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Originally published In Press as doi:10.1074/jbc.M311103200 on January 13, 2004

J. Biol. Chem., Vol. 279, Issue 14, 14307-14314, April 2, 2004
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Bidirectional Regulation of Neuronal Nitric-oxide Synthase Phosphorylation at Serine 847 by the N-Methyl-D-aspartate Receptor*

Gerald A. Rameau{ddagger}, Ling-Yu Chiu, and Edward B. Ziff§

From the Howard Hughes Medical Institute, Department of Biochemistry, New York University School of Medicine, New York, New York 10016

Received for publication, October 8, 2003 , and in revised form, December 22, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
At glutamatergic synapses, the scaffolding protein PSD95 links the neuronal isoform of nitric-oxide synthase (nNOS) to the N-methyl-D-aspartate (NMDA) receptor. Phosphorylation of nNOS at serine 847 (Ser847) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca2+-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser847 phosphorylation. nNOS phosphorylated at Ser847 colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 µM glutamate stimulated CaMKII phosphorylation of nNOS at Ser847, whereas excitotoxic concentrations of glutamate, 100 and 500 µM, induced Ser847-PO4 dephosphorylation by protein phosphatase 1. Strong NMDA receptor stimulation was likely to activate nNOS under these conditions because protein nitration to form nitrotyrosine, a marker of nNOS activity, correlated in individual neurons with Ser847-PO4 dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser847 could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser847 by different doses of glutamate suggests two mechanisms with opposite effects: 1) a time-dependent negative feedback induced by physiological concentrations of glutamate that limits nNOS activation and precludes the overproduction of NO; and 2) a pathological stimulation by high concentrations of glutamate that leads to unregulated nNOS activation and production of toxic levels of NO. These mechanisms may share pathways, respectively, with NMDA receptor-induced forms of synaptic plasticity and excitotoxicity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
nNOS1 is an enzyme expressed in brain which catalyzes the conversion of arginine to citrulline and NO (1-4), the latter a novel diffusible second messenger with multiple physiologic and pathologic effects (3, 5-7). One regulator of nNOS is the NMDAR, a tetrameric cation channel consisting of NR1 and NR2 subunits which is targeted to excitatory synapses where it functions in neural plasticity (8). Stimulation of the NMDAR by glutamate and glycine induces the influx of Ca2+ through the receptor pore, thereby activating Ca2+-dependent NMDAR functions (9-12). PSD95, a scaffolding protein, binds both the NMDAR and nNOS at excitatory synapses and assembles them into a macromolecular signaling complex in which nNOS is under NMDAR control (13-19). Suppression of PSD95 expression blocks NMDAR and Ca2+-dependent nNOS activation, and uncoupling of the NMDAR from PSD95 suppresses NMDAR signaling (14, 20).

Transient elevations in intracellular [Ca2+] following NMDAR activation stimulate nNOS by promoting the binding of Ca2+-calmodulin (Ca2+-CaM). In addition, it has been shown that the activity of nNOS undergoes complex regulation by phosphorylation (21-23). Of particular note, the protein kinase CaMKII phosphorylates recombinant nNOS at Ser847, which reduces nNOS activity by inhibiting the binding of Ca2+-CaM (23, 24). However, the NMDAR-induced mechanism of regulation of nNOS by phosphorylation at specific residues remains largely unknown.

We have shown previously that mutations at the apex of the pore of the NMDAR NR1 subunit which block Ca2+ entry through the channel reduce NMDAR-dependent excitotoxicity in heterologous cells and neurons (25). Because Ca2+-dependent activation of nNOS by the NMDAR has been linked to NMDAR excitotoxic effects (6, 7, 20, 26-30), we have also analyzed the NMDAR-mediated mechanism of modulation of phosphorylation of nNOS. We have shown that after excitotoxic activation of the NMDAR in cultured primary cortical neurons, nNOS undergoes an overall dephosphorylation by a pathway dependent on the phosphatases calcineurin and PP1/PP2A (30).

It is well established that the effectiveness of synapses and even the viability of the neuron can be altered by NMDAR-dependent activity that can be achieved by various patterns of stimulation. Here, we analyze the effects of NMDAR activation on the level of phosphorylation of nNOS at Ser847, a modification implicated in the regulation of nNOS. We show that the NMDAR induces a novel bidirectional control of phosphorylation of nNOS at Ser847. Using an antibody specific for Ser847-PO4 nNOS, GR847, we show that nNOS phosphorylated at this site is localized in proximity to NMDAR·PSD95 complexes at synapses. Differential activation of the NMDAR in dissociated cortical neurons in culture with varying concentrations of glutamate leads to opposing effects on nNOS phosphorylation at Ser847. Stimulation with a low concentration of glutamate resulted in a time-dependent phosphorylation of nNOS at Ser847 by CaMKII. In contrast, high pathological concentrations of glutamate stimulated dephosphorylation of Ser847-PO4. The first of these effects may share similarity with a form of long term potentiation induced by theta pulse stimulation of hippocampal neurons which promotes the activation of CaMKII (31, 32), whereas the second may relate to excitotoxicity after stroke and spinal cord injury which has been shown to involve induction of phosphatases (33-36). Strikingly, after the 5 µM glutamate induction of phosphorylation of nNOS at Ser847, nNOS remains sensitive to subsequent dephosphorylation when neurons are exposed to the excitotoxic glutamate concentration. These results support bidirectional regulation of NMDAR-mediated phosphorylation of nNOS at Ser847.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cortical and Hippocampal Cell Cultures—Cultured E18 rat cortical and hippocampal neurons were prepared as described previously (25). Briefly, after dissociation, cortical cells were plated on poly-L-lysine-coated dishes or coverslips in minimum Eagle's medium supplemented with glutamine, 10% fetal bovine serum, 0.45% glucose, and 1 mM pyruvate. Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO2 for 3 h, and the medium was exchanged for neurobasal medium plus B-27 supplement plus 0.5 mM glutamine. For immunocytochemistry, the cultures were treated with AraC until non-neuronal cells were absent. Under these conditions 99% of the cells are neurons, which could be identified reliably by morphology and by immunolabeling of microtubule-associated protein-2 (MAP-2).

Immunocytochemistry—Hippocampus-derived neurons on coverslips were fixed in 4% paraformaldehyde plus 4% sucrose for 15 min and permeabilized in 0.1% Triton X-100 for 10 min at room temperature, or they were fixed with 100% methanol at 0 °C for 15 min. After several washes with phosphate-buffered saline, the cells were incubated in 2% bovine serum albumin for 1 h and subsequently stained with a polyclonal antibody to nitrotyrosine (NT) (Upstate Biotechnology, Lake Placid, NY) and a monoclonal antibody to MAP-2 (Upstate Biotechnology). Detection was performed using a goat anti-mouse antibody conjugated to Cy5 (blue channel) and goat anti-rabbit antibody conjugated to either rhodamine or fluorescein (Jackson ImmunoResearch and Molecular Probes) (red or green channel). Immunoreactivity was examined using a Nikon PCM2000 confocal laser scanning microscope.

Quantitation of NT and pSer847 in Individual Neurons—C-imaging Systems software was used to quantify the fluorescence intensity of the antibody-conjugated fluorochromes in images acquired by confocal microscopy. Identical confocal settings were used for each set of experiments. Neurons were first selected by establishing a threshold of MAP-2 fluorescence intensity (blue channel). Dendritic NT or Ser847-PO4 nNOS was measured by eliminating the cell body for quantitation from the image, using a software-based "erasure" method. The levels of Ser847-PO4 nNOS and NT were calculated for each identified cell by determining the magnitudes of red and green channel signals within the selected dendrites. The signal from an average of 200 neurons/coverslip was averaged to obtain a population mean (presented as the mean ± S.E.). Statistical significance of differences between means was calculated using Student's t test.

Immunoblotting—The cells were washed twice with cold phosphate-buffered saline on ice, scraped, and transferred into 1.5-ml microcentrifuge tubes that were spun for 1 min at maximum speed. The pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1% (v/v) 2-mercaptoethanol, 1% Triton X-100, plus 0.1% deoxycholate and 0.1% SDS) plus tablets of protease inhibitors (Roche Applied Science) plus the phosphatase inhibitors, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 10 mM sodium {beta}-glycerophosphate, 1 µM microcystin, 1 mM sodium orthovanadate. The cells were lysed for 30 min at 4 °C with rocking. Cell lysates were centrifuged, and the supernatant fractions were collected, 300-µg aliquots were resuspended in Laemmli sample buffer (final concentration 1x). Equivalent amounts of total protein were electrophoresed on SDS-polyacrylamide gels and transferred to Hydrobond-C nitrocellulose blots (Amersham Biosciences).

Antibodies—Anti-Ser847-PO4 nNOS serum was generated by immunizing rabbits with a synthetic phosphopeptide synthesized by the Tufts University Core Facility (Boston) corresponding to 11 amino acids in nNOS NH2-CKVRFN(S-PO4)VSSYS-COOH. Antibodies were generated and affinity purified by Covance (Princeton, NJ). Anti-PSD95 monoclonal antibody was purchased from Upstate Biotechnology, and monoclonal anti-synaptophysin from Sigma and polyclonal anti-MAP-2 from (Santa Cruz Biotechnology; Santa Cruz, CA).

Statistical Analysis—Data are presented as the means ± S.E. Statistical analysis was performed using the unpaired Student's t test and analysis of variance post hoc test Bonferroni/Dunn, using Stat View (Abacus Concepts, Inc., Berkeley, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of S847-PO4 nNOS and Phosphorylation of nNOS by CaMKII—The phosphorylation of nNOS at Ser847 has been detected by Western blotting of brain lysates (23), and CaMKII{alpha} has been implicated in the phosphorylation of nNOS at this position (23, 24). We confirmed by an in vitro [{gamma}-32P]ATP labeling assay that CaMKII{alpha} phosphorylates recombinant nNOS expressed in Escherichia coli (Fig. 1A). To study the mechanism of NMDAR-induced regulation of nNOS phosphorylation at Ser847, we used an affinity-purified anti-phosphopeptide antiserum, GR847, specific to the Ser847-PO4 nNOS form. GR847 recognizes nNOS phosphorylated in vitro by CaMKII{alpha} (Fig. 1B, lane 2) but did not detect either unphosphorylated nNOS (Fig. 1B, lane 1) or nNOS phosphorylated by Akt or protein kinase C (Fig. 1B, lanes 3 and 4). Reprobing the blot for total nNOS showed that each lane contained an equal amount of nNOS and demonstrated that GR847 does not recognize unphosphorylated nNOS. GR847 specifically recognized in neuronal lysates two nNOS forms identified previously in rodent brain, the 155-kDa nNOS{alpha} and to a lesser extent the 135-kDa nNOS{beta} (1, 23) (Fig. 1C). We confirmed the identity of the major 155 kDa {alpha} band as nNOS by reprobing the blot with nNOS antibody (Transduction Laboratories) (Fig. 1D), thereby demonstrating the specificity of GR847 for detecting Ser847-PO4 nNOS.



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  		FIG. 1.
Phosphorylation of nNOS at Ser847 by CaMKII and detection with a phosphopeptide antiserum specific for Ser847-PO4. A, autoradiogram of SDS-PAGE of 32P-nNOS labeled in the presence of [{gamma}-32P]ATP and CaMKII{alpha}. B, Western blots showing the specificity of the antibody GR847 for nNOS phosphorylated by CaMKII{alpha}. nNOS was incubated with the designated kinases plus nonradioactive ATP, or without any enzyme as a control. Lane 1, no enzyme, control; lane 2, CaMKII{alpha}; lane 3, AKT; lane 4, protein kinase C. A Western blot for total nNOS is given for each kinase reaction showing equivalent levels of nNOS protein loading. The specificity of the antibody GR847 for phosphorylated nNOS was demonstrated using extracts from cultured primary cortical neurons. Western blots of cultured cortical neurons using antibody GR847, in which the cell lysate was fractionated by 10% SDS-PAGE (C) and reprobing of C using a nNOS antibody (Transduction Laboratories) (D), are shown.

 
Localization of Ser847-PO4 nNOS at Synapses in Hippocampal Neurons—Interaction of nNOS with PSD95 localizes nNOS{alpha} at synapses (19, 20, 37, 38). nNOS{alpha} also interacts in the cytoplasm with the protein Capon (39) and with carboxyl-terminal-binding protein (CtBP) (40) and synapsin (41). The nNOS splice variant, nNOS{beta}, has full catalytic activity and is expressed predominantly during development (42, 43). In contrast to nNOS{alpha}, nNOS{beta} lacks the PDZ targeting domain that interacts with PSD95, and thus its subcellular distribution is likely to be mainly nonsynaptic and cytoplasmic. Previous studies have shown that synaptic puncta, identified by synaptophysin staining, were associated with nNOS{alpha} (37). We extended these studies by determining the extent of colocalization of Ser847-PO4 nNOS with PSD95 and the synaptic marker, synaptophysin, in E18 rat hippocampus-derived neuronal cultures 21 days in vitro.

Ser847-PO4 nNOS and PSD95 colocalized in spine-like structures on dendrites (arrows, Fig. 2, A-C), consistent with an interaction between Ser847-PO4 nNOS and PSD95. In cultured neurons with a pyramidal morphology, Ser847-PO4 nNOS appeared as brightly stained puncta on dendritic structures that resemble spines. Ser847-PO4 nNOS also colocalized with the synaptic marker, synaptophysin (arrow, Fig. 2, D-F). The labeling also occurred on the edges of the soma and diffusely in the cytoplasm. Therefore, in addition to an apparent concentration at synapses in spine-like structures along the dendrites, Ser847-PO4 nNOS was also observed in the cytoplasm. In agreement with tethering of nNOS to the NMDAR by PSD95, staining for PSD95 and total nNOS also revealed (Fig. 2, G-I) the punctate localization of these proteins in spine-like structures along dendrites (arrows).



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  		FIG. 2.
Synaptic localization of Ser847-PO4 nNOS. Confocal micrograph images of hippocampal neurons 21 days in vitro labeled with GR847 and costained for synaptic markers PSD95 and synaptophysin are shown. A-C show, respectively, Ser847-PO4 nNOS (red), PSD95 (green), and the colocalization of PSD95 and Ser847-PO4 nNOS (yellow). Magnifications of dendritic segments shown below reveal the colocalization of PSD95 and Ser847-PO4 nNOS in spine-like structures, as indicated by the arrows. D-F show, respectively, Ser847-PO4 nNOS (red), synaptophysin (green), and the colocalization of synaptophysin and Ser847-PO4 nNOS (yellow). Sites of colocalization of Ser847-PO4 nNOS and synaptophysin are indicated by arrows. G-I show, respectively, nNOS (red), PSD95 (green), and the colocalization of PSD95 and nNOS (Zymed Laboratories) (yellow). Magnifications of dendritic segments shown below reveal colocalization of PSD95 and nNOS in spine-like structures, as indicated by the arrows. Sites of colocalization of nNOS and PSD95 are indicated by arrows. Scale bar = 20 µm.

 
NMDAR-dependent Phosphorylation of Ser847 by CaMKII—The manner of stimulation of the NMDAR with glutamate determines distinct types of synaptic changes or excitotoxicity (44-46). Also, NMDAR activation in cultured neurons by glutamate induces a Ca2+-dependent activation of CaMKII that regulates gene expression (47). This raised the possibility that glutamate acting through the NMDAR might induce CaMKII phosphorylation of Ser847. We examined the regulation of Ser847-PO4 nNOS after neuron treatment with 5 µM glutamate, a concentration that is considered to fall within a physiological range (48). Neuron stimulation with 5 µM glutamate showed a robust increase in phosphorylation at Ser847 (Fig. 3A, lanes 4 and 5). Phosphorylation at Ser847 was observed 15 min after stimulation and continued to increase from 30 o 60 min (Fig. 3A, lanes 3-5) relative to the unstimulated control (Fig. 3B, lane 1). Reprobing of this blot for total nNOS confirmed equal loading of proteins. Quantification of several experiments showed that at 60 min of treatment, Ser847-PO4 had increased up to 5-fold relative to its constitutive level (Fig. 3B).



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  		FIG. 3.
Glutamate stimulates nNOS phosphorylation at Ser847 dependent on the NMDAR and CaMKII. A, activity-dependent time course of increase in phosphorylation of nNOS at Ser847. Lane 1, no glutamate control; lane 2, 5 µM glutamate, 5 min; lane 3, 5 µM glutamate, 15 min; lane 4, 5 µM glutamate, 30 min; lane 5, 5 µM glutamate, 60 min. B, quantitation of A from experiments that were repeated four times with similar results. Asterisks represent the means ± S.E. n = 4; p < 0.001 compared with no glutamate control. C, treatment with glutamate for 60 min increased phosphorylation at Ser847. Lane 1, no glutamate control; lane 2, 5 µM glutamate; lane 3, 5 µM glutamate plus 300 µM KN93; lane 4, 300 µM KN93; lane 5, 5 µM glutamate plus 100 µM D-2-amino-5-phosphonopentanoate. A Western blot of nNOS for each sample, shown below, was used to determine the level of nNOS. D, OA inhibition of PP1 but not PP2A elevates endogenous levels of Ser847-PO4 nNOS. Neuronal cultures were incubated for 1 h in the presence or absence of OA. Lane 1, no OA, control; lane 2, 1 nM OA; lane 3, 10 nM OA; lane 4, 100 nM OA.

 
We next distinguished between two possibilities that could account for the NMDAR-mediated increase in phosphorylation at Ser847: 1) the increase in phosphorylation may be caused by the elevated activity of kinases, or 2) the increase could result from the inhibition of the activities of phosphatases. We distinguished these possibilities by blocking the activity of CaMKII, a kinase that was likely to mediate NMDAR-dependent phosphorylation at Ser847. KN93, a specific inhibitor of CaMKII, blocked the 5 µM glutamate-induced increase in Ser847-PO4 (Fig. 3C, lane 3) relative to the control, stimulation with 5 µM glutamate alone (Fig. 3C, lane 2). As a further control, KN93 alone did not have any effect on the phosphorylation levels at Ser847 (Fig. 3C, lane 4), suggesting that the constitutive pool of Ser847-PO4 nNOS is either relatively stable or not dependent on CaMKII for its maintenance. D-2-Amino-5-phosphonopentanoate, an NMDAR antagonist, blocked the increase, indicating that this phosphorylation of Ser847 is mediated by an NMDAR-dependent mechanism (Fig. 3C, lane 5). The NMDAR-dependent modulation of phosphorylation at Ser847 observed here is consistent with our previous studies (30) that showed that NMDAR modulation of phospho-nNOS levels was independent of AMPA receptor or voltage-sensitive calcium channel activity.

Protein phosphorylation is maintained in a steady state by the concurrent actions of kinases and phosphatases, and the levels of Ser847-PO4 observed in control cells may reflect the outcome of opposing phosphorylation and dephosphorylation. To understand the basis of Ser847 constitutive phosphorylation, we measured Ser847-PO4 levels in the presence of okadaic acid (OA), an inhibitor that blocks two phosphatases, PP1 and PP2A. We took advantage of the fact that different OA concentrations are required to inhibit PP2A (Ki = 0.1 nM) and PP1 (Ki = 15 nM). We exposed cultured neurons to different levels of OA for 1 h. Concentrations of OA that inhibit PP1 (10 and 100 nM) increased Ser847-PO4 up to 4-fold (Fig. 3D, lanes 3 and 4) relative to an untreated control (Fig. 3D, lane 1). OA at 1 nM, which inhibits only PP2A, in contrast did not induce an increase in Ser847-PO4 nNOS levels (Fig. 3D, lane 2). These results indicate that PP1 decreases the level of constitutive phosphorylation of nNOS at Ser847.

NMDAR-dependent Ser847 Dephosphorylation by PP1—To prevent excitotoxic death of cultured neurons, it is necessary to limit the exposure of these neurons to concentrations of glutamate in the low micromolar ranges, although it is estimated that the peak concentration of glutamate at synapses in vivo to be in the millimolar range (49, 50). Exposure of NMDARs to elevated levels of glutamate is the primary cause of neuronal death after traumatic injuries, including stroke, seizures, and mechanical trauma (51, 52). Stimulation of cultured neurons with 100 µM glutamate and to higher glutamate concentrations mimics these pathological effects (14, 25, 53-56). A pathological dose of glutamate, 100 µM, induced the dephosphorylation of nNOS at Ser847 (Fig. 4A, lane 2) relative to the control (Fig. 4A, lane 1). Dephosphorylation of Ser847-PO4 by this excitotoxic dose of glutamate was blocked by either 30 µM MK801 (dizocilpine) or 10 nM OA, indicating a dependence of the dephosphorylation on NMDAR and PP1 activity (Fig. 4A, lanes 3 and 4). These results are consistent with our previous studies of a decrease in the overall level of nNOS phosphorylation after a pathological stimulation by glutamate involves PP1 (30). Quantification of the data revealed that glutamate treatment at 100 µM for 60 min reduced the phosphorylation of nNOS at Ser847 up to 50% of its constitutive level (values represent means ± S.E., n = 4, p < 0.001) (Fig. 4B). We conclude that in contrast to the effects of physiological levels of glutamate, the activation of the NMDAR by a pathological concentration of glutamate leads to nNOS dephosphorylation at Ser847 via NMDAR regulation of phosphatase activity.



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  		FIG. 4.
PP1 dephosphorylation of Ser847-PO4. A, neuron treatment with 100 µM glutamate reduced the basal level of Ser847-PO4 nNOS, and this reduction was blocked when treatment with 100 µM glutamate was accompanied by 30 µM MK801 or 0.01 µM OA. B, quantitation of the effects in A of glutamate and OA. The levels of nNOS phosphorylation were normalized to the level of the no NMDA control. The asterisk represents the means ± S.E. n = 4; p < 0.001 compared with the no glutamate control.

 
Bidirectional Regulation of Phosphorylation at Ser847 by Glutamate—Stimulation of the NMDAR by low and high concentrations of glutamate resulted in a differential regulation of phosphorylation of nNOS at Ser847 dependent on the concentration of glutamate. To determine the concentration of glutamate at which dephosphorylation rather than phosphorylation is the outcome, we examined the levels of Ser847-PO4 nNOS after treatment of neurons with concentrations of glutamate increasing from 5 to 500 µM. In this experiment and all other experiments, we supplemented the medium with 20 µM glycine, a coagonist. This eliminated any ambiguity that varying glycine concentrations, rather than glutamate determined the transition point. Stimulation of cortical neurons with 5 and 50 µM glutamate increased phosphorylation of nNOS at Ser847 (Fig. 5A, lanes 2 and 3) relative to the control (Fig. 5A, lane 1), whereas stimulation with 100-500 µM glutamate decreased phosphorylation (Fig. 5A, lanes 4-6). Treatment with 50 µM glutamate appears to be less stimulatory than with 5 µM glutamate, suggesting that the transition lies between 5 and 50 µM glutamate. Dephosphorylation was clearly manifested by treatment with 100 µM glutamate (Fig. 5A, lane 3). Analysis of Ser847-PO4 levels from several Western blots (Fig. 5B) confirmed that stimulation of the NMDAR by high glutamate dephosphorylated Ser847-PO4.



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  		FIG. 5.
Excitotoxic stimulation of the NMDAR reverses the increase in phosphorylation resulting from physiologic stimulation. A, glutamate modulates the phosphorylation of nNOS at Ser847. The levels of Ser847-PO4 were determined by Western blotting after 60 min of neuron stimulation with no drug (lane 1), 5 µM glutamate (lane 2), 50 µM glutamate (lane 3), 100 µM glutamate (lane 4), 250 µM glutamate (lane 5), or 500 µM glutamate (lane 6). B, quantitation of A from experiments that were repeated three times, with similar results. C, phosphorylation of nNOS at Ser847 induced by NMDAR stimulation by physiologic glutamate is reversed by glutamate overstimulation of the NMDAR. Treatment of cells with 5 µM glutamate increased phosphorylation at nNOS Ser847 during the period 15-60 min (lanes 2-6). The addition of 500 µM glutamate for 15 min (lane 7) or 30 min (lane 8) to cells pretreated for 30 min with 5 µM glutamate reversed the low agonist induced increase of phosphorylation at Ser847. D, we divided the results of the Westerns into two groups, those experiments for 5 µM glutamate for 60 min and those experiments for 5 µM glutamate for 30 min plus 500 µM glutamate for an additional 30 min. Quantification showed that the treatment with 5 µM glutamate displayed an increase in phosphorylation of nNOS at Ser847 which was significantly reversed by treatment with 500 µM glutamate. The asterisk represents the means ± S.E., n = 4*, p < 0.0024 by analysis of variance.

 
Sequential Phosphorylation and Dephosphorylation of Ser847—Activation of the NMDAR by low and high concentrations of glutamate induces increases and decreases, respectively, in nNOS phosphorylation which involve different signaling pathways. We therefore asked whether the increase in Ser847-PO4 nNOS induced by 5 µM glutamate could be reversed by subsequent treatment with 500 µM glutamate. Treatment with 500 µM glutamate was chosen instead of the 100 µM glutamate used in the previous experiments because this concentration showed a more robust dephosphorylation of nNOS at Ser847. We added 500 µM glutamate to cells that had been prestimulated with 5 µM glutamate for 30 min. Ser847-PO4 levels in these cells were compared with phosphorylation in cells that had been maintained in 5 µM glutamate for 45 and 60 min (Fig. 5C, lanes 5 and 6 compared with lanes 7 and 8). Strikingly, the increase in phosphorylation of Ser847 which was induced by 5 µM glutamate persisted in the low glutamate-treated control cells but was reversed to the basal level after treatment with 500 µM glutamate. Fig. 5D confirmed that the reversal of phosphorylation at Ser847 by 500 µM glutamate for 30 min was highly significant relative to stimulation with 5 µM glutamate.

Cellular Analysis of NOS Activity and Levels of Ser847-PO4 nNOS—To analyze the role of Ser847 phosphorylation in the NMDAR-mediated activation of nNOS, a confocal microscope-based assay was used to correlate the production of NO with the level of Ser847-PO4 nNOS in individual neurons. We demonstrated previously that NT, detected by an anti-NT antibody, can be used as a measure of NO accumulation after the activation of nNOS by the NMDAR (30). The levels of Ser847-PO4 nNOS and NT were measured in individual cells by quantitative fluorometry, as described previously (30). We focused on NMDAR-mediated changes in NT and Ser847-PO4 in dendrites and spines because these appear to be the more physiologically relevant subcellular compartments for NO actions.

Neurons in the hippocampal cultures 15-17 days in vitro were stimulated with 500 µM NMDA. NT and Ser847-PO4 levels in individual cells were compared under three conditions: 1) in unstimulated control cells; 2) in cells stimulated with NMDA either for 1 h or with NMDA for 1 h followed by a 2-h incubation without agonist; and 3) in cells stimulated for 1 h with NMDA in the presence MK801 to block NMDAR activity. In NMDA-treated cells (Fig. 6, D-I), NT levels were increased, and Ser847-PO4 nNOS levels were decreased relative to untreated controls (Fig. 6, A-C). MK801 significantly reduced labeling for NT and prevented the decrease in Ser847-PO4 nNOS levels after NMDA treatment (Fig. 6, J-L). Furthermore, NT and Ser847-PO4 nNOS showed strong colocalization in dendrites and in the cell bodies of NMDA-treated neurons (Fig. 6, E and H) compared with control or NMDA plus MK801-treated cells (Fig. 6, B and K). This was true for cells that were positive for MAP-2, a neuronal marker, indicating that the dependence of NT positivity and Ser847-PO4 levels on the activation of NMDAR had been observed in neurons rather than other cell types in the culture (Fig. 6, A, D, G, and J).



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  		FIG. 6.
Correlation of reduced nNOS Ser847-PO4 phosphorylation and increased NT positivity in NMDA-treated hippocampal neurons. A-L, confocal micrograph images of representative hippocampal neurons, including neurons treated with NMDA, control cells (no NMDA), and cells treated with NMDA plus MK801. The neuronal marker MAP-2 (blue), Ser847-PO4 nNOS (red), and NT (green) were visualized as described under "Experimental Procedures." A, D, G, and J show all three markers, whereas B, E, H, and K show Ser847-PO4 nNOS and NT. C, F, I, and L show NT only. NT levels significantly increased, and Ser847-PO4 significantly decreased for cells stimulated with NMDA (E and H). In control cells and in cells treated with NMDA plus MK801 NT was reduced (scale bar = 20 µm). M and N, quantitation of markers in dendrites of cells identified as neurons by MAP-2 staining showed that NMDA induction of NT and Ser847-PO4 nNOS was significantly reduced by MK801. The graph presents the averages of four repetitions of the experiment.

 
Quantification of NT positivity showed 2.5 and 8-fold increases for neurons treated with NMDA for 1 h and for neurons treated for 1 h followed by a 2-h incubation without agonist, respectively (Fig. 6N). In these same cells Ser847-PO4 decreased 50 and 60%, respectively, relative to the control (Fig. 6M). These results demonstrate that nNOS activation, as reflected by NT formation, correlates with NMDAR-mediated dephosphorylation of Ser847-PO4 nNOS. A dependence of these changes on NMDAR function in inducing these changes was confirmed by MK801 blockage of the effects. These results demonstrate that following NMDAR treatment, nNOS activation as reflected by NT formation is accompanied by dephosphorylation of nNOS at Ser847.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Synaptic activity regulates production of NO, catalyzed by nNOS through pathways involving the phosphorylation of nNOS. Protein kinase A, protein kinase C, and CaMKII have been demonstrated to phosphorylate nNOS protein (21-23). The NMDAR increases the activity of CaMKII (57) which in turn may phosphorylate nNOS at Ser847, within an inhibitory hinge loop region adjacent to a binding sequence for Ca2+-CaM. The inhibitory loop links the nNOS carboxyl-terminal reductase domain to the amino-terminal oxygenase domain. Binding of Ca2+-CaM adjacent to the inhibitory loop is proposed to displace the loop and to increase the flow of electrons from the reductase domain to the oxygenase domain, increasing the rate of enzyme catalysis. Hinge loop phosphorylation by CaMKII may inhibit binding of Ca2+-CaM (58-60) and reduces enzyme activity. Because both the activator, Ca2+-CaM, and the inhibitory kinase, CaMKII, are Ca2+-regulated, nNOS activity is likely to be under a complex form of Ca2+ control. Furthermore, because Ser847-PO4 is located within the autoinhibitory loop, it may function to prevent the displacement of the loop even in the presence of high concentrations of Ca2+-CaM. These Ca2+-dependent regulatory mechanisms suggest that fluxes of Ca2+ through the NMDAR may regulate nNOS that is tethered in proximity to the channel.

In this study, we show that stimulation of the NMDAR provides a complex, bidirectional control of phosphorylation at Ser847, with the outcome dependent on the concentration of the agonist. We have shown previously that NMDAR activation by excitotoxic concentrations of agonist induces an overall dephosphorylation of nNOS (30). Here, we extend these results by demonstrating that stimulation of culture primary cortical neurons with a physiologic level of glutamate results in an NMDAR-mediated increase in phosphorylation at Ser847 of nNOS. In contrast, an excitotoxic stimulus decreases phosphorylation. We also show that the increase after the low agonist stimulation could be reversed by the pathological concentration of glutamate, indicating bidirectional control.

A significant proportion of Ser847-PO4 nNOS is synaptic and thus in proximity to the NMDAR, which is implicated directly in the control of nNOS phosphorylation. However, substantial levels of Ser847-PO4 nNOS are also observed in the cell body. The pattern of localization of Ser847-PO4 nNOS also appears to be complex; nevertheless its synaptic colocalization with PSD95 and the NMDAR supports a role for synaptically induced changes. Analyses by immunocytochemistry of dynamic changes in Ser847-PO4 and NT levels in individual cells treated with glutamate or glutamate plus MK801 also support such an NMDAR regulatory function.

Several studies have examined the increase in intracellular free Ca2+ in cultured neurons under varying conditions of excitatory stimulation (10, 11, 56, 61, 62). The magnitude of the increase of Ca2+ correlated with the increase in the concentration of glutamate (56). We have demonstrated previously that NMDAR regulation of the overall phosphorylation of nNOS is independent of AMPA receptors or L-type voltage-sensitive calcium channels (30). In the current studies, we have demonstrated an NMDAR-dependent increase of phosphorylation at Ser847 in cortical cultured neurons treated with 5 µM glutamate. Because phosphorylation at this site is thought to reduce the activity of nNOS, we speculate that the increase of phosphorylation of nNOS at Ser847 limits the activity of the enzyme following a physiologic excitatory stimulus and prevents the accumulation of toxic levels of NO. Activation of both the NMDAR and CaMKII is required to increase phosphorylation of nNOS at Ser847 because phosphorylation was blocked by D-2-amino-5-phosphonopentanoate or KN93. Given the established regulation of CaMKII by Ca2+, this suggests that low agonist stimulation of the NMDAR activates CaMKII via the formation and the binding of Ca2+-CaM, enabling CaMKII to phosphorylate Ser847. This is the first direct demonstration of an activity-regulated control of nNOS Ser847 phosphorylation in cultured neurons dependent on NMDAR activation of the downstream NMDAR effector, CaMKII.

The use of the inhibitors OA and KN93 revealed the dynamic and differential contributions of PP1 and CaMKII to the regulation of Ser847-PO4 steady-state levels. Because Ser847-PO4 levels were elevated by OA treatment, pools of PP1 and a constitutively active kinase, potentially CaMKII, capable of phosphorylating Ser847, must operate in the steady state. A second but not mutually exclusive possibility is that treatment with OA could enhance the autocatalytic activation of phosphorylated CaMKII.

Overstimulation of the NMDAR leads to pathological accumulation of NT (28, 30, 63-65). NT accumulation might result if a pathologic stimulation of NMDAR overrides the inhibitory action of Ser847 phosphorylation. Indeed, we show that strong (100-500 µM glutamate) stimulation of the NMDAR decreases the constitutive levels of nNOS Ser847-PO4. MK801 and OA blocked the dephosphorylation of Ser847, indicating an NMDAR-associated mechanism working through the activity of PP1. Thus the NMDAR can exert a bidirectional regulation of phosphorylation of nNOS at Ser847, depending on the magnitude of the agonist stimulus. We show that in cultured neurons, the transition from phosphorylation to dephosphorylation takes place in the 5-50 µM glutamate range. This makes it likely that pathological, excitotoxic stimulation of the NMDAR in vivo may also override inhibitory effects of Ser847 phosphorylation. However, further studies in vivo will be necessary to establish this point for excitatory insult in tissues.

Our work shows that Ser847 phosphorylation is limited in the steady state by PP1. The activity of PP1 is controlled by inhibitory proteins (DARPP32 and Inhibitor-1) whose activities themselves are controlled by phosphorylation (66-71). The NMDAR-mediated dephosphorylation of nNOS at Ser847 may result from Ca2+ influx through the NMDAR pore which activates PP2B/calcineurin, which may in turn dephosphorylate DARPP32 or Inhibitor-1, disinhibiting PP1 (69).

The NMDAR-mediated increase in phosphorylation at Ser847 after low agonist treatment was reversed by a subsequent high agonist stimulus. The mechanism that underlies this interesting phenomenon is currently under investigation and may involve differential activation of kinases and phosphatases following differential stimulation of the NMDAR.

A microscope-based immunocytochemical assay was used to assess NMDAR-mediated changes in NO levels in individual neurons after the agonist-induced decrease of Ser847-PO4. A stimulation of the NMDAR sufficient to decrease Ser847-PO4 resulted in a time-dependent accumulation of NT, a marker for NO production. The time-dependent increase of NO, as reflected in elevated levels of NT in individual cells, correlated with the decrease of nNOS Ser847 phosphorylation measured in the same cell. Thus, the activation of nNOS after Ser847-PO4 dephosphorylation by high agonist treatment could account for the overproduction of NO during pathogenesis. Although a moderate activation of the NMDAR may briefly induce the activity of nNOS, the subsequent accumulation of NO is likely to be limited by the eventual phosphorylation at Ser847, which represses catalysis and curtails NO production. The current work shows that this limitation is nonetheless vulnerable to high, excitotoxic stimulation.


   FOOTNOTES
 
* This work was supported in part by Grant R01 AG13620 from the NIA, National Institutes of Health, and by pilot project funds from New York University/NIEHS, National Institutes of Health, Grant ES 00260. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Aaron Diamond Foundation postdoctoral research fellow, United Negro College Fund-Merck fellow, and an associate of the Howard Hughes Medical Institute. Supported by National Institutes of Health Training Grant NS 07457-04. Back

§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, Dept. of Biochemistry, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-5774; Fax: 212-683-8453; E-mail: edward.ziff{at}med.nyu.edu.

1 The abbreviations used are: nNOS, neuronal nitric-oxide synthase; Ca2+-CaM, calcium-calmodulin; CaMKII, calcium-calmodulin protein kinase II; MAP, microtubule-associated protein; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; NO, nitric oxide; NT, nitrotyrosine; OA, okadaic acid; PP1 protein phosphatase 1; PP2B, protein phosphatase 2B; PSD95, postsynaptic density 95; AMPA, D-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Dennis Stuehr for providing the affinity-purified recombinant nNOS protein and Drs. Amanda M. Brown-Rameau and Charu Misra for helpful comments and careful reading of the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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