Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn

In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed. The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody. Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein. Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110. Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site. Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice. Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized. In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization. In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate. These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.     

Lithium-induced inhibition of Src tyrosine kinase in rat cerebral cortical neurons: a role in neuroprotection against N-methyl-D-aspartate receptor-mediated excitotoxicity

The neuroprotective effects of lithium, a mood stabilizer, against glutamate-induced excitotoxicity in rat cortical neurons were associated with a decrease in Tyr1472 phosphorylation of the N-methyl-D-aspartate (NMDA) receptor NR2B subunit and a loss of receptor activity. Since this receptor tyrosine phosphorylation is mediated by the Src-family tyrosine kinases, we investigated the effects of lithium on the Src kinase activity. Levels of phosphorylated Src kinase at Tyr416, an index of Src activation, were reduced after treatment with LiCl (1 mM) for more than 3 days. Protein levels of Src-family kinases such as Src, Fyn, and Yes were unchanged by lithium treatment. The activities of cytosolic protein tyrosine kinase and protein phosphatase were also unchanged by lithium treatment, indicating the selectivity and the modulation. Moreover, the levels of postsynaptic densities (PSD) and SynGAP, the scaffolding proteins of the NMDA receptor complex, were unaltered by lithium. A Src kinase inhibitor, SU6656, and an NR2B antagonist, ifenprodil, partially blocked glutamate excitotoxicity. Our results suggest that lithium-induced inactivation of Src kinase contributes to this drug-induced NMDA receptor inhibition and neuroprotection against excitotoxicity.     

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.     

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.     

Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with v-Src

The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v-Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v-Src, the general region of v-Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site-directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src-independent regulation of receptor currents.     

mGluR1 antagonist decreases tyrosine phosphorylation of NMDA receptor and attenuates infarct size after transient focal cerebral ischemia

The contribution of metabotropic glutamate receptors to brain injury after in vivo cerebral ischemia remains to be determined. We investigated the effects of the metabotropic glutamate receptor 1 (mGluR1) antagonist LY367385 on brain injury after transient (90 min) middle cerebral artery occlusion in the rat and sought to explore their mechanisms. The intravenous administration of LY367385 (10 mg/kg) reduced the infarct volume at 24 h after the start of reperfusion. As the Gq-coupled mGluR1 receptor is known to activate the PKC/Src family kinase cascade, we focused on changes in the activation and amount of these kinases. Transient focal ischemia increased the amount of activated tyrosine kinase Src and PKC in the post-synaptic density (PSD) at 4 h of reperfusion. The administration of LY367385 attenuated the increases in the amounts of PSD-associated PKCγ and Src after transient focal ischemia. We further investigated phosphorylation of the NMDA receptor, which is a major target of Src family kinases to modulate the function of the receptor. Transient focal ischemia increased the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B. Tyrosine phosphorylation of NR2A, but not that of NR2B, in the PSD at 4 h of reperfusion was inhibited by LY367385. These results suggest that the mGluR1 after transient focal ischemia is involved in the activation of Src, which may be linked to the modification of properties of the NMDA receptor and the development of cerebral infarction.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2 (NR2B) subunit of the N-methyl-D-aspartate receptor

The N-methyl-d-aspartate (NMDA) receptors play critical roles in synaptic plasticity, neuronal development, and excitotoxicity. Tyrosine phosphorylation of NMDA receptors by Src-family tyrosine kinases such as Fyn is implicated in synaptic plasticity. To precisely address the roles of NMDA receptor tyrosine phosphorylation, we identified Fyn-mediated phosphorylation sites on the GluR epsilon 2 (NR2B) subunit of NMDA receptors. Seven out of 25 tyrosine residues in the C-terminal cytoplasmic region of GluR epsilon 2 were phosphorylated by Fyn in vitro. Of these 7 residues, Tyr-1252, Tyr-1336, and Tyr-1472 in GluR epsilon 2 were phosphorylated in human embryonic kidney fibroblasts when co-expressed with active Fyn, and Tyr-1472 was the major phosphorylation site in this system. We then generated rabbit polyclonal antibodies specific to Tyr-1472-phosphorylated GluR epsilon 2 and showed that Tyr-1472 of GluR epsilon 2 was indeed phosphorylated in murine brain using the antibodies. Importantly, Tyr-1472 phosphorylation was greatly reduced in fyn mutant mice. Moreover, Tyr-1472 phosphorylation became evident when hippocampal long term potentiation started to be observed, and its magnitude became larger in murine brain. Finally, Tyr-1472 phosphorylation was significantly enhanced after induction of long term potentiation in the hippocampal CA1 region. These data suggest that Tyr-1472 phosphorylation of GluR epsilon 2 is important for synaptic plasticity.     

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.     

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.     

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.     

Fyn is required for haloperidol-induced catalepsy in mice

Fyn-mediated tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D(2) receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D(2) receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D(2) receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response.     

The Epstein-Barr virus protein, latent membrane protein 2A, co-opts tyrosine kinases used by the T cell receptor

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and is associated with several human malignancies. The EBV protein latent membrane protein 2A (LMP2A) promotes viral latency in memory B cells by interfering with B cell receptor signaling and provides a survival signal for mature B cells that have lost expression of surface immunoglobulin. The latter function has suggested that LMP2A may enhance the survival of EBV-positive tumors. EBV is associated with several T cell malignancies and, since LMP2A has been detected in several of these disorders, we examined the ability of LMP2A to transmit signals and interfere with T cell receptor signaling in T cells. We show that LMP2A is tyrosine-phosphorylated in Jurkat TAg T cells, which requires expression of the Src family tyrosine kinases, Lck and Fyn. Lck and Fyn are recruited to the tyrosine-phosphorylated Tyr112 site in LMP2A, whereas phosphorylation of an ITAM motif in LMP2A creates a binding site for the ZAP-70/Syk tyrosine kinases. LMP2A also associates through its two PPPPY motifs with AIP4, a NEDD4 family E3 ubiquitin ligase; this interaction results in ubiquitylation of LMP2A and serves to regulate the stability of LMP2A and LMP2A-kinase complexes. Furthermore, stable expression of LMP2A in Jurkat T cells down-regulated T cell receptor levels and attenuated T cell receptor signaling. Thus, through recruiting tyrosine kinases involved in T cell receptor activation, LMP2A may provide a survival signal for EBV-positive T cell tumors, whereas LMP2A-associated NEDD4 E3 ligases probably titer the strength of this signal.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

Increased tyrosine phosphorylation of PSD-95 by Src family kinases after brain ischaemia

PSD (postsynaptic density)-95, a scaffold protein that tethers NMDA (N-methyl-D-aspartate) receptors to signal molecules, is implicated in pathological events resulting from excitotoxicity. The present study demonstrates that brain ischaemia and reperfusion increase the tyrosine phosphorylation of PSD-95 in the rat hippocampus. PP2, a specific inhibitor of SrcPTKs (Src family protein tyrosine kinases), prevents the ischaemia-induced increases not only in the tyrosine phosphorylation of PSD-95, but also in the interaction between PSD-95 and Src kinases. PSD-95 is phosphorylated either by purified Src/Fyn kinases in vitro or by co-expression of constitutively active Src/Fyn in COS7 cells. The results suggest that SrcPTKs are involved in PSD-95 phosphorylation. The single Tyr(523) mutation to phenylalanine (Y523F) reduces the Src/Fyn-mediated phosphorylation of PSD-95 in COS7 cells and in vitro. As shown with a rabbit polyclonal antibody against phospho-PSD-95 (Tyr(523)), Tyr(523) phosphorylation is responsible for the increased tyrosine phosphorylation of PSD-95 induced by ischaemia in the rat hippocampus. In cultured hippocampal neurons, overexpression of PSD-95 Y523F, but not PSD-95 Y533F, abolishes the facilitating effect of PSD-95 on the glutamate- or NMDA-mediated currents, implying that PSD-95 Tyr(523) phosphorylation contributes to the post-ischaemic over-activation of NMDA receptors. Thus the present study reveals an additional mechanism for the regulation of PSD-95 by tyrosine phosphorylation. This mechanism may be of pathological significance since it is associated with excitotoxicity in the ischaemic brain.     

酪氨酸蛋白激酶Fyn突变体FynY531F与FynK299M活性的鉴定

Fyn是Src酪氨酸蛋白激酶家族（Src family of protein tyrosine kinases;Src PTKs）中的一员,在神经元的增殖、分化和存活中起着重要作用.本文利用分子克隆手段构建Fyn突变体FynY531F（酪氨酸突变为苯丙氨酸）和FynK299M（赖氨酸突变为甲硫氨酸）的真核表达载体,并通过检测突变体对NMDA受体亚基NR2A的磷酸化作用以鉴定其激酶活性.根据GenBank提供的大鼠Fyn的cDNA序列（NM\--012755）,采用RT-PCR扩增野生型 Fyn（wFyn）目的基因,应用定点突变技术扩增活化型Fyn（FynY531F）目的基因,并用重叠延伸PCR定点突变技术扩增失活型Fyn（FynK299M）目的基因,分别克隆入真核表达载体pcDNA3.1(+).免疫印迹分析显示,wFyn、FynY531F及FynK299M重组体均能在COS-7细胞表达.将构建的3个重组体分别与NMDA受体亚基NR2A的表达质粒共转染COS-7细胞.结果显示,活化型Fyn能够使NR2A酪氨酸磷酸化,而失活型Fyn则没有表现出激酶活性.结果表明,本文成功构建了野生型、活化型及失活型Fyn的真核表达载体,为进一步揭示Fyn在中枢神经系统中的病理生理意义奠定了基础.     

Regulation of TRPC6 channel activity by tyrosine phosphorylation

Various hormonal stimuli and growth factors activate the mammalian canonical transient receptor potential (TRPC) channel through phospholipase C (PLC) activation. However, the precise mechanism of the regulation of TRPC channel activity remains unknown. Here, we provide the first evidence that direct tyrosine phosphorylation by Src family protein-tyrosine kinases (PTKs) is a novel mechanism for modulating TRPC6 channel activity. We found that TRPC6 is tyrosine-phosphorylated in COS-7 cells when coexpressed with Fyn, a member of the Src family PTKs. We also found that Fyn interacts with TRPC6 and that the interaction is mediated by the SH2 domain of Fyn and the N-terminal region of TRPC6 in a phosphorylation-independent manner. In addition, we demonstrated the physical association of TRPC6 with Fyn in the mammalian brain. Moreover, we showed that stimulation of the epidermal growth factor receptor induced rapid tyrosine phosphorylation of TRPC6 in COS-7 cells. This epidermal growth factor-induced tyrosine phosphorylation of TRPC6 was significantly blocked by PP2, a specific inhibitor of Src family PTKs, and by a dominant negative form of Fyn, suggesting that the direct phosphorylation of TRPC6 by Src family PTKs could be caused by physiological stimulation. Furthermore, using single channel recording, we showed that Fyn modulates TRPC6 channel activity via tyrosine phosphorylation. Thus, our findings demonstrated that tyrosine phosphorylation by Src family PTKs is a novel regulatory mechanism of TRPC6 channel activity.     

Expression of polyglutamine-expanded huntingtin induces tyrosine phosphorylation of N-methyl-D-aspartate receptors

In our previous studies, we found that expression of polyglutamine-expanded huntingtin in HN33 cells induced sensitization of N-methyl-D-aspartate (NMDA) receptors (Sun, Y., Savinainen, A., and Liu, Y. F. (2001) J. Biol. Chem. 276, 24713-24718). Following this study, we investigated whether tyrosine phosphorylation of NMDA receptors might contribute to the altered property of the receptors. Expression of polyglutamine-expanded huntingtin induced elevation of phosphorylated or activated Src and increased targeting of PSD-95 (post-synaptic density 95) and activated Src to cell surface membrane. Expression of the mutated huntingtin also induced tyrosine phosphorylation of NR2B (NMDA receptor 2B) subunits, and co-expression of PSD-95 enhanced the phosphorylation. Treatment of SU6656 (a specific Src inhibitor) or co-expression of a mutated NR2B subunit with mutations of all three major tyrosine phosphorylation sites significantly attenuated neuronal toxicity induced by the mutated huntingtin. Addition of AP-5 did not further inhibit the neuronal toxicity. Taken together, our studies show that polyglutamine-expanded huntingtin increases tyrosine phosphorylation of NMDA receptors via PSD-95 and Src, and increased tyrosine phosphorylation may contribute to the sensitization of the receptors mediated by polyglutamine-expanded huntingtin.     

Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.     

Effect of Src kinase phosphorylation on disordered C-terminal domain of N-methyl-D-aspartic acid (NMDA) receptor subunit GluN2B protein

NMDA receptors are ligand-gated ion channels with a regulatory intracellular C-terminal domain (CTD). In GluN2B, the CTD is the largest domain in the protein but is intrinsically disordered. The GluN2B subunit is the major tyrosine-phosphorylated protein in synapses. Src kinase phosphorylates the GluN2B CTD, but it is unknown how this affects channel activity. In disordered proteins, phosphorylation can tip the balance between order and disorder. Transitions can occur in both directions, so it is not currently possible to predict the effects of phosphorylation. We used single molecule fluorescence to characterize the effects of Src phosphorylation on GluN2B. Scanning fluorescent labeling sites throughout the domain showed no positional dependence of the energy transfer. Instead, efficiency only scaled with the separation between labeling sites suggestive of a relatively featureless conformational energy landscape. Src phosphorylation led to a general expansion of the polypeptide, which would result in greater exposure of known protein-binding sites and increase the physical separation between contiguous sites. Phosphorylation makes the CTD more like a random coil leaving open the question of how Src exerts its effects on the NMDA receptor.