(Received for publication, September 10, 1996, and in revised form, February 21, 1997)
From the Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532
Ca2+ influx through N-methyl-D-aspartate (NMDA)-type glutamate receptors plays a pivotal role in synaptic plasticity during brain development as well as in mature brain. Cyclic AMP-dependent protein kinase (PKA) and members of the protein kinase C (PKC) family are also essential for various forms of synaptic plasticity and regulate the activity of different ion channels including NMDA and non-NMDA receptors. We now demonstrate that PKA and various PKC isoforms phosphorylate the NMDA receptor in vitro. The stoichiometry of [32P]phosphate incorporation per [3H]MK-801 binding site is greater than 1 for both PKA and PKC. Double immunoprecipitation experiments show that all three NMDA receptor subunits that are prevalent in the cortical structures, NR1, NR2A, and NR2B, are substrates for PKA as well as PKC. Two-dimensional phosphopeptide mapping reveals that the major phosphorylation sites for PKA and PKC differ for all three subunits. We provide evidence that some if not most of these sites are phosphorylated in the central nervous system of rats in vivo. The results presented in this article together with earlier electrophysiological experiments demonstrating that PKA and PKC activation increases the activity of NMDA receptors indicate that NMDA receptor potentiation can be mediated by direct phosphorylation by PKA and PKC. Collectively, these results strongly suggest that NMDA receptor functions such as control of neuronal development or expression of synaptic plasticity are modulated by PKA- and PKC-mediated phosphorylation of NMDA receptors.
Ionotropic glutamate receptors mediate fast synaptic transmission by glutamate, the prevailing excitatory neurotransmitter in the mammalian brain (1, 2). These glutamate receptors can be divided into two major families, N-methyl-D-aspartate (NMDA)1 receptors and non-NMDA receptors, based on their pharmacological and electrophysiological properties as well as sequence identity (1-3). NMDA receptors are key participants in brain development (4, 5) and synaptic plasticity, which may underlie learning and memory (6-8).
Analogous to other ligand-gated ion channels of the nicotinic acetylcholine receptor superfamily, glutamate receptors are thought to consist of five different subunits that are homologous to each other (3, 9, 10). NMDA receptors are formed by NR1 subunits in various combinations with NR2A-NR2D subunits (11-13). The apparent mass, as determined by immunoblotting, is 110-120 kDa for NR1 and 160-190 kDa for NR2A-D (14-19). Deglycosylation in vitro reduces the apparent Mr of glutamate receptor subunits, usually by 10-20 kDa. Immunoprecipitation experiments suggest that native NMDA receptors are pentamers made of one or two NR1 and two or three NR2A-D subunits (15, 19, 20). Functional NMDA receptors are formed when NR1 is expressed alone or in combination with NR2 subunits, but not when NR2 subunits are expressed without NR1 (11, 12). Targeted disruption of the NR1 gene results in a lethal effect (21), while the disruption of the NR2A gene has been implicated in reduced hippocampal long term potentiation (LTP) in mice (22). As expected from their role in synaptic transmission, biochemical (23) and immunohistochemical studies demonstrate that glutamate receptors are specifically localized at postsynaptic sites of excitatory, but not inhibitory, synapses (17, 18, 24, 25).
Hydrophobicity plots identified four hydrophobic regions in NMDA and
non-NMDA receptors termed M1-M4. By comparison with nicotinic acetylcholine receptors, these regions were originally thought to form
four transmembrane segments so that the
NH3+- and COO-terminal
ends would both be located extracellularly. Recent studies on non-NMDA
receptor subunits, however, demonstrated that only the
NH3+-terminal end is extracellular,
followed by the transmembrane region M1; the M2 region, which loops
only partially into the plasma membrane and back into the cytosol; the
transmembrane regions M3 and M4; and finally the cytosolic
COO
terminus (26-28). NMDA receptor subunits probably
adopt a similar membrane topology (16).
NMDA receptor-mediated currents were potentiated by protein kinase C (PKC) activators (phorbol esters or oleoyl-acetylglycerol), and this potentiation was prevented by PKC inhibitors (H7, staurosporin, or sphingosine) in trigeminal (29, 30), spinal cord dorsal horn (31), and hippocampal neurons (Ref. 32, but see Ref. 33). Similar results were obtained in oocytes injected with total mRNA (34, 35), with RNA encoding different splicing variants of NR1 (36), or with RNA encoding NR1 and NR2A or 2B (11, 37). Cyclic AMP analogs and forskolin were employed to show that cyclic AMP-dependent protein kinase (PKA) enhances the NMDA response in spinal cord dorsal horn neurons (38) and in hippocampal microcultures (39). Finally, the effects of phosphatase inhibitors and direct application of phosphatase 1 and 2A suggest that phosphatases 1, 2A, and 2B are involved in the down-regulation of NMDA receptor activity under various conditions (40-42). Most of these studies are based on electrophysiological methods that do not reveal whether the NMDA receptor itself or another protein or enzyme modifying NMDA receptor activity has been phosphorylated.
The work presented in this study addresses the question of whether the NMDA receptor itself is a substrate for PKA and PKC and whether the corresponding phosphorylation sites are targeted in vivo. No previous evidence has demonstrated that NMDA receptors are directly phosphorylated by PKA, although it has been shown earlier by Tingley et al. (16) that NR1 is phosphorylated by PKC in vitro and in cell culture upon stimulation with phorbol ester. However, it has been unknown whether PKC also phosphorylates NR2 subunits. We now demonstrate that PKA as well as PKC phosphorylate NR1, NR2A, and NR2B. Two-dimensional phosphopeptide mapping reveals, however, different patterns for PKA and PKC phosphorylation, suggesting differential regulation of NMDA receptors by PKA and PKC. Back-phosphorylation of NMDA receptors immunoisolated from rapidly collected and homogenized rat brain in the absence and presence of phosphatase inhibitors shows that NR1 and NR2 subunits are phosphorylated in vivo at those sites recognized by PKA and PKC in vitro.
MK-801
((+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine
maleate) was obtained from RBI (Natick, MA), and [3H]MK-801 (884 GBq/mmol) and [-32P]ATP
(111 TBq/mmol) were from DuPont NEN. The ECL detection kit was
purchased from Amersham Corp., protein A-Sepharose (PAS) and bovine
serum albumin (BSA; IgG-free) from Sigma, microcystin LR from
Calbiochem, 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc) from
Boehringer (Mannheim, Germany), sequencing grade trypsin from Promega
(Madison, WI), and control antibodies from Zymed (South San Francisco,
CA). PKA and PKC isoforms were isolated, and the absence of activity of
other, contaminating, protein kinases was confirmed by established
procedures (43-46). PKA and Phc isoforms were kindly supplied by Dr.
E. I. Rotman, Department of Pharmacology, University of Washington
(Seattle, WA) and S.-M. Huang and Dr. P. J. Bertics, Department of
Biomolecular Chemistry, University of Wisconsin (Madison, WI),
respectively. All other reagents were purchased from commercial
suppliers and were of standard biochemical quality.
The monoclonal antibody 54.2 (R1) recognizes
specifically NR1 (15) and was generously supplied by Dr. R. Jahn,
Howard Hughes Medical Institute, Yale University (New Haven, CT).
Polyclonal anti-peptide antibodies produced against the synthetic
peptide corresponding to the COO
-terminal 20 amino acid
residues of NR2A recognize NR2A and NR2B (
2A/B) (18, 20). Antibodies
raised against the synthetic peptide corresponding to amino acid
residues 1408-1429 of NR2A interact specifically with NR2A (
2Ap)
(20). For the production of antibodies against polyhistidine fusion
proteins containing the NR2A region spanning amino acids 934-1203
(
2A) or the NR2B region encompassing amino acids 935-1856 (
2B),
oligonucleotides flanking the respective region and containing
appropriate restriction sites were used to amplify corresponding
sequences from cDNA clones of NR2A and NR2B by PCR. The DNA
products were ligated into pQE30 (Qiagen, Chatsworth, CA), allowing the
expression of respective polyhistidine fusion proteins that were
subsequently purified on Ni2+ chelate resin
nickel-nitrolotriacetic acid and injected into rabbits. All antibodies
against NR2 subunits including those produced against fusion proteins
(
2A,
2B) were kindly provided by Dr. R. J. Wenthold, NIDCD,
National Institutes of Health (Bethesda, MD).
Forebrains (without the
cerebellum) from adult Sprague Dawley rats were prepared as a 10%
homogenate in ice-cold 320 mM sucrose, 10 mM
Tris-Cl, pH 7.4, 10 mM EDTA, 10 mM EGTA, with a
glass/Teflon homogenizer (12 strokes at approximately 800 rpm).
Pepstatin A (1 µM), leupeptin and aprotinin (10 µg/ml),
Pefabloc (0.2 mM), benzamidine (0.1 mg/ml), and calpain
inhibitors I and II (8 µg/ml each) were present in all buffers. After
a short centrifugation (5000 rpm, 2 min, SS 34-rotor, 4 °C) to
remove larger cell fragments, membranes were sedimented (50,000 rpm, 30 min, 70.1 Ti rotor, 4 °C) and solubilized in 1% deoxycholate, 50 mM Tris-Cl, pH 7.4, 10 mM EDTA, 10 mM EGTA, for 20 min on ice (usually 10 ml per rat brain)
(20). Unsolubilized material was sedimented by ultracentrifugation as
before, and the supernatant was collected and stored at 80 °C.
To determine the phosphorylation status of NMDA receptor subunits in vivo, rats were decapitated without anesthesia, and the heads were put into liquid nitrogen for 8-10 s. Using this procedure, brains were immediately cooled down to 0-5 °C without freezing. The brains were then dissected, and cortex, hippocampus, and cerebellum were processed separately. To reduce dephosphorylation, ultracentrifuge rotors were precooled on ice, and all procedures were carried out on ice or at 4 °C. Half of each tissue was processed in the absence of phosphatase inhibitors, allowing dephosphorylation of phosphoproteins by endogenous phosphatases. For the processing of the other half of the tissue, the following phosphatase inhibitors were present in the homogenization and solubilization buffer: 50 mM NaF, 50 mM sodium pyrophosphate, 20 mM 2-glycerolphosphate, 1 mM p-nitrophenylphosphate, and 2 µM microcystin LR.
Immunoisolation and Immunoblotting of NMDA Receptors0.5 ml
of deoxycholate extract containing at least 20 fmol of NMDA receptor
was preadsorbed with 150 µl of Sepharose CL-4B and 5 mg of PAS for 30 min. After centrifugation for 1 min in a table top centrifuge, the
supernatant was collected and incubated on a tilting mixer for 1.5 h with 0.5 µl of R1 or control ascites or with 0.5 µl of
2A/B,
2A,
2B, or control antiserum at 4 °C or on ice when
phosphatase inhibitors were present. Then 3-5 mg of PAS, preswollen
and washed three times with TBS (150 mM NaCl, 10 mM Tris-Cl, pH 7.4) containing 1% Triton X-100, were
added, and the samples were mixed for 2.5 h as before. The
immunocomplexes were sedimented by centrifugation and washed three
times with 1% Triton X-100 in TBS and once with 10 mM
Tris-Cl, pH 7.4, before being extracted with 20 µl of SDS-sample
buffer (2% SDS, 20 mM dithiothreitol, 10% sucrose, and
125 mM Tris-Cl, pH 6.8) for 20 min at 60 °C. In some
cases, brain homogenates were directly solubilized in SDS-sample
buffer.
After separation by SDS-polyacrylamide gel electrophoresis (SDS-PAGE;
the gels were polymerized using 6% acrylamide), the proteins were
transferred onto nitrocellulose, which was blocked by incubation for
2 h with 10% skim milk powder in TBS (TBS-milk). Blots were
incubated with R1 or control ascites (1:1000 in TBS-milk) or with
2A,
2B,
2A/B, or control antiserum (1:100 in TBS-milk) for
1-2 h, washed five times with TBS-milk, incubated with horseradish peroxidase-labeled sheep anti-mouse IgG (ascites) or protein A (sera)
(both diluted 1:1000 in TBS-milk), washed for 5-6 h with 0.05% Tween
20 in TBS (8-10 changes), and developed with the ECL reagent.
For double immunoprecipitations, the resins with antibody-NMDA receptor complexes attached were treated with 20 µl of 50 mM Tris-Cl, pH 8, 1.5% SDS, 5 mM dithiothreitol, 200 µM Pefabloc, 1 µM pepstatin A, 20 µg of leupeptin, and 40 µg aprotinin for 20 min at 60 °C in a thermomixer, diluted with 300 µl of 1% Triton X-100, 0.2% bovine serum albumin, 200 µM Pefabloc, 1 µM pepstatin A, 2 µg of leupeptin, and 4 µg of aprotinin in TBS and centrifuged. Supernatants were incubated with various antibodies and PAS as described above. PAS immunocomplexes were washed, extracted with SDS sample buffer, and loaded onto SDS-polyacrylamide gels as detailed above.
NMDA Receptor PhosphorylationNMDA receptors were
solubilized and immunoprecipitated as described above. The resins with
the receptor were washed three times with 1% Triton X-100 in TBS and
once with basic phosphorylation buffer (50 mM HEPES-NaOH,
pH 7.4, 10 mM MgCl2, 1 mM EGTA).
Samples were phosphorylated with 0.5-1 µg of PKA or PKC in 50 µl
of basic phosphorylation buffer containing 0.4 mM
dithiothreitol, 1 µM pepstatin A, 2 µg of leupeptin, 4 µg of aprotinin, and 0.2 µM [-32P]ATP.
This buffer was supplemented with 1.5 mM CaCl2,
50 µg of diolein, and 2.5 mg of phosphatidylserine for PKC. The
samples were incubated for 30 min at 32 °C in a thermomixer, washed
four times with 0.1% Triton X-100 in radioimmune assay buffer (10 mM Tris-HCl, pH 7.4, 75 mM NaCl, 20 mM EDTA, 10 mM EGTA, 20 mM sodium pyrophosphate, 50 mM NaF, 20 mM
2-glycerolphosphate, 1 mM
p-nitrophenylphosphate) and once in 10 mM
Tris-HCl, pH 7.4. For quantitative phosphorylation as required for the
measurement of the stoichiometry of [32P]phosphate
incorporation by PKA or PKC, 10 µM unlabeled ATP was added to the phosphorylation reaction. At this ATP concentration, phosphorylation of NMDA receptor subunits by PKA or PKC was saturated. Extending the incubation time to 1 h also did not result in an increase in [32P]phosphate incorporation by PKA or PKC.
The pellets were extracted with SDS sample buffer (see above) and used
directly for SDS-PAGE. For double immunoprecipitations, pellets were
treated as described in the previous paragraph except that radioimmune
assay buffer was used rather than TBS for the dilution of the SDS
extracts and the washes. Phosphorylated protein bands were visualized
by autoradiography, and incorporated 32P was quantified by
Cerenkov counting or scintillation counting.
The method for the
generation of phosphopeptide maps (47) was modified as follows: NMDA
receptors were solubilized from rat brain membranes with deoxycholate,
immunoprecipitated with R1, phosphorylated with PKA or PKC, and
dissociated with SDS, and NR1, NR2A, or NR2B was reprecipitated with
R1,
2A, or
2B, respectively, as described above. After
SDS-PAGE and autoradiography, gel pieces containing
32P-labeled NMDA receptor subunits were excised from the
gels, washed three times for 2-5 h with acetic
acid/2-propanol/H2O (1:1:8) and twice for 2 h with
50% methanol, dried in a vacuum concentrator, rehydrated with 800 µl
of 25 mM NH4HCO3 containing 0.5 µg trypsin (which is modified by reductive methylation to make it
resistant to autoproteolyis) and a trace amount of phenol red, and
digested overnight at 37 °C. After sedimentation for 1 min in a
table top centrifuge, supernatants were removed, and gel slices were
treated a second time with 0.5 µg of trypsin in 800 µl of 25 mM NH4HCO3 omitting phenol red in
the digestion buffer, permitting a control for the extraction
efficiency of the gel slices during the second cycle. In addition,
32P was quantified by Cerenkov counting of the gel pieces
before and after the digests. Only samples with an extraction efficacy of at least 80% of 32P were further processed.
Supernatants from both digests were combined, dried in a vacuum
concentrator, resuspended in 200-300 µl of H2O, and
dried again. Resuspension and evaporation was repeated until phenol red
changed its color from red to yellow, reflecting a nearly complete
removal of the basic NH4HCO3 salt. Tryptic
phosphopeptides were solubilized in 5 µl of 1% pyridine, 10% acetic
acid, pH 3.5, spotted onto thin layer chromatography cellulose plates,
and subjected to electrophoresis until phenol red had migrated 5 cm
toward the cathode (usually 2 h at 400 V). Plates were dried,
developed in the second dimension by ascending chromatography in
pyridine/1-butanol/acetic acid/H2O (10:15:3:12), and dried,
and the pattern of tryptic phosphopeptides was visualized by
autoradiography or PhosphorImager analysis. To ensure that tryptic
digest was complete, in 2-4 independent experiments tryptic digests
were performed for 3 full days rather than overnight, and each day 0.5 µg of trypsin was added to the reaction mixture. Phosphopeptide maps
obtained from NR1, 2A, and 2B subunits phosphorylated with PKA or PKC
were virtually identical after short and extended digests.
[3H]MK-801 binding was
performed with membrane fractions as described (48, 49). In brief, 500 µg of membrane protein in 0.1 ml of 5 mM Tris-Cl (pH 7.4)
and 1 mM EGTA were incubated with 100 nM
[3H]MK-801 to equilibrium (24 h, 4 °C). The
KD for [3H]MK-801-binding to NMDA
receptors in membrane fractions is 2.6 nM (49). A
concentration of 100 nM [3H]MK-801 will,
therefore, saturate [3H]MK-801 binding to NMDA receptors.
Nicotinic acetylcholine receptors are blocked by MK-801 with a low
affinity. Therefore, at a concentration of 100 nM, MK-801
binding to nicotinic acetylcholine receptors is negligible, since the
KD value of this receptor for MK-801 is 70-fold
higher than this concentration (50). Membranes were filtered through
Whatman GF/C membranes and washed three times with 3 ml of ice-cold
binding buffer. Nonspecific binding was defined by incubations in the
presence of 10 µM unlabeled MK-801 and subtracted from
total [3H]MK-801 binding to obtain specific binding. To
determine the amount of NMDA receptor immunoprecipitated by R1 for
quantitative phosphorylation experiments, 33% of the precipitates were
subjected to immunoblotting with
R1 as probing antibody. For
comparison, various amounts of membrane fractions (e.g. 25, 50, 100, and 200 µg of protein) containing defined amounts of NMDA
receptors as determined by [3H]MK-801 binding were loaded
in parallel lanes.
R1 binding to the NMDA receptors on the membrane
was detected with the ECL method and quantified by densitometry. ECL
signals resulting from various exposure times were compared with each
other to ensure that they were within the linear range of the film.
As
described earlier (15), R1 recognized a single band of about 110 kDa
by immunoblotting of total brain homogenate (Fig. 1A, lane 1). This band appears
elongated, probably reflecting the existence of closely comigrating NR1
isoforms as they are generated by differential splicing (19). The same
band was detected when solubilized NMDA receptors were
immunoprecipitated with
R1 (Fig. 1A, lane 2)
and absent after immunoprecipitation with control antibody (Fig.
1A, lane 3), excluding a nonspecific
precipitation of NMDA receptors under the experimental conditions.
Double immunoprecipitation utilizing
R1 for both precipitation steps
also yielded the immunoreactive 110-kDa band (Fig. 1A,
lane 4), demonstrating that NR1 can be dissociated from the
NMDA receptor complex with SDS and subsequently reprecipitated with
R1.
The specificity of antibodies directed against NR2 subunits was also
demonstrated by immunoblotting. Similar to earlier observations (20),
2A/B labeled a band of about 190 kDa when either total brain
homogenate (Fig. 1B, lane 1) or NMDA receptors
immunoprecipitated with
R1 (Fig. 1B, lane 2)
had been blotted. Similar results were obtained with the novel
antibodies
2A and
2B. Both antibodies recognized a single band of
about 190 kDa in total brain homogenate (Fig. 1B,
lanes 3 and 5). The same bands were present after
immunoprecipitation with
R1 (Fig. 1B, lanes 4 and 6), corroborating the finding that both antibodies
specifically react with NR2 subunits.
Expression of NR2C and NR2D is very low in the cortex and the other
brain areas used for the experiments described here in mature rat
brains (11-13). Therefore, they were not included in our studies. To
test, however, the possibility that 2A cross-reacts with NR2B and
vice versa, NMDA receptors were immunoprecipitated with
R1, dissociated with SDS, and reprecipitated with
2A or
2B.
Probing the immunoblots with
2A showed that this antibody bound to
the 190-kDa protein reprecipitated with
2A, but not the
2B
precipitate (Fig. 1C, lanes 2 and 3).
The opposite was true for
2B (Fig. 1C, lanes 5 and 6). These results indicate that
2A is specific for
NR2A and
2B for NR2B and that both NR2 subunits can be
reprecipitated with the respective antibody after SDS treatment of the
NMDA receptor complex.
NMDA receptors were solubilized with deoxycholate,
immunoprecipitated, phosphorylated with PKA, and analyzed by SDS-PAGE
and autoradiography. Two 32P-containing bands with
molecular masses of 110 and 190 kDa, corresponding to NR1 and NR2
subunits, were detectable after single precipitation with R1 (Fig.
2A, lane 1),
2A/B (Fig.
2A, lane 4),
2A (Fig. 2A,
lane 5), or
2B (Fig. 2A, lane 6).
When NMDA receptors were precipitated with
R1 and incubated in the
absence of PKA, no bands appeared (Fig. 2A, lane
2), indicating that the phosphorylation observed in the presence
of PKA is mediated by the PKA added for the in vitro
phosphorylation and not by an endogenous protein kinase associated with
the NMDA receptor. In addition, NR1 was also phosphorylated when
incubated with purified PKA after dissociation of the NMDA receptor
complex and after a second immunoprecipitation with
R1, resulting in
highly purified NR1 (see below; Fig. 2B, lane 3).
No bands were visible by autoradiography after precipitation with
control antibody (Fig. 2A, lane 3), showing that
the precipitations of the NMDA receptor had been specific.
NMDA receptor-associated proteins such as PSD-95 (51) or minor
contaminants could coprecipitate with the receptor complex and
comigrate during SDS-PAGE with one of the receptor subunits. If those
proteins are preferred kinase substrates, they may appear as one of the
major phosphorylated bands. Putative contaminating proteins can be
removed efficiently, and the different subunits can be separated by a
double immunoprecipitation method (46, 52). NMDA receptors were
immunoprecipitated with R1, phosphorylated with PKA, and dissociated
with SDS. NR1 and NR2 subunits were specifically and separately
reprecipitated with the corresponding antibodies. Reprecipitation with
R1 resulted in a 32P-containing band of about 110 kDa
(Fig. 2B, lanes 1 and 3),
corroborating the finding that this band corresponds to the NR1
subunit. Reprecipitation with
2A/B yielded a 32P-labeled
band of 190 kDa, which may contain NR2A or -B (Fig. 2B,
lane 4). More specifically, similar 32P-labeled
bands were apparent after reprecipitation with
2A or with
2Ap
(Fig. 2B, lanes 6 and 8), indicating
that these bands correspond to NR2A. Also
2B reprecipitated a
32P-labeled band of 190 kDa, identifying this band as NR2B
(Fig. 2B, lane 7). None of these bands were
detectable when precipitations were performed with control antibodies
(Fig. 2B, lanes 2 and 5). These
results clearly show that NR1, NR2A, and NR2B are phosphorylated in vitro by PKA and constitute, therefore, potential PKA
substrates in intact neurons.
After one-step immunoprecipitation of NMDA receptors with
R1 and phosphorylation with either a PKC preparation containing PKC
, -
, and -
or with purified PKC
or -
isoforms, two
phosphoprotein bands are apparent at 110 and 190 kDa (Fig.
3A, lanes 1, 3, and 5). Phosphorylation with the PKC mix yielded significantly
more 32P incorporation than the incubations with the other
two PKC isoforms, although the total amount of PKC was similar in all
tests. These findings suggest that PKC
may phosphorylate NMDA
receptors in vitro more efficiently than the other two
isoforms. Immunoprecipitation with nonspecific antibody did not result
in these 32P-containing bands (Fig. 3A,
lanes 2, 4, and 6), indicating the specificity of the precipitation with NR1.
To confirm the identity of NR1 and to identify which of the NR2
subunits is actually phosphorylated in these experiments, we
dissociated the NMDA receptor complex with SDS after phosphorylation with the PKC mix. As expected for the NR1 subunit, a phosphorylated 110-kDa band was obtained by reprecipitation with R1 (Fig.
3B, lane 1). Reprecipitation of
32P-labeled NR2A with either
2A or
2Ap showed that
this subunit is phosphorylated by PKC in vitro (Fig.
3B, lanes 2 and 4). NR2B also emerged
as a substrate for PKC as demonstrated by reprecipitation with
2B
(Fig. 3B, lane 3). Nonspecific precipitation was
excluded with control antibodies (Fig. 3B, lane
5). To rule out cross-reactivity between
2A with NR2B, and
2B with NR2A, NMDA receptors were immunoprecipitated with
R1,
phosphorylated with PKC, and dissociated with SDS. One sample was
completely depleted of NR2A by immunoprecipitation with
2A (Fig.
3C, lane 1), as demonstrated by a second
incubation with
2A that did not result in additional NR2 subunit
precipitation (Fig. 3C, lane 2). The presence of
NR2B in the supernatant of the sample twice extracted by
2A
precipitations was subsequently confirmed by precipitation with
2B
(Fig. 3C, lane 3). Analogous results were
obtained for a second sample, which was depleted of NR2B twice with
2B before precipitation of NR2A with
2A (Fig. 3C,
lanes 4-6).
Two-dimensional tryptic phosphopeptide mapping was used to
determine if PKA and PKC phosphorylate the NMDA receptor subunits at
the same or different sites. NMDA receptors were solubilized with
deoxycholate, immunoprecipitated with R1, washed, and split into two
equal samples. One sample was phosphorylated by PKA and the other by
PKC. Immunoprecipitates were washed, and the receptor subunits were
dissociated with SDS. NR1, NR2A, and NR2B were specifically reprecipitated with
R1,
2A, or
2B, respectively, subjected to
SDS-PAGE, and digested by trypsin. Fig. 4 shows the
resulting two-dimensional phosphopeptide maps. For all three subunits,
the pattern of the major phosphopeptides looks quite different after phosphorylation with PKA and PKC. For example, the three main phosphopeptides of PKA-phosphorylated NR1 are left of the origin toward
the cathode and barely migrated during the ascending chromatography (Fig. 4A, spots 1, 3, and
4). The prevailing phosphopeptide of PKC-phosphorylated NR1,
however, moved fairly far during this chromatography step (Fig.
4B, spot 5). Analogous considerations are true
for NR2A and NR2B. However, spot 3 of PKA-phosphorylated NR2A could be
identical with spot 1, 2, or 3 of PKC-phosphorylated NR2A. Similarly,
spot 1 of PKC-phosphorylated NR2B could be identical with spot 2 or 3 of PKA-phosphorylated NR2B.
To further confirm that PKA and PKC phosphorylate NMDA receptor subunits at different sites, phosphorylation reactions were performed with PKA alone, PKC alone, or PKA and PKC together under assay conditions under which the incorporation of [32P]phosphate by PKA or PKC was saturated, followed by SDS-PAGE, autoradiography, and PhosphorImager analysis. For NR1 as well as NR2 subunits, the incorporation of [32P]phosphate was as high during incubation with a combination of PKA and PKC as the total incorporation of [32P]phosphate by PKA and PKC during separate incubation reactions taken together. Accordingly, phosphorylation of NMDA receptor subunits is additive, corroborating the findings described in the previous paragraph that indicate that PKA and PKC target different sites.
Stoichiometry of PKA and PKC Phosphorylation of NMDA Receptors[3H]MK-801 binds specifically and with
high affinity to NMDA receptors in membrane fractions as well as after
solubilization with deoxycholate (48, 49). The KD
for [3H]MK-801 binding to NMDA receptors in rat brain
membranes is 2.6 ± 0.7 nM, and
Bmax is 2.2 pmol/mg protein (49). However, it is
unclear whether [3H]MK-801 labels all NMDA receptors when
solubilized. The following strategy was, therefore, developed to
measure the amount of NMDA receptor in solubilized fractions. First, we
determined the number of [3H]MK-801 binding sites in a
rat brain membrane fraction that had been prepared as described by
McKernan et al. (49). The membrane fractions were incubated
with 100 nM [3H]MK-801. At this
concentration, [3H]MK-801 binding to NMDA receptors is
saturated (48, 49), but MK-801 binding to nicotinic acetylcholine
receptors (50) is negligible because the KD for the
latter interaction (7 µM) is nearly 2 orders of magnitude
higher than the [3H]MK-801 concentration used in our
experiments. Nonspecific binding was obtained in the presence of a
100-fold excess of unlabeled MK-801 and subtracted from the total
amount of [3H]MK-801 binding. Similar to earlier studies
(48, 49), our membrane fractions specifically bound 1.3 ± 0.3 pmol [3H]MK-801/mg of protein (average of seven
experiments ± S.E.). To determine the amount of NMDA receptors
precipitated for phosphorylation by R1, part of the precipitates
were analyzed by immunoblotting. The amount of NMDA receptor as
detected by ECL using
R1 as primary antibody was compared by
densitometry with those of membrane fractions loaded onto the same gel.
Membrane protein of 25, 50, 100, and 200 µg had been applied to
control for linearity of the ECL signals. The results show that
typically 30-60 fmol of [3H]MK-801 binding sites were
present during the in vitro phosphorylations. Incorporation
of [32P]phosphate into NMDA receptors by PKA and PKC was
determined after SDS-PAGE and autoradiography by scintillation counting
of NR1- and NR2-containing gel pieces.
Assuming that one NMDA receptor complex binds one [3H]MK-801 molecule, these experiments indicate a molar ratio of about 1 for the incorporation of [32P]phosphate per receptor complex into NR1 by PKA as well as by PKC (Table I). The molar ratio of [32P]phosphate incorporation into NR2 subunits by PKA was 0.27, and the ratio for incorporation by PKC was 1.48. The latter ratio may reflect the fact that one NMDA receptor complex probably contains several NR1 and NR2 subunits. In addition, each subunit may possess more than one phosphorylation site. Taken together, these data indicate that the in vitro phosphorylation efficiency by PKA and PKC is high, suggesting that the NMDA receptor is a good substrate for both kinases in vitro and possibly in vivo. If one NMDA receptor binds more than one [3H]MK-801 molecule, the 32P incorporation rate per receptor would be even higher. Similar considerations are valid if a fraction of [3H]MK-801 associated during the binding assay to a receptor different from the NMDA receptor (e.g. nicotinic acetylcholine receptors; see above).
|
Recent
electrophysiological studies indicate that NMDA receptor currents are
down-regulated during stimulation of synaptic transmission and that
this effect is reversed by PKA (39). Accordingly, either NMDA receptors
(by themselves or associated) or other, regulatory, proteins are
tonically phosphorylated by PKA before stimulation of synaptic
transmission. To test if NMDA receptors are constitutively
phosphorylated in the rat central nervous system in vivo,
rat brains were cooled down to 0-4 °C immediately after decapitation, prepared from the skull, and cut in half. One half was
solubilized in the absence and the other half in the presence of
phosphatase inhibitors (see "Experimental Procedures" for more details). We expected that phosphorylated NMDA receptors would be
dephosphorylated by endogenous phosphatases when no phosphatase inhibitors were present. For example, the PKA site of class C L-type
Ca2+ channels is completely dephosphorylated during
isolation in the absence, but not in the presence, of phosphatase
inhibitors (53).2 After dephosphorylation,
the number of phosphorylation sites available for in vitro
phosphorylation (in this context referred to as
"back-phosphorylation") will be increased, and subsequent in
vitro phosphorylation using [-32P]ATP will lead
to a larger incorporation of [32P]phosphate. Solubilized
NMDA receptors were immunoprecipitated, phosphorylated with PKA or PKC,
and analyzed by SDS-PAGE. [32P]phosphate incorporation
into NR1 as well as NR2 subunits by both PKA and PKC during the
in vitro phosphorylation was clearly higher when NMDA
receptors were prepared without phosphatase inhibitors (Fig.
5). Similar results were obtained in three different
experiments. To ensure that identical amounts of NMDA receptors were
immunoprecipitated and present during the in vitro
phosphorylation step, one-third of each precipitate was used for
immunoblotting. Probing with
R1 or antibodies against NR2 subunits
demonstrated that exactly the same amounts of NR1 and NR2 subunits were
present in samples prepared with and without phosphatase inhibitors
(data not shown).
Expression of NMDA receptors in adult rats is highest in brain cortex,
hippocampus, and cerebellum (12, 13). These brain areas were processed
separately, and 32P incorporation into NMDA receptors
during in vitro phosphorylation was quantified after
SDS-PAGE by scintillation counting of excised gel pieces containing NR1
and NR2 subunits. [32P]phosphate incorporation into NR1
by PKA and into NR2 subunits by PKC was significantly higher in all
three brain regions when samples were prepared without phosphatase
inhibitors (Fig. 6). The same results were observed for
PKA phosphorylation of NR2 subunits and PKC phosphorylation of NR1 in
cortical and hippocampal preparations (Fig. 6). These results suggest
that the in vitro PKA as well as the in vitro PKC
phosphorylation sites had been phosphorylated in vivo before
homogenization of the tissue. During solubilization and
immunoprecipitation, a significant portion of these phosphorylation
sites had obviously been dephosphorylated when no phosphatase
inhibitors were utilized, thereby becoming available for the in
vitro phosphorylation with [32P]phosphate. However,
the differences in back-phosphorylation with PKA or PKC of cerebellar
NR1 or NR2 subunits, respectively, obtained in the absence and presence
of phosphatase inhibitors, were not significant (Fig. 6), although they
were highly significant for NR2 and NR1 subunits within the very same
samples back-phosphorylated with PKA and PKC, respectively, ruling out
sample-to-sample variation in the amount of NMDA receptors or
phosphatase activity. The latter results may reflect a difference in
NMDA receptor composition in the cerebellum when compared with cortex
and hippocampus. In fact, in contrast to NR2B, NR2C is strongly
expressed in the mature cerebellum but hardly detectable in cortical
structures (12, 13). NR2C may not be tonically phosphorylated by PKA
and may also influence the phosphorylation of NR1 by PKC.
Although electrophysiological studies indicate that NMDA receptor activity is regulated by the action of norepinephrine, and more directly by PKA (38, 39), PKA phosphorylation of NMDA receptors has not yet been reported. Our results demonstrate direct phosphorylation of NR1, NR2A, and NR2B. This conclusion is based on double immunoprecipitation protocols involving the dissociation of NMDA receptor complexes after specific immunoprecipitation and in vitro phosphorylation followed by reprecipitation of NR1, NR2A, and NR2B with subunit-specific antibodies.
An increasing number of electrophysiological investigations show that PKC regulates NMDA receptor activity (11, 29-37; see Introduction). Earlier investigations indicated that NR1 is phosphorylated upon stimulation of PKC in cortical cultures and in HEK 293 cells transfected with NR1 (16). In that study NR1 had been isolated by a single immunoprecipitation step. Employing single and double immunoprecipitation protocols, we unequivocally identify NR1 as a PKC substrate. We extended these investigations to demonstrate that both NR2A and NR2B are phosphorylated by PKC.
NMDA receptors were phosphorylated by PKA and PKC isoforms to a stoichiometry greater than 1 mol of phosphate incorporated per mol of [3H]MK-801 binding sites, although the concentration of ATP during the in vitro phosphorylation reaction (10 µM) was lower than under normal physiological conditions (around 1 mM). The phosphorylation stoichiometries, therefore, suggest that NMDA receptor subunits are efficient substrates for PKA and PKC and may readily be phosphorylated under physiological conditions. Two-dimensional phosphopeptide mapping of NMDA receptor subunits that had been immunoprecipitated twice before tryptic digestion to minimize the risk of contaminating phosphoproteins demonstrated that the main phosphorylation sites of PKA and PKC are substantially different for NR1, NR2A, and NR2B under our conditions (Fig. 4). Furthermore, [32P]phosphate incorporation by PKA and PKC was additive, supporting this conclusion.
In Vivo Phosphorylation of NMDA ReceptorsTo test the
physiological relevance of the in vitro PKA and PKC
phosphorylation sites, we asked whether these sites are phosphorylated in vivo. NMDA receptors were immunoisolated in the presence
and absence of phosphatase inhibitors and subjected to in
vitro phosphorylation by PKA and PKC in the presence of
[-32P]ATP. At least three scenarios are immediately
obvious for this kind of back-phosphorylation strategy: 1) NMDA
receptors are not phosphorylated at the sites probed by
back-phosphorylation with PKA or PKC; 2) NMDA receptors are
phosphorylated in vivo, but not efficiently dephosphorylated
by endogenous phosphatases; 3) NMDA receptors are phosphorylated
in vivo and effectively dephosphorylated after
homogenization in phosphatase inhibitor-free buffer. No difference in
[32P]phosphate incorporation into NMDA receptors prepared
in the absence and presence of phosphatase inhibitors would have been expected in the first two scenarios. However, the difference in [32P]phosphate incorporation was quite striking. Most
samples obtained in the presence of phosphatase inhibitors showed
40-70% less [32P]phosphate incorporation than those
isolated in the absence of those inhibitors (Fig. 6). These results
suggest that a large portion of the in vitro PKA and PKC
phosphorylation sites had been phosphorylated in vivo before
homogenization of the tissue.
A phosphatase inhibitor-induced decrease in back-phosphorylation of NR1 as well as NR2 subunits by PKA and PKC was consistently observed in hippocampal and cortical samples. However, back-phosphorylation by PKA of NR2 subunits isolated from the cerebellum was only slightly decreased when phosphatase inhibitors were present during the preparation (Fig. 6). This result cannot be explained by a variability in the amount of NMDA receptor present during the back-phosphorylation or in the efficacy of the back-phosphorylation reaction itself, because back-phosphorylation of NR1 was strongly reduced by phosphatase inhibitors in the very same samples. These observations may rather reflect differences in the subunit composition of NMDA receptors. NR2C is nearly exclusively expressed in the cerebellum of adult rats (11-13). It may substitute for NR2A and NR2B in many cerebellar NMDA receptor complexes and may not be extensively phosphorylated under normal conditions by PKA. In fact, when NR1 is coexpressed with NR2C in Xenopus oocytes, the resulting NMDA receptor is not modulated by PKC stimulation with phorbol esters, although NR1 when coexpressed with NR2A or NR2B in the same system is potentiated by phorbol esters (37).
Similarly, phosphatase inhibitors had only a slight effect on the back-phosphorylation by PKC of NR1 in NMDA receptor complexes isolated from the cerebellum. NR2 back-phosphorylation by PKC, however, was strongly reduced by these inhibitors in the same samples (Fig. 6). NR2C, which is specifically expressed in the cerebellum (Refs. 11-13; see above) may prevent phosphorylation of PKC sites in NR1 despite the fact that NR2C itself may be phosphorylated by PKC in vivo. Moreover, a number of splice variants exist for NR1 that exhibit differential distribution in the rat brain (54) and may be phosphorylated to different degrees in vivo (16). It is therefore possible that splicing isoforms of NR1 that are not significantly phosphorylated by PKC prevail in the cerebellum.
PKA and PKC appear to target different sites during in vitro phosphorylation as demonstrated by two-dimensional phosphopeptide mapping (Fig. 4), and both kinases are equally efficient in phosphorylating NR1 in vitro (Table I). Taken together, these findings suggest that the PKA sites had been phosphorylated by a PKA-like activity rather than by PKC isoforms in the intact brain and vice versa. However, it is not possible to rule out that some cross-phosphorylation between PKA and PKC sites occurs in vivo or that other arginine/lysine-guided serine/threonine protein kinases, such as Ca2+- and calmodulin-dependent protein kinase II and cyclic GMP-dependent protein kinase, may have contributed to the phosphorylation in vivo.
We did not investigate the phosphorylation of NR2C and NR2D. Coexpression of NR1 with NR2C or NR2D resulted in the expression of NMDA receptors that are not responsive to PKC stimulation by phorbol esters (37). In addition, expression of NR2D is generally very low and that of NR2C is only clearly detectable in the cerebellum in adult rats (11-13).
Physiological Relevance of NMDA Receptor PhosphorylationCa2+ influx through NMDA receptors is
crucial for brain development (4, 5) and synaptic plasticity (6-8).
The timing and probably the magnitude of Ca2+ influx are
critical in determining which form of plasticity will occur. For
example, homosynaptic LTP at the Schaffer collateral CA1 synapse in the
hippocampus usually requires several high frequency tetani. In
contrast, low frequency stimulations (1 Hz) over minutes lead to a
lasting depression of signal transmission at this synapse (8). This
phenomenon, which is known as long term depression, also requires
Ca2+ influx. Since PKA and PKC increase the activity of
NMDA receptors and thereby Ca2+ influx (11, 29-39),
phosphorylation of NMDA receptors by these two kinases may promote the
establishment of LTP. In this context it is noteworthy that various PKC
inhibitors, including a PKC-specific pseudosubstrate peptide inhibitor,
when injected postsynaptically prevent LTP in CA1 (6) and that
inhibition of PKA by Rp-cyclic adenosine 3,5
-monophosphorythioate
blocks the late phase of CA1 LTP (55). These effects may in part be due
to the inhibition of tonic phosphorylation of the NMDA receptor by PKA
and PKC. On the other hand, activation of phosphatases reduces the
activity of NMDA receptors (40-42) and is necessary for long term
depression (56, 57). Interestingly, NMDA receptor activity is reduced during a train of four stimuli in hippocampal cell cultures, and this
down-regulation is prevented by noradrenergic stimulation of PKA (39).
Noradrenergic input may, therefore, reverse this phosphorylation-sensitive depotentiation of NMDA receptors, thereby fostering the induction of LTP. Collectively, these observations suggest that the induction of NMDA receptor-dependent
synaptic plasticity is fine-tuned by PKA and PKC activities that are
opposed by phosphatase activity.
Another potential function of PKA and PKC phosphorylation of the NMDA
receptors is to control their subcellular distribution. Certain splice
variants of the NR1 subunit formed clusters when expressed in QT6 quail
fibroblasts, and these clusters were disrupted upon treatment with
PKC-stimulating phorbol esters (58). Phosphorylation of NMDA receptor
subunits by PKC may mediate this PKC-induced disruption of receptor
clusters. NMDA receptor subunits bind with their
COO-terminal ends to PSD-95/SAP90 (59), a protein
specifically localized at postsynaptic sites (60). Coexpression of NMDA
receptors and PSD-95/SAP90 in heterologous cell lines causes clustering of NMDA receptors that are evenly distributed in the plasma membrane when expressed alone (61). Several K+ channels also
interact with and are clustered by PSD-95/SAP90 (62). One of the
K+ channels binding to PSD-95/SAP90 is the inward rectifier
Kir 2.3. PKA phosphorylation disrupts the interaction between this channel and PSD-95/SAP90 in vitro (63). It is therefore
tempting to speculate that PKA or PKC phosphorylation controls the
postsynaptic clustering of NMDA receptors by regulating their
interaction with cytoskeletal structures.
Regulation of gene transcription by NMDA receptor-mediated activation of the transcription factors serum response factor and Elk-1 has recently been shown by Xia et al. (64). An increase of NMDA receptor activity caused by phosphorylation by PKA or PKC may therefore modulate the expression of genes under the control of serum response factor or Elk-1.
Ca2+-permeable glutamate receptors also play a critical role during neuropathological conditions. Overstimulation of glutamate receptors, especially the NMDA subtype, has been implicated in neuropathologies caused by ischemia as occurring during stroke, by status epilepticus, and by brain trauma (65-67). PKA and PKC phosphorylation of NMDA receptors may enhance NMDA receptor-induced neurotoxicity. Inhibiting PKA during or immediately after ischemic episodes, possibly by blocking noradrenergic receptors, may therefore be one strategy to alleviate subsequent neuronal damage.
In summary, our data demonstrate that NMDA receptors are substrates for PKA and PKC. Since NMDA receptor activity is modulated by PKA- and PKC-mediated phosphorylation in intact neurons, direct phosphorylation of NMDA receptors by PKA or PKC may modulate NMDA receptor-regulated neuronal functions including the induction of synaptic plasticity and gene expression.
We thank Dr. Reinhard Jahn for the monoclonal
antibody R1 (54.2), Dr. R. J. Wenthold for the antibodies against
NR2 subunits (
2A/B,
2A,
2B,
2Ap), S.-M. Huang and Dr. P. J. Bertics for the PKC isoforms, and Dr. E. I. Rotman for PKA. We also
thank Dr. R. J. Wenthold and Dr. P. J. Bertics for critically reading the manuscript.