
purified PKC in presence of [ -32P]ATP
as phosphate donor (2 µCi/tube; 3000 Ci/mmol). Proteins were
separated by SDS-PAGE (running gel: 11% acrylamide) and
phosphoproteins revealed by autoradiograph.
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To analyze whether PKC-mediated NR2A(Ser1416)
phosphorylation in the C-terminal region was sufficient to affect
CaMKII binding, control and mutated GST-NR2A-(1349-1464)S1416A
fusion proteins were cold-phosphorylated by PKC and then subjected to
pull-out assay in the presence of hippocampal PSD previously
radiolabeled in conditions known to activate
CaMKII
autophosphorylation. It is well known that this condition fosters
maximal
CaMKII/NR2A association (17). Fig.
4 shows a representative autoradiograph of this pull-out assay. A phosphorylated band at 50 kDa corresponding to bound 32P-
CaMKII is present. S1416A mutation
(first lane) is able to prevent the modulation of
CaMKII
binding to NR2A-(1349-1464) induced by PKC phosphorylation when
compared with the nonmutated NR2A fusion protein (middle
lanes;
59.6% ± 8.3%, mean ± S.D.; n = 6, p < 0.05).

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Fig. 4.
Effect of NR2A (S1416A) point mutation on
NR2A- CaMKII association. Control and
GST-NR2A-(1349-1464)S1416A fusion proteins were phosphorylated in a
PKC-dependent manner in the presence or absence of ATP and
then incubated in a pull-out assay with purified radiolabeled
hippocampal PSD; following extensive washes, the bound proteins were
eluted from the beads with SDS sample buffer, separated by SDS-PAGE,
and autoradiographed.
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The binding of PKC-phosphorylated NR2A C-terminal tail to either
PSD-associated
CaMKII or purified
CaMKII was confirmed by means
of the overlay procedure using control and mutated
GST-NR2A-(1349-1464) fusion proteins. PSD proteins or purified
CaMKII were separated in SDS-PAGE and electroblotted; blocked
nitrocellulose was incubated with eluted control and mutated
GST-NR2A-(1349-1464) fusion proteins previously phosphorylated in a
PKC-dependent manner. As shown in Fig.
5A (first
and second lanes), PKC phosphorylation of eluted control
fusion products is sufficient to decrease significatively the binding
of NR2A-(1349-1464) to PSD-associated
CaMKII (upper panel;
77.2% ± 7.3%, mean ± S.D.; n = 8, p < 0.01) and to purified
CaMKII (lower
panel;
70.3% ± 9.3%, mean ± S.D.; n = 8, p < 0.01), confirming the results obtained using
the pull-out assay (Fig. 2). S1416A mutation restore
CaMKII-NR2A
binding to control levels (third lane); additional S1403A
and S1404A mutations do not further increase the
CaMKII/GST-NR2A
association (fourth and fifth lanes). In
addition, S1416D mutant qualitatively and quantitatively mimics the
binding of the PKC phosphorylated S1416-NR2A to
CaMKII both in
pull-out (Fig. 5C) and overlay assays using either PSD (Fig. 5B, left panel) or purified
CaMKII (Fig.
5B, right panel).

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Fig. 5.
Overlay (A and B) and
pull-out assay (C). A, GST-NR2A-(1349-1464),
GST-NR2A-(1349-1464)S1416A, GST-NR2A-(1349-1464)S1403A/S1416A and
GST-NR2A-(1349-1464)S1403A/S1404A/S1416A bind in overlay experiments
both PSD-associated (upper panel; 50-kDa band) and purified
CaMKII (lower panel). Either PSD proteins or purified
CaMKII were subjected to SDS-PAGE and electroblotted; blocked
nitrocellulose was incubated with eluted control and mutated
GST-NR2A-(1349-1464) fusion proteins previously phosphorylated in a
PKC-dependent manner. The nitrocellulose sheet is revealed
by Western blotting (WB) using anti-GST polyclonal antibody.
B, control and S1416D mutant GST-NR2A-(1349-1464) fusion
proteins binding in overlay experiments to both PSD-associated
(left panel, 50-kDa band) and purified CaMKII
(right panel). C, control and S1416D mutant
GST-NR2A-(1349-1464) fusion proteins binding in pull-out assay to
CaMKII.
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To determine whether the PKC phosphorylation consensus site surrounding
NR2A-Ser1416 was necessary for
CaMKII binding,
site-directed mutagenesis was applied to introduce deletions in the
NR2A C-terminal tail to produce GST-NR2A-(1349-1411) and
GST-NR2A-(1349-1464)
1412-1419 fusion proteins. Deletion of the
NR2A-(1411-1464) C-terminal domain completely prevents the binding to
CaMKII both in pull-out (Fig. 6B) and overlay assays using
either PSD (Fig. 6A, left panel) or purified
CaMKII (Fig. 6A, right panel).
1412-1419
deletion is not sufficient to preclude but significatively decreases
the association of NR2A to
CaMKII both in pull-out (Fig.
6B) and overlay assays (Fig. 6A) using either PSD
(left panel;
67.2% ± 6.3%, mean ± S.D.;
n = 8, p < 0.01) and purified
CaMKII (right panel;
62.1% ± 5.9%, mean ± S.D.; n = 8, p < 0.01), thus
suggesting an involvement of both NR2A-(1412-1419) and the downstream
NR2A-(1420-1464) domain in the association with
CaMKII.

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Fig. 6.
Overlay (A) and pull-out
assay (B). Deletion of NR2A-(1411-1464)
C-terminal domain completely prevents the binding to CaMKII;
1412-1419 deletion is not sufficient to preclude, but
significatively decreases (p < 0.01), the
NR2A- CaMKII association in all experimental conditions.
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To further confirm the specific role of NR2A domain amino acids
1412-1419 in
CaMKII binding, phospho-SRDS(p)RGH and nonphosphorylated SRDSRGH peptides corresponding to
NR2A-(1412-1419) were used in in vitro competition
experiments with GST-NR2A-(1349-1464) and hippocampal PSD. The
competition experiment with phosphopeptide SRDS(p)RGH (Fig.
7B) does not result in a
change of NR2A-(1349-1464)/
CaMKII binding even at high
concentrations. On the other hand, the nonphosphorylated peptide
SRDSRGH (Fig. 7A) competes with NR2A-(1349-1464) for
CaMKII binding in a concentration-dependent manner
(0-100 nM), indicating a role of the NR2A-(1412-1419)
domain in the modulation of
CaMKII binding (SRDSRGH, 50 nM:
78.4% ± 8.9%, mean ± S.D.; n = 8 p < 0.01). In addition, the competition experiment strengthens the relevance of Ser1416 phosphorylation in
CaMKII binding modulation.

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Fig. 7.
NR2A-(1412-1419) Competes with
GST-NR2A-(1349-1464) in the binding with PSD-associated
CaMKII. In vitro competition experiments between
GST-NR2A-(1349-1464) and hippocampal PSD in the presence of either
nonphosphorylated SRDSRGH (A: 0, 5, 10, 50, and 100 nm) or
phospho-SRDS(p)RGH peptides (B: 0, 5, 10, 50, and 100 nm) corresponding
to NR2A-(1412-1419); following extensive washes, the bound proteins
were eluted, separated by SDS-PAGE and analyzed on Western blotting
with anti- CaMKII monoclonal
antibodies.
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To investigate in a more physiological context the modulation of
CaMKII·NMDA receptor complex association mediated by PKC activation, we used hippocampal slices prepared as described previously (26); in a first set of experiments, slices were incubated with PKC
activator PDBu (10
7 M), with the
PKC inhibitor H-7 (10
5 M), and
with vehicle alone. After incubation, a Triton X-100-insoluble fraction
(TIF) was obtained, and Western blotting analysis and NMDA receptor
coprecipitation studies were performed (27). Western blotting analysis
performed in the TIF fraction with anti-
CaMKII and anti-active
p286-
CaMKII (Fig. 8A)
monoclonal antibodies shows that the incubation with either PDBu or H-7
does not influence both the concentration and the autophosphorylation
degree of
CaMKII in the TIF compartment. On the other hand, when TIF
proteins were immunoprecipitated with a polyclonal antibody raised
against NR2A/B subunits of NMDA receptor complex, Western blot analysis
performed in the cointraperitoneal material (Fig. 8B) shows
a significative increase of
CaMKII·NMDA receptor
association in presence of the PKC inhibitor (+82.7% ± 9.2%,
mean ± S.D.; n = 6, p < 0.01)
and a decrease of
CaMKII cointraperitoneal in presence of the PKC activator (
84.7% ± 11.2%, mean ± S.D.; n = 6, p < 0.01). In a second set of experiments
hippocampal slices were incubated in presence of t-ACPD to induce PKC
activation mediated by mGluR; also under these experimental conditions,
immunoprecipitation of NMDA receptor complex leads to a significant
reduction in
CaMKII cointraperitoneal, even if at a lower extent
when compared with the results obtained in presence of PDBu (
54.7% ± 9.2%, mean ± S.D.; n = 6, p < 0.05).

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Fig. 8.
Modulation of CaMKII
binding to NMDA receptor complex in acute hippocampal slices is
modulated by PKC activation. A, Western blotting
analysis performed in the TIF fraction with anti- CaMKII and
anti-active p286- CaMKII; B, proteins from the TIF were
immunoprecipitated with a polyclonal antibody raised against NR2A/B
subunits of NMDA receptor complex; Western blot analysis was performed
in the intraperitoneal material with anti-NR2A/B, anti- CaMKII.
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 |
DISCUSSION |
In the last few years, it has been shown that CaMKII shuttles
between the cytosol and the postsynaptic compartment and that CaMKII
localization is under the dynamic regulation of neuronal activity (28).
Although in the resting state CaMKII is highly localized in the cytosol
and partially bound to actin, glutamate stimulation translocates the
kinase to the PSD region. Once bound to the PSD, CaMKII still remains
active, suggesting that PSD-trapped CaMKII can serve as a molecular tag
involved in synapse potentiation for subsequent long term modifications
(1). Different groups have supported the idea that
CaMKII
translocation to the PSD fraction is probably due to a high affinity
binding interaction with NMDA receptor subunits (13, 16-18).
We show here that the activation of PKC pathway in the postsynaptic
compartment promotes
CaMKII dissociation from NMDA receptor complex.
The mechanism shown in this study is not mediated by alteration of the
autophosphorylation state of the kinase (Fig. 8) and does not affect
the total amount of
CaMKII present in the Triton-insoluble fraction
(that mimics the PSD; Fig. 8), suggesting a mechanism intrinsic to this
cellular compartment and therefore involving
CaMKII translocation to
the cytosolic fraction.
Moreover, we show here that stimulation of mGluR through t-ACPD,
inducing NMDA receptor potentiation (29), decreases the affinity of
CaMKII for the NMDA receptor complex. Since stimulation of mGluR1
and mGluR5 leads to activation of PKC, and since PKC-mediated phosphorylation of NR2A decreases the affinity of NR2A for
CaMKII, it is likely that phosphorylation of NR2A by activated PKC through mGluR results in inhibition of
CaMKII-NR2A interaction.
In particular, we identify NR2A-Ser1416 as
PKC-dependent phosphosite partially responsible of
CaMKII-NR2A binding modulation. Either deletion of NR2A-(1412-1419)
or PKC-mediated phosphorylation of NR2A-Ser1416 are
sufficient to decrease, but not to prevent, the
CaMKII-NR2A association. On the other hand, deletion of the C-terminal tail NR2A-(1411-1464) renders the receptor subunit completely unable to
bind the kinase, suggesting the presence of at least two domains involved in
CaMKII-NR2A binding.
In the last decade different experimental approaches have suggested a
functional cross-talk between Ca2+/CaM and PKC pathways
during long term potentiation induction (19, 30), even if the precise
interaction between CaMKII and PKC during synaptic potentiation were
not elucidated. PKC activity has been indicated to be required for
Ca2+/CaM-induced potentiation, as it has been shown for
tetanus-induced long term potentiation (19), suggesting a positive
communication between the CaMKII and PKC pathways. On the other hand,
other data suggest that CaMKII and PKC pathways can run in parallel: these findings are supported by the observation that PKC or CaMKII activation can be independent and sufficient for synaptic potentiation (31). Very recently, the group of MacDonald and coworkers (24) demonstrated a PKC-mediated modulation of NMDA receptor assembly, suggesting a PKC phosphorylation-dependent regulation of
the interactions occurring between NMDA receptor subunits, CaM and PSD
proteins. Therefore, the role of PKC in modulating the NMDA receptor
can be viewed as independent from a simple mechanism of channel
potentiation due to subunits phosphorylation (23).
The role of PKC-dependent phosphorylation of NR2A in
affecting the clustering between NMDA channel and other PSD proteins and the functional consequences of CaMKII dissociation on NMDA channel
activity needs further elucidation. However, our data showing a
dissociation of the complex CaMKII·NMDA receptor after PKC
activation without affecting the total amount of CaMKII localized in
the Triton-insoluble fraction shed light on a new fine biochemical mechanism intrinsic to this specialized postsynaptic compartment. Recent studies indicating
CaMKII binding to other PSD proteins, i.e. NR2B and Densin-180 (32), suggest a dynamic modulation of
CaMKII localization inside the PSD fraction of relevance in modulating NMDA-dependent potentiation of the postsynaptic
compartment. The dissociation of
CaMKII·NMDA complex modulated by
t-ACPD increases our knowledge on the mechanisms regulating
metabotropic and ionotropic glutamate receptors in the postsynaptic compartment.
In conclusion, our results demonstrate a fine tuning of NMDA receptor
complex assembly mediated by PKC and propose new molecular aspects of
the cross-talk occurring between CaMKII and PKC in a specialized
postsynaptic compartment involved in synaptic potentiation.
 |
ACKNOWLEDGEMENT |
We are grateful to S. Nakanishi for kindly
providing NR2A cDNA.
 |
FOOTNOTES |
*
This work was supported in part by Telethon Grant 946 and
MURST 40 and 60% (to M. D. L.) and MURST 60% and CNR
96.02075.PS04 (to F. C.).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: Inst. of
Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. Tel.: 39-02-20488374; Fax: 39-02-29404961; E-mail: fabrizio.gardoni@unimi.it.
Published, JBC Papers in Press, December 4, 2000, DOI 10.1074/jbc.M009922200
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ABBREVIATIONS |
The abbreviations used are:
PSD, postsynaptic density;
PKC, protein kinase C;
GST, glutathione
S-transferase;
PAGE, polyacrylamide gel electrophoresis;
t-ACPD, trans-1-amino-1,3-cyclopentanedicarboxylic acid;
PDBu, phorbol 12,13-dibutyrate;
TIF, Triton X-100-insoluble fraction;
NMDA, N-methyl-D-aspartate;
CaMKII,
-caldmodulin kinase II.
 |
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