Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A

Synapse Associated Protein 97 (SAP97), a member of membrane-associated guanylate kinase (MAGUK) protein family, has been involved in the correct targeting and clustering of ionotropic glutamate receptors (iGluRs) at postsynaptic sites. Calcium/calmodulin kinase II (CaMKII) phosphorylates SAP97 on two major sites in vivo; one located in the N-terminal domain (Ser39) and the other in the first postsynaptic density disc large ZO1 (PDZ) domain (Ser232). CaMKII-mediated phosphorylation of SAP97-Ser39 is necessary and sufficient to drive SAP97 to the postsynaptic compartment in cultured hippocampal neurons. CaMKII-dependent phosphorylation of Ser232 disrupts SAP97 interaction with NR2A subunit, thereby regulating synaptic targeting of this NMDA receptor subunit. Here we show by means of phospho-specific antibodies that SAP97-Ser39 phosphorylation represents the driving force to release SAP97/NR2A complex from the endoplasmic reticulum. Ser39 phosphorylation does not interfere with SAP97 capability to bind NR2A. On the contrary, SAP97-Ser232 phosphorylation occurs within the postsynaptic compartment and is responsible for both the disruption of NR2A/SAP97 complex and, consequently, for NR2A insertion in the postsynaptic membrane. Thus, CaMKII-dependent phosphorylation of SAP97 in different time frames and locations within the neurons controls both NR2A trafficking and insertion.     

Calcium/calmodulin-dependent protein kinase II phosphorylation drives synapse-associated protein 97 into spines

Synapse-associated protein 97 (SAP97) has been involved in the correct delivery and clustering of glutamate ionotropic receptors to the postsynaptic compartment. Here we demonstrate that synaptic trafficking of SAP97 itself was modulated by calcium/calmodulin-dependent protein kinase II (CaMKII) in cultured hippocampal neurons. CaMKII activation led to increased targeting of SAP97 into dendritic spines, whereas CaMKII inhibition was responsible for SAP97 high colocalization in the cell soma with the endoplasmic reticulum protein disulfide-isomerase. No effect was detected for other members of the membrane-associated guanylate kinase protein family, such as SAP102 and PSD-95. Transfection of activated alphaCaMKII T286D dramatically increased concentration of both endogenous and transfected SAP97 at postsynaptic terminals. In vitro CaMKII phosphorylation of the SAP97 N-terminal fusion protein and metabolic labeling of transfected COS7 cells indicated SAP97-Ser-39 as a CaMKII phosphosite in the SAP97 protein sequence. Moreover, transfection in hippocampal neurons of SAP97 mutants that blocked or mimicked Ser-39 phosphorylation had effects similar to those observed upon inhibiting or constitutively activating CaMKII. Further, CaMKII-dependent SAP97-Ser-39 phosphorylation determined a redistribution of the glutamate receptor subunit (GluR1) of the AMPA receptor. In conclusion, our data show that CaMKII-dependent SAP97-Ser-39 phosphorylation regulates the association of SAP97 with the postsynaptic complex, thus providing a fine molecular mechanism responsible for the synaptic delivery of SAP97 interacting proteins, i.e. ionotropic glutamate receptor subunits.     

Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Differential interaction of NMDA receptor subtypes with the post-synaptic density-95 family of membrane associated guanylate kinase proteins

NMDA receptors are a subclass of ionotropic glutamate receptors. They are trafficked and/or clustered at synapses by the post-synaptic density (PSD)-95 membrane associated guanylate kinase (MAGUK) family of scaffolding proteins that associate with NMDA receptor NR2 subunits via their C-terminal glutamate serine (aspartate/glutamate) valine motifs. We have carried out a systematic study investigating in a heterologous expression system, the association of the four major NMDA receptor subtypes with the PSD-95 family of MAGUK proteins, chapsyn-110, PSD-95, synapse associated protein (SAP) 97 and SAP102. We report that although each PSD-95 MAGUK was shown to co-immunoprecipitate with NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptor subtypes, they elicited differential effects with regard to the enhancement of total NR2 subunit expression which then results in an increased cell surface expression of NMDA receptor subtypes. PSD-95 and chapsyn-110 enhanced NR2A and NR2B total expression which resulted in increased NR1/NR2A and NR1/NR2B receptor cell surface expression whereas SAP97 and SAP102 had no effect on total or cell surface expression of these subtypes. PSD-95, chapsyn-110, SAP97 and SAP102 had no effect on either total NR2C and NR2D subunit expression or cell surface NR1/NR2C and NR1/NR2D expression. A comparison of PSD-95α, PSD-95β and PSD-95α^C3S,C5S showed that PSD-95-enhanced cell surface expression of NR1/NR2A receptors was dependent upon the PSD-95 N-terminal C3,C5 cysteines. These observations support differential interaction of NMDA receptor subtypes with different PSD-95 MAGUK scaffolding proteins. This has implications for the stabilisation, turnover and compartmentalisation of NMDA receptor subtypes in neurones during development and in the mature brain.     

AlphaCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Tyrosine phosphorylation of ionotropic glutamate receptors by Fyn or Src differentially modulates their susceptibility to calpain and enhances their binding to spectrin and PSD-95

Both tyrosine phosphorylation and calpain-mediated truncation of ionotropic glutamate receptors are important mechanisms for synaptic plasticity. Previous work from our laboratory has shown that calpain activation results in truncation of the C-terminal domains of several glutamate receptor subunits. To test whether and how tyrosine phosphorylation of glutamate ionotropic receptor subunits modulates calpain susceptibility, synaptic membranes were phosphorylated by Fyn or Src, two members of the Src family tyrosine kinases. Tyrosine phosphorylation of synaptic membranes by Src significantly reduced calpain-mediated truncation of both NR2A and NR2B subunits of NMDA receptors, but not of GluR1 subunits of AMPA receptors. In contrast, phosphorylation with Fyn significantly protected calpain-mediated truncation of GluR1 subunits of AMPA receptors, but enhanced calpain-mediated truncation of NR2A subunits of NMDA receptors. Similar results were observed with NR2A and NR2B C-terminal domain fusion proteins phosphorylated by Fyn or Src before incubation with calpain and calcium. In addition, phosphorylation of NR2A and NR2B C-terminal fusion proteins by Fyn or Src enhanced their binding to spectrin and PSD-95. Thus, tyrosine phosphorylation impairs or facilitates calpain-mediated truncation of glutamate receptor subunits, depending on which tyrosine kinase is activated. Such mechanisms could serve to regulate receptor integrity and location, in addition to modulating channel properties.     

Relative extent of tyrosine phosphorylation of the NR2A and NR2B subunits in the rat forebrain postsynaptic density fraction

The activity of the N-methyl-D-aspartate (NMDA) receptor, a subclass of ionotropic glutamate receptor, is modulated by a complex network of phosphorylation and dephosphorylation. I investigated the relative extent of tyrosine phosphorylation of NMDA receptor subunit 2A (NR2A) and 2B (NR2B) subunits in the rat forebrain postsynaptic density (PSD) fraction. Immunoblot analysis of immunoprecipitates with antiphosphotyrosine antibodies indicated that tyrosine phosphorylation of NR2A was only 28.6% of that of NR2B. When phosphotyrosine-containing peptides were isolated by affinity-purification or immunoprecipitation, and probed for the two subunits, NR2B was detected but not NR2A. Furthermore, depletion of NR2B removed the phosphotyrosine-containing 180 kDa peptide from the solution while the converse was not true. The small extent of tyrosine phosphorylation of NR2A in the unstimulated condition may explain the dramatic increase in tyrosine phosphorylation in various physiological and pathological conditions.     

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats

In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.     

Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.     

Activation of Ca2+/calmodulin-dependent protein kinase II and protein kinase C by glutamate in cultured rat hippocampal neurons.

In cultured rat hippocampal neurons, glutamate elevated the Ca(2+)-independent activity of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) through autophosphorylation when the neurons were incubated in Mg(2+)-free buffer, and this response was blocked by specific antagonists of the N-methyl-D-aspartate (NMDA) receptor. In addition, glutamate stimulated the transient translocation of protein kinase C (PKC) from the cytosol to the membrane fraction. This effect was not blocked by NMDA receptor antagonists but was partially blocked by DL-2-amino-3-phosphonopropionate. Quisqualate or trans-1-amoinocyclopentane-trans1,3-dicarboxylate produced a similar effect on the translocation of PKC. In the experiments with 32P-labeled cells, the phosphorylation of microtuble-associated protein 2 and synapsin I, as well as autophosphorylation of CaM kinase II, were found to be stimulated by exposure to glutamate. These results suggest that glutamate can activate CaM kinase II through the ionotropic NMDA receptor, which in turn increases the phosphorylation of microtuble-associated protein 2 and synapsin I. PKC was activated through the metabotropic glutamate receptor in the hippocampal neurons.     

Displacement of alpha-actinin from the NMDA receptor NR1 C0 domain By Ca2+/calmodulin promotes CaMKII binding

Ca2+ influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor triggers activation and postsynaptic accumulation of Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII, calmodulin, and alpha-actinin directly bind to the short membrane proximal C0 domain of the C-terminal region of the NMDA receptor NR1 subunit. In a negative feedback loop, calmodulin mediates Ca2+-dependent inactivation of the NMDA receptor by displacing alpha-actinin from NR1 C0 upon Ca2+ influx. We show that Ca2+-depleted calmodulin and alpha-actinin simultaneously bind to NR1 C0. Upon addition of Ca2+, calmodulin dislodges alpha-actinin. Either the N- or C-terminal half of calmodulin is sufficient for Ca2+-induced displacement of alpha-actinin. Whereas alpha-actinin directly antagonizes CaMKII binding to NR1 C0, the addition of Ca2+/calmodulin shifts binding of NR1 C0 toward CaMKII by displacing alpha-actinin. Displacement of alpha-actinin results in the simultaneous binding of calmodulin and CaMKII to NR1 C0. Our results reveal an intricate mechanism whereby Ca2+ functions to govern the complex interactions between the two most prevalent signaling molecules in synaptic plasticity, the NMDA receptor and CaMKII.     

Neuroprotection of GluR5-containing kainate receptor activation against ischemic brain injury through decreasing tyrosine phosphorylation of N-methyl-D-aspartate receptors mediated by Src kinase

Previous studies indicate that cerebral ischemia breaks the dynamic balance between excitatory and inhibitory inputs. The neural excitotoxicity induced by ionotropic glutamate receptors gain the upper hand during ischemia-reperfusion. In this paper, we investigate whether GluR5 (glutamate receptor 5)-containing kainate receptor activation could lead to a neuroprotective effect against ischemic brain injury and the related mechanism. The results showed that (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a selective GluR5 agonist, could suppress Src tyrosine phosphorylation and interactions among N-methyl-D-aspartate (NMDA) receptor subunit 2A (NR2A), postsynaptic density protein 95 (PSD-95), and Src and then decrease NMDA receptor activation through attenuating tyrosine phosphorylation of NR2A and NR2B. More importantly, ATPA had a neuroprotective effect against ischemia-reperfusion-induced neuronal cell death in vivo. However, four separate drugs were found to abolish the effects of ATPA. These were selective GluR5 antagonist NS3763; GluR5 antisense oligodeoxynucleotides; CdCl(2), a broad spectrum blocker of voltage-gated calcium channels; and bicuculline, an antagonist of gamma-aminobutyric acid A (GABA(A)) receptor. GABA(A) receptor agonist muscimol could attenuate Src activation and interactions among NR2A, PSD-95 and Src, resulting the suppression of NMDA receptor tyrosine phosphorylation. Moreover, patch clamp recording proved that the activated GABA(A) receptor could inhibit NMDA receptor-mediated whole-cell currents. Taken together, the results suggest that during ischemia-reperfusion, activated GluR5 may facilitate Ca(2+)-dependent GABA release from interneurons. The released GABA can activate postsynaptic GABA(A) receptors, which then attenuates NMDA receptor tyrosine phosphorylation through inhibiting Src activation and disassembling the signaling module NR2A-PSD-95-Src. The final result of this process is that the pyramidal neurons are rescued from hyperexcitability.     

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.     

Substrate localization creates specificity in calcium/calmodulin-dependent protein kinase II signaling at synapses

Calcium/calmodulin-dependent protein kinase II (CaMKII), a major component of the postsynaptic density (PSD) of excitatory synapses, plays a key role in the regulation of synaptic function in the mammalian brain. Although many postsynaptic substrates for CaMKII have been characterized in vitro, relatively little is known about their phosphorylation in vivo. By tagging synaptic proteins with a peptide substrate specific for CaMKII and expressing them in cultured neurons, we have visualized substrate phosphorylation by CaMKII at intact synapses. All substrates tested were strongly phosphorylated by CaMKII in HEK293 cells. However, activity-dependent phosphorylation of substrates at synapses was highly selective in that the glutamate receptor subunits NR2B and GluR1 were poorly phosphorylated whereas PSD-95 and Stargazin, proteins implicated in the scaffolding and trafficking of AMPA receptors, were robustly phosphorylated. Phosphatase activity limited phosphorylation of Stargazin but not NR2B and GluR1. These results suggest that the unique molecular architecture of the PSD results in highly selective substrate discrimination by CaMKII.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Mechanism and regulation of calcium/calmodulin-dependent protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor

Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689-20692). Here, we show that residues 1290-1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys(1292), Leu(1298), Arg(1299), Arg(1300), Gln(1301), and Ser(1303)). Phosphorylation of NR2B at Ser(1303) by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII.NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260-1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets.     

Decreased calcium/calmodulin-dependent protein kinase II and protein kinase C activities mediate impairment of hippocampal long-term potentiation in the olfactory bulbectomized mice

Olfactory bulbectomized (OBX) mice showed significant impairment of learning and memory-related behaviors 14 days after olfactory bulbectomy, as measured by passive avoidance and Y-maze tasks. We here observed a large impairment of hippocampal long-term potentiation (LTP) in the OBX mice. Concomitant with decreased acetylcholinesterase expression, protein kinase C (PKC)alpha autophosphorylation and NR1(Ser-896) phosphorylation significantly decreased in the hippocampal CA1 region of OBX mice. Both PKCalpha and NR1(Ser-896) phosphorylation significantly increased following LTP in the control mice, whereas increases were not observed in OBX mice. Like PKC activities, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in the hippocampal CA1 region of OBX mice as compared with that of control mice. In addition, increased CaMKII autophosphorylation following LTP was not observed in OBX mice. Finally, the impairment of CaMKII autophosphorylation was closely associated with reduced pGluR1(Ser-831) phosphorylation, without change in synapsin I (site 3) phosphorylation in the hippocampal CA1 region of OBX mice. Taken together, in OBX mice NMDA receptor hypofunction, possibly through decreased PKCalpha activity, underlies decreased CaMKII activity in the post-synaptic regions, thereby impairing LTP induction in the hippocampal CA1 region. Both decreased PKC and CaMKII activities with concomitant LTP impairment account for the learning disability observed in OBX mice.     

Bidirectional regulation of neuronal nitric-oxide synthase phosphorylation at serine 847 by the N-methyl-D-aspartate receptor

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 (Ser(847)) by the calcium-calmodulin protein kinase II (CaMKII) inhibits nNOS activity, possibly by blocking the binding of Ca(2+)-CaM. Here we show that the NMDA mediates a novel bidirectional regulation of Ser(847) phosphorylation. nNOS phosphorylated at Ser(847) colocalizes with the NMDA receptor at spines of cultured hippocampal neurons. Treatment of neurons with 5 microm glutamate stimulated CaMKII phosphorylation of nNOS at Ser(847), whereas excitotoxic concentrations of glutamate, 100 and 500 microm, induced Ser(847)-PO(4) 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 Ser(847)-PO(4) dephosphorylation. Of particular note, stimulation with low glutamate that increased phosphorylation of nNOS at Ser(847) could be reversed by subsequent high glutamate treatment which induced dephosphorylation. The reversibility of NMDA receptor-induced phosphorylation at Ser(847) 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.     

Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1

We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.