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To whom correspondence should be addressed: Edward B. Ziff, Howard Hughes Medical Institute, NYU School of Medicine, Dept. of Biochemistry, 550 First Avenue, New York, NY 10016. Tel: (212) 263-5774. Fax: (212) 683-8453. Email: edward.ziff@med.nyu.edu The abbreviations used are: nNOS, neuronal nitric oxide synthase; NO, nitric oxide; NMDAR, N-methyl-D-aspartate receptor; NT, nitrotyrosine; Ca, calcium; Ca-CaM, calcium calmodulin; OA, Okadaic acid; PP1 protein phosphatase 1; PP2B, protein phosphatase 2B; CaMKII, calciumcalmodulin protein kinase II; PSD95, post synaptic density 95; APV, D-2-amino-5-phosphonopentanoate; MK801, Dizocilpine, LTP, long term potentiation; LTD, long term depression. JBC Papers in Press. Published on January 13, 2004 as Manuscript M311103200

Suppression of PSD95 expression blocks NMDAR and Ca 2+ -dependent nNOS activation, and uncoupling of the NMDAR from PSD-95 suppresses NMDAR signaling (14,20). 2+ ] following NMDAR activation stimulate nNOS by promoting the binding of Ca 2+ -calmodulin (Ca 2+ -CaM). In addition, it has been shown that the activity of nNOS undergoes complex regulation by phosphorylation (21)(22)(23). Of particular note, the protein kinase, CaMKII, phosphorylates recombinant nNOS at S847, which reduces nNOS activity by inhibiting the binding of Ca 2+ -CaM (23,24). However, the NMDAR-induced mechanism of regulation of nNOS by phosphorylation at specific residues remains largely unknown.

Transient elevations in intracellular [Ca
We have shown previously that mutations at the apex of the pore of the NMDAR NR1 subunit that block Ca 2+ entry through the channel reduce NMDAR-dependent excitotoxicity in heterologous cells and neurons (25). Since Ca 2+ -dependent activation of nNOS by the NMDAR has been linked to NMDAR excitotoxic effects (6,7,20,(26)(27)(28)(29)(30), we by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 5 have also analyzed the NMDAR-mediated mechanism of modulation of phosphorylation of nNOS. We have shown that following 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 S847, a modification implicated in the regulation of nNOS.
We show that the NMDAR induces a novel bidirectional control of phosphorylation of nNOS at S847. Using an antibody specific for S847-PO 4 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 S847. Stimulation with a low concentration of glutamate resulted in a time-dependent phosphorylation of nNOS at S847 by CaMKII. In contrast high pathological concentrations of glutamate stimulated dephosphorylation of S847-PO4. The first of these effects may share similarity with a form of long term potentiation (LTP) induced by theta pulse stimulation of hippocampal neurons that promotes the activation of CaMKII (31,32) while the second may relate to excitotoxicity following stroke and 7

EXPERIMENTAL PROCEDURES
Cortical and Hippocampal Cell Cultures -Cultured E18 rat cortical and hippocampal neurons were prepared as described previously (Rameau et al., 2000). Briefly, after dissociation, cortical cells were plated on poly-L-lysine-coated dishes or coverslips in MEM medium supplemented with glutamine, 10% fetal bovine serum, 0.45% glucose and 1mM pyruvate. Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 for 3 hr, 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 reliably identified 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 were fixed with 100% methanol at 0°C for 15 min.
After several washes with PBS, the cells were incubated in 2% BSA for 1 hr and subsequently stained with a polyclonal antibody to NT (Upstate Biotechnology) and a monoclonal antibody to MAP2 (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). Immuno-reactivity was examined using a Nikon PCM2000 confocal laser-scanning microscope.
Quantitation of NT and pS847 in Individual Neurons -C-imaging Systems software was used to quantify the fluorescence intensity of the antibody-conjugated-fluorochromes in 8 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 S847-PO 4 nNOS were measured by eliminating the cell body for quantitation from the image, using a software-based "erasure" method. The levels of S847-PO 4 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 per coverslip was averaged to obtain a population mean (presented as mean ±SEM). Statistical significance of differences between means was calculated using Student's t-test.
Immunoblotting -The cells were washed twice with cold PBS on ice, scraped and transferred into 1.5 ml microcentrifuge tubes that were spun for 1 min at maximum speed. Antibodies -Anti-S847-PO 4 nNOS serum was generated by immunizing rabbits with a synthetic phosphopeptide synthesized by the Tufts University Core Facility (Boston) 9 corresponding to 11 amino acids in nNOS NH 2 -CKVRFN(S-PO 4 )VSSYS-COOH.
Antibodies were generated and affinity purified by Covance (Princeton, NJ). Anti-PSD95 monoclonal antibody was purchased from Upstate (Lake Placid, NY), and monoclonal anti-synaptophysin from Sigma and polyclonal anti-MAP-2 from (Santa Cruz; Santa Cruz, CA).

Detection of S847-PO 4 nNOS and phosphorylation of nNOS by CaMKII -
The phosphorylation of nNOS at S847 has been detected by Western blotting of brain lysates (23), and the Ca 2+ -CaM-activated protein kinase IIα (CaMKIIα) has been implicated in the phosphorylation of nNOS at this position (23,24). We confirmed by an in vitro γ-32 P-ATP labeling assay that CaMKIIα phosphorylates recombinant nNOS expressed in E. coli (Fig. 1A). To study the mechanism of NMDAR-induced regulation of nNOS phosphorylation at S847, we used an affinity purified anti-phosphopeptide antiserum, GR847 specifically recognized in neuronal lysates two nNOS forms previously identified in rodent brain, the 155-kDa nNOSα, and to a lesser extent the 135-kDa nNOSβ (1,23) ( Fig. 1C). We confirmed the identity of the major 155 kDα band as nNOS by reprobing the blot with nNOS antibody (Transduction Lab) (Fig. 1D), thereby demonstrating the specificity of GR847 for detecting S847-PO 4 nNOS.

Localization of S847-P0 4 nNOS at synapses in hippocampal neurons -Interaction
of nNOS with PSD95 localizes nNOSα at synapses (19,20,37,38). nNOSα 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, nNOSβ, has full catalytic activity and is expressed predominantely during development (42,43). In contrast to nNOSα, nNOSβ lacks the PDZ targeting domain that interacts with PSD95, and thus its subcellular distribution is likely to be mainly non-synaptic and cytoplasmic. Previous studies have shown that synaptic puncta, identified by synaptophysin staining, were associated with nNOSα (37). We extended these studies by determining the extent of colocalization of S847-PO 4 nNOS with PSD95 and the synaptic marker, synaptophysin, in We next distinguished between two possibilities that could account for the NMDAR mediated increase in phosphorylation at S847: 1) the increase in phosphorylation may be due to 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 S847. KN93, a specific inhibitor of CaMKII, blocked the 5 µM glutamate-induced increase in S847-PO 4 (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 S847 (Fig. 3C, lane 4), suggesting that the constitutive pool of S847-PO 4 nNOS is either relatively stable or not dependent on CaMKII for its maintenance. APV, an NMDAR antagonist, blocked the increase, indicating that this phosphorylation of S847 is mediated by an NMDARdependent mechanism (Fig. 3C, lane 5). The NMDAR-dependent modulation of 13 phosphorylation at S847 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 (VSCC) activity.
Protein phosphorylation is maintained in a steady state by the concurrent actions of kinases and phosphatases, and the levels of S847-PO 4 observed in control cells may reflect the outcome of opposing phosphorylation and dephosphorylation. To understand the basis of S847 constitutive phosphorylation, we measured S847-PO 4 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 (K i =0.1 nM) and PP1 (K i =15 nM). We exposed cultured neurons to different levels of OA for 1 hr. Concentrations of OA that inhibit PP1 (10 nM and 100 nM) increased S847-PO 4 up to 4 fold (Fig. 3D, lane 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 S847-PO 4 nNOS levels (Fig. 3D, lane 2). These results indicate that PP1 decreases the level of constitutive phosphorylation of nNOS at S847.
NMDAR-dependent S847 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 following 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)(54)(55)(56). A pathological 14 dose of glutamate, 100 µM, induced the dephosphorylation of nNOS at S847, (Fig. 4A, lane 2) relative to the control (Fig. 4A, lane 1). Dephosphorylation of S847-PO 4 by this excitotoxic dose of glutamate was blocked by either 30 µM MK801 or 10nM 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 following a pathological stimulation by glutamate involves PP1 (30). Quantification of the data revealed that glutamate treatment at 100 µM for 60 minutes reduced the phosphorylation of nNOS at S847 up to 50% of its constitutive level (values represent means ±SEM, 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 S847 via NMDAR regulation of phosphatase activity.  (Fig. 5A, lanes 2 and 3) relative to the control (Fig. 5A, lane 1), whereas by guest on March 24, 2020 http://www.jbc.org/ Downloaded from stimulation with 100 to 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 S847-PO 4 levels from several Western blots (Fig. 5B)  We added 500 µM glutamate to cells that had been pre-stimulated with 5 µM glutamate for 30 min. S847-PO 4 levels in these cells were compared to phosphorylation in cells that had been maintained in 5 µM glutamate for 45 and 60 min (Fig. 5C, lanes 5 and 6 compared to, lanes 7 and 8). Strikingly, the increase in phosphorylation of S847 that was induced by 5 µM glutamate persisted in the low glutamate treated control cells, but was reversed to the basal level following treatment with 500 µM glutamate. Figure 5D confirmed that the reversal of phosphorylation at S847 by 500 µM glutamate for 30 min was highly significant relative to stimulation with 5 µM glutamate . were decreased relative to untreated controls ( Fig. 6A-C). MK801 significantly reduced labeling for NT and prevented the decrease in S847-PO 4 nNOS levels following NMDA treatment (Fig. 6 J-L). Furthermore, NT and S847-PO 4 nNOS showed strong colocalization in dendrites and in the cell bodies of NMDA treated neurons ( Fig. 6E and H) compared to control or NMDA plus MK801 treated cells (Fig. 6B and 6K). This was true for cells that were positive for MAP-2, a neuronal marker, indicating that the dependence 17 of NT positivity and S847-PO 4 levels on the activation of NMDAR had been observed in neurons rather than other cell types in the culture (Fig. 6A, D, G and J).
Quantification of NT positivity showed 2.5 and 8-fold increases for neurons treated with NMDA for 1h, and for neurons treated for 1h followed by a 2h incubation without agonist, respectively (Fig. 6N). In these same cells S847-P0 4 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 S847-PO 4 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 S847. In this study, we show that stimulation of the NMDAR provides a complex, bidirectional control of phosphorylation at S847, with the outcome dependent on the concentration of the agonist. We have previously shown 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 S847 of nNOS. In contrast, an excitotoxic stimulus decreases phosphorylation. We also show that the increase following the low agonist stimulation could be reversed by the pathological concentration of glutamate, indicating bidirectional control.
A significant proportion of S847-PO 4 nNOS is synaptic, and thus in proximity to the NMDAR, which is directly implicated in the control of nNOS phosphorylation.
However, substantial levels of S847-PO 4 nNOS are also observed in the cell body. The pattern of localization of S847-PO 4 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 S847-PO4 and NT levels in individual cells treated with glutamate or glutamate plus MK801 also support such a NMDAR regulatory function.
Several studies have examined the increase in intracellular free Ca 2+ in cultured neurons under varying conditions of excitatory stimulation (10,11,56,61,62). The magnitude of the increase of Ca 2+ correlated with the increase in the concentration of glutamate (56). We have previously demonstrated that NMDAR regulation of the overall phosphorylation of nNOS is independent of AMPA receptors or L-type voltage sensitive calcium channels (VSCC) (30). In the current studies, we have demonstrated an NMDAR-dependent increase of phosphorylation at S847 in cortical cultured neurons treated with 5 µM glutamate. Since phosphorylation at this site is thought to reduce the activity of nNOS, we speculate that the increase of phosphorylation of nNOS at S847 limits the activity of the enzyme following a physiologic excitatory stimulus and prevents the accumulation of toxic levels of NO. Both activation of the NMDAR and CaMKII are 20 required to increase phosphorylation of nNOS at S847 since phosphorylation was blocked by APV or KN93. Given the established regulation of CaMKII by Ca 2+ , this suggests that low agonist stimulation of the NMDAR activates CaMKII via the formation and the binding of Ca 2+ -CaM, enabling CaMKII to phosphorylate S847. This is the first direct demonstration of an activity regulated control of nNOS S847 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 S847-PO 4 steady state levels.
Because S847-PO 4 levels were elevated by OA treatment, pools of PP1 and a constitutively active kinase, potentially CaMKII, capable of phosphorylating S847 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 This makes likely that pathological, excitotoxic stimulation of the NMDAR in vivo may 21 also override inhibitory effects of S847 phosphorylation. However further studies in vivo will be necessary to establish this point for excitatory insult in tissues.
Our work shows that S847 phosphorylation is limited in the steady state by PP1.
The NMDAR-mediated increase in phosphorylation at S847 following 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-