Divergent regulation of Pyk2/CAKbeta phosphorylation by Ca2+ and cAMP in the hippocampus

Proline-rich tyrosine kinase 2 (Pyk2) is activated in neurones following NMDA receptor stimulation via PKC. Pyk2 is involved in hippocampal LTP and acts to potentiate NMDA receptor function. Elevations of intracellular Ca2+ and cAMP levels are key NMDA receptor-dependent triggering events leading to induction of hippocampal LTP. In this study, we compared the ability of A23187 (Ca2+ ionophore) or forskolin (adenylate cyclase activator) to modulate the phosphorylation of Pyk2 in rat hippocampal slices. Using an immunoprecipitation assay, phosphorylated Pyk2 levels were increased following treatment with A23187, levels peaking at around 10 min. Staurosporine, at concentrations inhibiting conventional and novel isoforms of PKC, and chelerythrine, at concentrations inhibiting the atypical PKC isoform PKMxi, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to either chelerythrine or staurosporine completely blocked enhanced phosphorylation of Pyk2 by A23187, suggesting a possible involvement of PKMxi and typical PKCs in Pyk2 activation by Ca2+. In contrast, application of forskolin reduced phosphorylated Pyk2 below basal levels, suggesting that cAMP inhibits Pyk2. These results implicate Ca2+ and multiple forms of PKC in the activation of Pyk2 downstream of NMDA receptors and suggest that cAMP-dependent processes exert a suppressive action on Pyk2.     

H-Ras modulates N-methyl-D-aspartate receptor function via inhibition of Src tyrosine kinase activity

Tyrosine phosphorylation of the NR2A and NR2B subunits of the N-methyl-d-aspartate (NMDA) receptor by Src protein-tyrosine kinases modulates receptor channel activity and is necessary for the induction of long term potentiation (LTP). Deletion of H-Ras increases both NR2 tyrosine phosphorylation and NMDA receptor-mediated hippocampal LTP. Here we investigated whether H-Ras regulates phosphorylation and function of the NMDA receptor via Src family protein-tyrosine kinases. We identified Src as a novel H-Ras binding partner. H-Ras bound to Src but not Fyn both in vitro and in brain via the Src kinase domain. Cotransfection of H-Ras and Src inhibited Src activity and decreased NR2A tyrosine phosphorylation. Treatment of rat brain slices with Tat-H-Ras depleted NR2A from the synaptic membrane, decreased endogenous Src activity and NR2A phosphorylation, and decreased the magnitude of hippocampal LTP. No change was observed for NR2B. We suggest that H-Ras negatively regulates Src phosphorylation of NR2A and retention of NR2A into the synaptic membrane leading to inhibition of NMDA receptor function. This mechanism is specific for Src and NR2A and has implications for studies in which regulation of NMDA receptor-mediated LTP is important, such as synaptic plasticity, learning, and memory and addiction.     

Iron mediates N-methyl-D-aspartate receptor-dependent stimulation of calcium-induced pathways and hippocampal synaptic plasticity

Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-D-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP.     

Interaction of the tyrosine kinase Pyk2 with the N-methyl-D-aspartate receptor complex via the Src homology 3 domains of PSD-95 and SAP102

The protein-tyrosine kinase Pyk2/CAKbeta/CADTK is a key activator of Src in many cells. At hippocampal synapses, induction of long term potentiation requires the Pyk2/Src signaling pathway, which up-regulates the activity of N-methyl-d-aspartate-type glutamate receptors. Because localization of protein kinases close to their substrates is crucial for effective phosphorylation, we investigated how Pyk2 might be recruited to the N-methyl-d-aspartate receptor complex. This interaction is mediated by PSD-95 and its homolog SAP102. Both proteins colocalize with Pyk2 at postsynaptic dendritic spines in the cerebral cortex. The proline-rich regions in the C-terminal half of Pyk2 bind to the SH3 domain of PSD-95 and SAP102. The SH3 and guanylate kinase homology (GK) domain of PSD-95 and SAP102 interact intramolecularly, but the physiological significance of this interaction has been unclear. We show that Pyk2 effectively binds to the Src homology 3 (SH3) domain of SAP102 only when the GK domain is removed from the SH3 domain. Characterization of PSD-95 and SAP102 as adaptor proteins for Pyk2 fills a critical gap in the understanding of the spatial organization of the Pyk2-Src signaling pathway at the postsynaptic site and reveals a physiological function of the intramolecular SH3-GK domain interaction in SAP102.     

Platelet-derived growth factor receptor-induced feed-forward inhibition of excitatory transmission between hippocampal pyramidal neurons.

Growth factor receptors provide a major mechanism for the activation of the nonreceptor tyrosine kinase c-Src, and this kinase in turn up-regulates the activity of N-methyl-D-aspartate (NMDA) receptors in CA1 hippocampal neurons (1). Unexpectedly, applications of platelet-derived growth factor (PDGF)-BB to cultured and isolated CA1 hippocampal neurons depressed NMDA-evoked currents. The PDGF-induced depression was blocked by a PDGF-selective tyrosine kinase inhibitor, by a selective inhibitor of phospholipase C-gamma, and by blocking the intracellular release of Ca(2+). Inhibitors of cAMP-dependent protein kinase (PKA) also eliminated the PDGF-induced depression, whereas a phosphodiesterase inhibitor enhanced it. The NMDA receptor-mediated component of excitatory synaptic currents was also inhibited by PDGF, and this inhibition was prevented by co-application of a PKA inhibitor. Src inhibitors also prevented this depression. In recordings from inside-out patches, the catalytic fragment of PKA did not itself alter NMDA single channel activity, but it blocked the up-regulation of these channels by a Src activator peptide. Thus, PDGF receptors depress NMDA channels through a Ca(2+)- and PKA-dependent inhibition of their modulation by c-Src.     

(alpha)v(beta)3 integrins and Pyk2 mediate insulin-like growth factor I activation of Src and mitogen-activated protein kinase in 3T3-L1 cells

IGF-I stimulates cell growth through interaction of the IGF receptor with multiprotein signaling complexes. However, the mechanisms of IGF-I receptor-mediated signaling are not completely understood. We have previously shown that IGF-I-stimulated 3T3-L1 cell proliferation is dependent on Src activation of the ERK-1/2 MAPK pathway. We hypothesized that IGF-I activation of the MAPK pathway is mediated through integrin activation of Src-containing signaling complexes. The disintegrin echistatin decreased IGF-I phosphorylation of Src and MAPK, and blocking antibodies to (alpha)v and beta3 integrin subunits inhibited IGF-I activation of MAPK, suggesting that (alpha)v(beta)3 integrins mediate IGF-I mitogenic signaling. IGF-I increased ligand binding to (alpha)v(beta)3 as detected by immunofluorescent staining of ligand-induced binding site antibody and stimulated phosphorylation of the beta3 subunit, consistent with inside-out activation of (alpha)v(beta)3 integrins. IGF-I increased tyrosine phosphorylation of the focal adhesion kinase (FAK) Pyk2 (calcium-dependent proline-rich tyrosine kinase-2) to a much greater extent than FAK, and increased association of Src with Pyk2 but not FAK. The intracellular calcium chelator BAPTA prevented IGF-I phosphorylation of Pyk2, Src, and MAPK, suggesting that IGF-I activation of Pyk2 is calcium dependent. Transient transfection with a dominant-negative Pyk2, which lacks the autophosphorylation and Src binding site, decreased IGF-I activation of MAPK, but no inhibition was seen with transfected wild-type Pyk2. These results indicate that IGF-I signaling to MAPK is dependent on inside-out activation of (alpha)v(beta)3 integrins and integrin-facilitated multiprotein complex formation involving Pyk2 activation and association with Src.     

Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses

The physiological conditions under which adenosine A2A receptors modulate synaptic transmission are presently unclear. We show that A2A receptors are localized postsynaptically at synapses between mossy fibers and CA3 pyramidal cells and are essential for a form of long-term potentiation (LTP) of NMDA-EPSCs induced by short bursts of mossy fiber stimulation. This LTP spares AMPA-EPSCs and is likely induced and expressed postsynaptically. It depends on a postsynaptic Ca2+ rise, on G protein activation, and on Src kinase. In addition to A2A receptors, LTP of NMDA-EPSCs requires the activation of NMDA and mGluR5 receptors as potential sources of Ca2+ increase. LTP of NMDA-EPSCs displays a lower threshold for induction as compared with the conventional presynaptic mossy fiber LTP; however, the two forms of LTP can combine with stronger induction protocols. Thus, postsynaptic A2A receptors may potentially affect information processing in CA3 neuronal networks and memory performance.     

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.     

Insulin modulates hippocampal activity-dependent synaptic plasticity in a N-methyl-d-aspartate receptor and phosphatidyl-inositol-3-kinase-dependent manner

Insulin and its receptor are both present in the central nervous system and are implicated in neuronal survival and hippocampal synaptic plasticity. Here we show that insulin activates phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), and results in an induction of long-term depression (LTD) in hippocampal CA1 neurones. Evaluation of the frequency-response curve of synaptic plasticity revealed that insulin induced LTD at 0.033 Hz and LTP at 10 Hz, whereas in the absence of insulin, 1 Hz induced LTD and 100 Hz induced LTP. LTD induction in the presence of insulin required low frequency synaptic stimulation (0.033 Hz) and blockade of GABAergic transmission. The LTD or LTP induced in the presence of insulin was N-methyl-d-aspartate (NMDA) receptor specific as it could be inhibited by alpha-amino-5-phosphonopentanoic acid (APV), a specific NMDA receptor antagonist. LTD induction was also facilitated by lowering the extracellular Mg(2+) concentration, indicating an involvement of NMDA receptors. Inhibition of PI3K signalling or discontinuing synaptic stimulation also prevented this LTD. These results show that insulin modulates activity-dependent synaptic plasticity, which requires activation of NMDA receptors and the PI3K pathway. The results obtained provide a mechanistic link between insulin and synaptic plasticity, and explain how insulin functions as a neuromodulator.     

Fyn-mediated phosphorylation of NR2B Tyr-1336 controls calpain-mediated NR2B cleavage in neurons and heterologous systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.     

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.     

Postsynaptic factors control the duration of synaptic enhancement in area CA1 of the hippocampus.

In area of CA1 of the hippocampus, at least two phases of long-term potentiation (LTP) can be isolated: an early decremental component referred to as short-term potentiation (STP), which precedes a long-lasting, nondecremental component commonly considered to be stable LTP. Utilizing the hippocampal slice preparation, experiments were performed to determine the physiological factors controlling the conversion of STP to LTP. The duration of NMDA receptor-dependent synaptic enhancement was influenced by several factors, including the degree of postsynaptic NMDA receptor activation and the magnitude and timing of postsynaptic membrane depolarization during synaptic transmission. It was possible to convert STP to LTP by manipulations that increased the influx of calcium into the postsynaptic cell. These results demonstrate that NMDA receptor activation can result in distinct forms of synaptic potentiation and imply that the magnitude of postsynaptic calcium increase is a critical variable controlling the duration of synaptic enhancement.     

Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade

The epidermal growth factor receptor (EGFR) and the non-receptor protein tyrosine kinases Src and Pyk2 have been implicated in linking a variety of G-protein-coupled receptors (GPCR) to the mitogen-activated protein (MAP) kinase signaling cascade. In this report we apply a genetic strategy using cells isolated from Src-, Pyk2-, or EGFR-deficient mice to explore the roles played by these protein tyrosine kinases in GPCR-induced activation of EGFR, Pyk2, and MAP kinase. We show that Src kinases are critical for activation of Pyk2 in response to GPCR-stimulation and that Pyk2 and Src are essential for GPCR-induced tyrosine phosphorylation of EGFR. By contrast, Pyk2, Src, and EGFR are dispensable for GPCR-induced activation of MAP kinase. Moreover, GPCR-induced MAP kinase activation is normal in fibroblasts deficient in both Src and Pyk2 (Src-/-Pyk2-/- cells) as well as in fibroblasts deficient in all three Src kinases expressed in these cells (Src-/-Yes-/-Fyn-/- cells). Finally, experiments are presented demonstrating that, upon stimulation of GPCR, activated Pyk2 forms a complex with Src, which in turn phosphorylates EGFR directly. These experiments reveal a role for Src kinases in Pyk2 activation and a role for Pyk2 and Src in tyrosine phosphorylation of EGFR following GPCR stimulation. In addition, EGFR, Src family kinases, and Pyk2 are not required for linking GPCRs with the MAP kinase signaling cascade.     

CaM kinase II and protein kinase C activations mediate enhancement of long-term potentiation by nefiracetam in the rat hippocampal CA1 region

Nefiracetam is a pyrrolidine-related nootropic drug exhibiting various pharmacological actions such as cognitive-enhancing effect. We previously showed that nefiracetam potentiates NMDA-induced currents in cultured rat cortical neurons. To address questions whether nefiracetam affects NMDA receptor-dependent synaptic plasticity in the hippocampus, we assessed effects of nefiracetam on NMDA receptor-dependent long-term potentiation (LTP) by electrophysiology and LTP-induced phosphorylation of synaptic proteins by immunoblotting analysis. Nefiracetam treatment at 1-1000 nM increased the slope of fEPSPs in a dose-dependent manner. The enhancement was associated with increased phosphorylation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) without affecting synapsin I phosphorylation. In addition, nefiracetam treatment increased PKCalpha activity in a bell-shaped dose-response curve which peaked at 10 nM, thereby increasing phosphorylation of myristoylated alanine-rich protein kinase C substrate and NMDA receptor. Nefiracetam treatment did not affect protein kinase A activity. Consistent with the bell-shaped PKCalpha activation, nefiracetam treatment enhanced LTP in the rat hippocampal CA1 region with the same bell-shaped dose-response curve. Furthermore, nefiracetam-induced LTP enhancement was closely associated with CaMKII and PKCalpha activation with concomitant increases in phosphorylation of their endogenous substrates except for synapsin I. These results suggest that nefiracetam potentiates AMPA receptor-mediated fEPSPs through CaMKII activation and enhances NMDA receptor-dependent LTP through potentiation of the post-synaptic CaMKII and protein kinase C activities. Together with potentiation of nicotinic acetylcholine receptor function, nefiracetam-enhanced AMPA and NMDA receptor functions likely contribute to improvement of cognitive function.     

Novel Role of Src in Priming Pyk2 Phosphorylation

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase (FAK) family of non-receptor tyrosine kinases and plays an important role in diverse cellular events downstream of the integrin-family of receptors, including cell migration, proliferation and survival. Here, we have identified a novel role for Src kinase in priming Pyk2 phosphorylation and subsequent activation upon cell attachment on the integrin-ligand fibronectin. By using complementary methods, we show that Src activity is indispensable for the initial Pyk2 phosphorylation on the Y402 site observed in response to cell attachment. In contrast, the initial fibronectin-induced autophosphorylation of FAK in the homologous Y397 site occurs in a Src-independent manner. We demonstrate that the SH2-domain of Src is required for Src binding to Pyk2 and for Pyk2 phosphorylation at sites Y402 and Y579. Moreover, Y402 phosphorylation is a prerequisite for the subsequent Y579 phosphorylation. While this initial phosphorylation of Pyk2 by Src is independent of Pyk2 kinase activity, subsequent autophosphorylation of Pyk2 in trans is required for full Pyk2 phosphorylation and activation. Collectively, our studies reveal a novel function of Src in priming Pyk2 (but not FAK) phosphorylation and subsequent activation downstream of integrins, and shed light on the signaling events that regulate the function of Pyk2.     

Activation of dopamine D1 receptors blocks phencyclidine-induced neurotoxicity by enhancing N-methyl-D-aspartate receptor-mediated synaptic strength

Early postnatal blockade of NMDA receptors by phencyclidine (PCP) causes cortical apoptosis in animals. This is associated with the development of schizophrenia-like behaviors in rats later in life. Recent studies show that the mechanism involves a loss of neurotrophic support from the phosphoinositol-3 kinase/Akt pathway, which is normally maintained by synaptic NMDA receptor activation. Here we report that activation of dopamine D1 receptors (D1R) with dihydrexidine (DHX) prevents PCP-induced neurotoxicity in cortical neurons by enhancing the efficacy of NMDAergic synapses. DHX increases serine phosphorylation of the NR1 subunit through protein kinase A activation and tyrosine phosphorylation of the NR2B subunit via Src kinase. DHX enhances recruitment of NR1 and NR2B, but not NR2A, into synapses. DHX also facilitated the synaptic response in cortical slices and this was blocked by an NR2B antagonist. DHX pre-treatment of rat pups prior to PCP on postnatal days 7, 9 and 11 inhibited PCP-induced caspase-3 activation on PN11 and deficits in pre-pulse inhibition of acoustic startle measured on PN 26-28. In summary, these data demonstrate that PCP-induced deficits in NMDA receptor function, neurotoxicity and subsequent behavioral deficits may be prevented by D1R activation in the cortex and further, it is suggested that D1R activation may be beneficial in treating schizophrenia.     

What mechanisms underlie involvement of different NMDA receptor subunits in the induction of hippocampal long-term potentiation and long-term depression?

There is a point of view that N-methyl-D-aspartate (NMDA) receptor subunit-specific signaling outcomes determine the direction of modifications of efficacy of synaptic transmission. Activation of NMDA receptors that contain the 2A subunit promotes LTP, while LTD requires activation of NMDA receptors containing 2B subunit. However, this hypothesis is inconsistent with some experimental data. For explanation of these data, we put forward an alternative hypothesis. According to this hypothesis, the activation of diverse subtypes of NMDA receptors can lead to ether LTP or LTD depending on the relation between posttetanic Ca2+ rise and increase in postsynaptic Ca2+ concentration produced by previous stimulation. Activation of NMDA receptors with 2B subunit can promote LTD of excitatory input to the pyramidal cell due to presence of these receptors on inhibitory interneurons, induction of the LTP in interneuron, and potentiation of inhibitory transmission between the interneuron and the target pyramidal cell.     

Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.     

Forebrain NR2B overexpression facilitating the prefrontal cortex long-term potentiation and enhancing working memory function in mice

Prefrontal cortex plays an important role in working memory, attention regulation and behavioral inhibition. Its functions are associated with NMDA receptors. However, there is little information regarding the roles of NMDA receptor NR2B subunit in prefrontal cortical synaptic plasticity and prefrontal cortex-related working memory. Whether the up-regulation of NR2B subunit influences prefrontal cortical synaptic plasticity and working memory is not yet clear. In the present study, we measured prefrontal cortical synaptic plasticity and working memory function in NR2B overexpressing transgenic mice. In vitro electrophysiological data showed that overexpression of NR2B specifically in the forebrain region resulted in enhancement of prefrontal cortical long-term potentiation (LTP) but did not alter long-term depression (LTD). The enhanced LTP was completely abolished by a NR2B subunit selective antagonist, Ro25-6981, indicating that overexpression of NR2B subunit is responsible for enhanced LTP. In addition, NR2B transgenic mice exhibited better performance in a set of working memory paradigms including delay no-match-to-place T-maze, working memory version of water maze and odor span task. Our study provides evidence that NR2B subunit of NMDA receptor in prefrontal cortex is critical for prefrontal cortex LTP and prefrontal cortex-related working memory.