H-Ras Modulates N-Methyl-D-aspartate Receptor Function via Inhibition of Src Tyrosine Kinase Activity
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m302389200
ISSN1083-351X
AutoresClaire Thornton, Rami Yaka, Son Ta Dinh, Dorit Ron,
Tópico(s)Ion channel regulation and function
ResumoTyrosine 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. 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. The N-methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDA, N-methyl-d-aspartate; LTP, long term potentiation; PSD, postsynaptic density; GST, glutathione S-transferase; PBS, phosphate-buffered saline; WT, wild type; HA, hemagglutinin; aCSF, artificial cerebrospinal fluid; fEPSP, Field excitatory post-synaptic potential. 1The abbreviations used are: NMDA, N-methyl-d-aspartate; LTP, long term potentiation; PSD, postsynaptic density; GST, glutathione S-transferase; PBS, phosphate-buffered saline; WT, wild type; HA, hemagglutinin; aCSF, artificial cerebrospinal fluid; fEPSP, Field excitatory post-synaptic potential. receptor is a ligand-gated calcium channel that plays an essential role in neuronal development, addiction, and learning and memory (1Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61Google Scholar, 2Kumari M. Ticku M.K. Prog. Drug Res. 2000; 54: 152-189Google Scholar). Tyrosine phosphorylation modulates NMDA receptor function; for example, inhibition of tyrosine kinase activity decreases NMDA receptor-mediated currents, whereas treatment with tyrosine phosphatase inhibitors increases these currents (3Wang Y.T. Salter M.W. Nature. 1994; 369: 233-235Google Scholar). Tyrosine phosphorylation of NR2A and NR2B occurs via Fyn and Src protein-tyrosine kinases (4Lau L.F. Huganir R.L. J. Biol. Chem. 1995; 270: 20036-20041Google Scholar, 5Rostas J.A. Brent V.A. Voss K. Errington M.L. Bliss T.V. Gurd J.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10452-10456Google Scholar), resulting in potentiation of NMDA channel activity (6Kohr G. Seeburg P.H. J. Physiol. 1996; 492: 445-452Google Scholar, 7Yu X.M. Askalan R. Keil II, G.J. Salter M.W. Science. 1997; 275: 674-678Google Scholar). NMDA receptor phosphorylation by Src and Fyn is modulated by many mechanisms including protein kinase C (8Lu W.Y. Xiong Z.G. Lei S. Orser B.A. Dudek E. Browning M.D. MacDonald J.F. Nat. Neurosci. 1999; 2: 331-338Google Scholar) and the scaffolding protein RACK1 (9Yaka R. Thornton C. Vagts A.J. Phamluong K. Bonci A. Ron D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5710-5715Google Scholar). Thus, the modulation of NMDA receptor function by Src protein-tyrosine kinases likely involves the convergence of diverse signaling pathways generating a complex mechanism of receptor regulation. Glutamate activation of the NMDA receptor and the subsequent increase in intracellular calcium are essential for the induction of long term potentiation (LTP), a candidate mechanism underlying synaptic plasticity (10Bliss T.V. Collingridge G.L. Nature. 1993; 361: 31-39Google Scholar) thought to mediate learning and memory (11Rogan M.T. Staubli U.V. LeDoux J.E. Nature. 1997; 390: 604-607Google Scholar, 12McKernan M.G. Shinnick-Gallagher P. Nature. 1997; 390: 607-611Google Scholar). NMDA receptor-mediated LTP is associated with an increase in tyrosine phosphorylation of NR2 subunits via a mechanism that requires Src family protein-tyrosine kinases (5Rostas J.A. Brent V.A. Voss K. Errington M.L. Bliss T.V. Gurd J.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10452-10456Google Scholar). In the CA1 region of the hippocampus, activation of Src occurs within 5 min of LTP induction, and LTP can be prevented by application of Src-specific inhibitors (13Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Google Scholar). In addition, Fyn knockout mice show impaired LTP, which can be rescued by introduction of a Fyn transgene (14Grant S.G. O'Dell T.J. Karl K.A. Stein P.L. Soriano P. Kandel E.R. Science. 1992; 258: 1903-1910Google Scholar, 15Kojima N. Wang J. Mansuy I.M. Grant S.G. Mayford M. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4761-4765Google Scholar). Thus, both Src and Fyn are necessary for the induction of LTP (13Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Google Scholar, 14Grant S.G. O'Dell T.J. Karl K.A. Stein P.L. Soriano P. Kandel E.R. Science. 1992; 258: 1903-1910Google Scholar, 15Kojima N. Wang J. Mansuy I.M. Grant S.G. Mayford M. Kandel E.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4761-4765Google Scholar, 16Salter M.W. Biochem. Pharmacol. 1998; 56: 789-798Google Scholar). Recently, increased tyrosine phosphorylation of NR2A and NR2B and subsequent enhanced LTP was observed in the hippocampus of H-Ras null mice (17Manabe T. Aiba A. Yamada A. Ichise T. Sakagami H. Kondo H. Katsuki M. J. Neurosci. 2000; 20: 2504-2511Google Scholar). H-Ras functions as a molecular switch, existing in an active GTP-bound or inactive GDP-bound form (18Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Google Scholar). It is an upstream initiator of the mitogen-activated protein kinase pathway, which in neurons can be activated by the influx of calcium resulting from activation of the NMDA receptor (19Iida N. Namikawa K. Kiyama H. Ueno H. Nakamura S. Hattori S. J. Neurosci. 2001; 21: 6459-6466Google Scholar, 20Yun H.Y. Gonzalez-Zulueta M. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5773-5778Google Scholar, 21English J.D. Sweatt J.D. J. Biol. Chem. 1997; 272: 19103-19106Google Scholar). Many components of the H-Ras-mitogen-activated protein kinase pathway are associated with the postsynaptic density (PSD (22Suzuki T. Mitake S. Murata S. Brain Res. 1999; 840: 36-44Google Scholar)), a region of the synapse packed with signal transduction complexes, including Src family protein-tyrosine kinases, which colocalize with the NMDA receptor (23Kennedy M.B. Science. 2000; 290: 750-754Google Scholar, 24Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Google Scholar, 25Yamauchi T. Mass Spectrom. Rev. 2002; 21: 266-286Google Scholar). A number of Ras family regulatory proteins have also been identified in the PSD, suggesting that the H-Ras-mitogen-activated protein kinase pathway may have varied synapse-specific functions (26Kim J.H. Liao D. Lau L.F. Huganir R.L. Neuron. 1998; 20: 683-691Google Scholar, 27Chen H.J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Google Scholar, 28Okabe T. Nakamura T. Nishimura Y.N. Kohu K. Ohwada S. Morishita Y. Akiyama T. J. Biol. Chem. 2003; 278: 9920-9927Google Scholar, 29Ye B. Liao D. Zhang X. Zhang P. Dong H. Huganir R.L. Neuron. 2000; 26: 603-617Google Scholar, 30Pak D.T. Yang S. Rudolph-Correia S. Kim E. Sheng M. Neuron. 2001; 31: 289-303Google Scholar). Because NMDA receptor channel activity is positively regulated by Src tyrosine phosphorylation that is essential for LTP, we hypothesized that H-Ras negatively regulates NMDA phosphorylation and function via inhibition of a Src protein-tyrosine kinase. Here we present data showing that Src and H-Ras interact in vitro and in the brain. The interaction is specific for Src because Fyn, another member of the Src family expressed in the PSD, was unable to bind H-Ras. H-Ras binds via the Src kinase domain and inhibits Src kinase activity, decreasing phosphorylation and, subsequently, the membrane level of NR2A. Furthermore, overexpression of H-Ras in hippocampal slices results in a decrease in NMDA-mediated LTP. Taken together our results imply that H-Ras negatively regulates NMDA receptor channel activity by decreasing the number of NR2A-containing NMDA receptors in the synaptic membrane. Materials—Active recombinant Src and Fyn tyrosine kinases, H-Ras-GST-agarose, pUSE-SrcWT, pUSE-H-RasWT, Raf-1 H-Ras binding Domain GST beads, anti-Src-agarose, monoclonal anti-Src, anti-Fyn, and anti-H-Ras antibodies were purchased from Upstate Biotechnologies (Lake Placid, NY). Anti-Src[pY418] antibodies were purchased from BIOSOURCE (Camarillo, CA). Phosphatase inhibitor cocktails, polyclonal anti-H-Ras, and anti-c-Src antibodies were purchased from Sigma. Anti-HA antibodies, all secondary antibodies, protease inhibitor tablets, and Expand PCR system were purchased from Roche Applied Science. Sequencing and generation of primers was carried out by the Gallo Center molecular biology core. Restriction enzymes and TNT in vitro translation kit were purchased from Promega (Madison, WI). pGBK-T7 was purchased from Clontech (Palo Alto, CA). [35S]methionine (15 mCi/ml, 3000 Bq) and Amplify were from Amersham Biosciences. LipofectAMINE PLUS was purchased from Invitrogen. L(-tk) cells stably transfected with NR1+NR2A were a generous gift from Merck Sharp and Dohme. Animals—Src+/– and Fyn–/– mice (129vImJ/C57BL6J hybrids) were purchased from Jackson Laboratories. Fyn–/– mice were mated in house with 129 wild type mice to generate Fyn+/– mice. Src+/– and Fyn+/– mice were mated to generate Src –/– and Fyn–/– mice. The genotyping of mice was determined by reverse transcription-PCR analysis of products derived from tail mRNA. The mean age of animals used in this study was 4 weeks. Male Sprague-Dawley rats, 3–4 weeks old, were purchased from Simonsen. In Vitro Translations—[35S]Methionine-labeled proteins were generated in rabbit reticulocyte lysates (TNT kit, Promega) using the appropriate cDNAs. The translation reactions were analyzed by SDS-PAGE and fluorography. In Vitro Pull-down Assay—30 units of Src kinase or 75 units of Fyn kinase were incubated with 5 μg of H-Ras-GST-agarose or GST-Sepharose for 2 h at 4 °C with mixing. Agarose pellets were washed (1× PBS, 1% Triton X-100), and proteins were resolved by SDS-PAGE and analyzed by Western blotting using anti-Src (1:500), anti-Fyn (1:500), or anti-H-Ras (1:5000) monoclonal antibodies. The H-Ras-GST-agarose pull-down and competition assays using radiolabeled in vitro translated proteins were incubated and resolved as above. Gels were fixed in 40% methanol, 10% acetic acid, incubated in Amplify, and dried down. Radiolabeled proteins were detected by overnight fluorography (–80 °C). Plasmid Construction—Src deletion mutant cDNAs, Src-SH3 (amino acids 84–145), Src-SH2 (amino acids 151–238), and Src-KD (amino acids 270–523), were amplified from pUSE-SrcWT using the Expand PCR system and cloned into pGBKT7. H-RasWT cDNA was amplified from mRNA generated from NT2 cells and cloned into pTAT-HA via EcoRI and XhoI sites. Preparation of Brain Homogenates—Src –/– mice, Fyn–/– mice, and 3–4-week-old male Sprague-Dawley rats were euthanized with halothane, whole brain-dissected, and homogenized in homogenization buffer (250 mm sucrose, 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 10 mm EGTA, and protease and phosphatase inhibitors as per manufacturer's instructions). Membrane and cytosolic fractions were prepared as described previously (31Dunah A.W. Standaert D.G. J. Neurosci. 2001; 21: 5546-5558Google Scholar). Briefly, homogenates were centrifuged at 1000 × g for 2 min, yielding pellet (P1) and supernatant (S1) fractions. The S1 fraction was further centrifuged (10,000 × g, 30 min, 4 °C) generating fractions P2 (crude synaptosomal membranes) and S2 (cytosol and light membranes). Pellets were washed in cold PBS then solubilized in solubilization buffer (1% deoxycholate, 10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 10 mm EGTA, protease and phosphatase inhibitors as per the manufacturer's instructions). Immunoprecipitation—Immunoprecipitation was performed with 5 μg of the appropriate antibodies and 500 μg of protein homogenate as described previously (9Yaka R. Thornton C. Vagts A.J. Phamluong K. Bonci A. Ron D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5710-5715Google Scholar). Transfection—L(-tk) cells were cultured on 100-mm plates, and expression of NR1 and NR2A was induced as previously described (32Grimwood S. Le Bourdelles B. Atack J.R. Barton C. Cockett W. Cook S.M. Gilbert E. Hutson P.H. McKernan R.M. Myers J. Ragan C.I. Wingrove P.B. Whiting P.J. J. Neurochem. 1996; 66: 2239-2247Google Scholar). When 60% confluent, cells were transfected with a total of 10 μg of pUSE-SrcWT and pUSE-H-RasWT cDNA using LipofectAMINE PLUS in accordance with the manufacturer's instructions. Preparation of Cell Homogenates—L(-tk) cells were washed once with cold PBS, harvested, and resuspended in lysis buffer (20 mm Tris-HCl, pH 7.5, 10 mm EGTA, 2 mm EDTA, 0.25 m sucrose, 1% deoxycholate, and protease and phosphatase inhibitors as per the manufacturer's instructions). Samples were sonicated briefly, and lysis was allowed to proceed for 30 min on ice. Determination of the protein concentration was made using a BCA kit (Pierce). In addition, samples were also normalized with respect to SrcWT by Western blot and densitometry (NIH Image Version 1.62). Generation of Phospho-NR2A-specific Polyclonal Antibody—The NR2A-derived peptide RLLEGNFY(PO3)GSLFSV corresponding to amino acids 1318–1331 was generated by SynPep and used to immunize rabbits. Test bleeds were made over the course of three months, after which terminal bleeds were taken. The antigenicity of the bleeds was analyzed by slot blot using the immunizing peptide and showed increasing recognition of the antigen over three months. The terminal bleed was tested in the same way and then subsequently purified using the phospho-peptide coupled to an inert support. Specificity for the phosphorylated NR2A epitope was analyzed by slot blotting the immunizing phospho-peptide and a non-phospho form of the same epitope. No cross-reaction with the non-phospho-peptide was detected. Raf-1 Binding Assay—Raf-1 H-Ras binding domain GST-agarose and rat total brain homogenate (250 μg) were diluted in PBS and incubated for 30 min at 4 °C as per the manufacturer's instructions. Raf-1-H-Ras complexes were resolved by SDS-PAGE and analyzed by Western blotting using anti-H-Ras antibodies. Preparation of Tat-H-Ras Fusion Protein—pTAT-H-RasWT was expressed in and purified from Escherichia coli as previously described (33He D.Y. Vagts A.J. Yaka R. Ron D. Mol. Pharmacol. 2002; 62: 272-280Google Scholar). Tat fusion protein was detected using anti-HA antibodies. Immunohistochemistry—After incubation with 1 μm Tat-H-Ras, L(-tk) cells were washed in cold wash buffer (PBS, 0.1% Triton X-100), fixed in ice-cold methanol for 3 min, and blocked in wash buffer containing 0.3% normal goat serum for 4 h at room temperature. Immunofluorescence was performed using rat monoclonal anti-HA antibodies (1:100) incubated overnight at 4 °C. Staining was detected with secondary antibodies conjugated to Texas Red (1:500) incubated for 2 h at room temperature in the dark. Slides were mounted using Vectashield and viewed with a Zeiss 510 meta laser-scanning confocal microscope. Images shown are individual middle sections of projected Z series and were processed using Adobe Photoshop (Adobe Systems Inc). Preparation of Tat-H-Ras-treated Brain Slices—Coronal whole brain slices were prepared from 3–4 week old male Sprague-Dawley rats (300 μm), Src–/–, and Fyn–/– mice (250 μm). The slices were allowed to recover for at least 1 h in an artificial cerebrospinal fluid (aCSF) perfusion medium saturated with 95% O2, 5% CO2 containing 126 mm NaCl, 1.2 mm KCl, 1.2 mm NaH2PO3, 1.2 mm MgCl2, 2.4 mm CaCl2, 18 mm NaHCO3, and 11 mm glucose. Slices were incubated in either aCSF (Control) or 1 μm Tat-H-Ras diluted in aCSF (treated) for 2 h at room temperature, after which homogenates were made and fractionated as described above. Electrophysiology—Transverse hippocampal slices (350 μm) were prepared from 3–5-week-old male Sprague-Dawley rats. Slices were maintained for at least 2 h in aCSF that contained 126 mm NaCl, 1.2 mm KCl, 1.2 mm NaH2PO4, 0.01 mm MgCl2, 2.4 mm CaCl2, 18 mm NaHCO3, and 11 mm glucose saturated with 95% O2, 5% CO2 at 25 °C. After recovery, slices were submerged and continuously superfused with aCSF at 25 °C. Field excitatory post-synaptic potentials (fEPSPs) were recorded from stratum-radiatum of CA1 region with glass microelectrodes filled with 2 m NaCl. Picrotoxin (100 μm) was added to the bath solution to block GABAA receptor-mediated inhibitory postsynaptic potentials. To evoke fEPSPs, Schaffer collateral/commissural afferents were stimulated with 0.1-Hz pulses using steel bipolar microelectrodes at intensities adjusted to produce an evoked response that was 40–50% that of the maximum-recorded fEPSP for each recording. LTP was induced by high frequency stimulation (100 Hz, 1-s duration, 2 trains at 10-s intervals) at the same intensity as the test stimulus, and synaptic responses were monitored for 60 min after LTP induction. Data were collected using an Axopatch-1D amplifier (Axon instruments), filtered at 2 kHz, and digitized at 5–10 kHz. Compiled data were analyzed and expressed as the mean percent of fEPSP slope ±S.E. over the base-line levels. Src and H-Ras Interact in Vitro and in Brain—We set out to determine whether H-Ras negatively regulates the NMDA receptor through a Src-dependent mechanism. Previously Stancato et al. (34Stancato L.F. Silverstein A.M. Owens-Grillo J.K. Chow Y.H. Jove R. Pratt W.B. J. Biol. Chem. 1997; 272: 4013-4020Google Scholar) observed that H-Ras coimmunoprecipitates with Src in Sf9 insect cells. Because both Src and H-Ras are present in the PSD (24Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Nat. Neurosci. 2000; 3: 661-669Google Scholar), we hypothesized that H-Ras may regulate Src activity via a direct interaction. To confirm that Src and H-Ras interact directly, purified Src was incubated with H-Ras-GST-agarose or GST-Sepharose alone. H-Ras-GST-agarose was capable of “pulling down” Src (Fig. 1a, lane 3), whereas no interaction was observed between Src and GST-Sepharose (Fig. 1a, lane 4), suggesting a direct interaction between Src and H-Ras in vitro. To determine the specificity of this interaction, the pull-down experiment was repeated using another member of the Src family, Fyn, which shares 84% sequence homology with Src and is also expressed in the PSD (35Suzuki T. Okumura-Noji K. Biochem. Biophys. Res. Commun. 1995; 216: 582-588Google Scholar). There was no significant interaction detected between Fyn and H-Ras (Fig. 1b, lane 3), suggesting that the interaction between H-Ras and Src is specific and not a general property of the Src family of protein-tyrosine kinases. Next, to confirm that the Src-H-Ras interaction occurred in brain, we performed co-immunoprecipitation studies in rat brain homogenate (Fig. 1c). We found that anti-Src antibodies were capable of co-immunoprecipitating H-Ras (lane 1) and, conversely, that anti-H-Ras antibodies formed an immune complex with Src (lane 2). In summary, we show that Src and H-Ras can interact in vitro and in rat brain. H-Ras Interacts with Src via Its Kinase Domain—Src is comprised of a unique N-terminal region, an SH3 domain, an SH2 domain, and a C-terminal catalytic domain (36Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Google Scholar). To further characterize the Src–H-Ras interaction, we made constructs of various domains and expressed them as [35S]methionine-labeled proteins by in vitro translation (Fig. 2a). Radiolabeled Src-SH3, Src-SH2, and Src-KD were incubated with H-Ras-GST-agarose and analyzed by fluorography. Src-KD, but not SrcSH2 or SrcSH3, bound to H-Ras-GST-agarose, indicating that the kinase domain of Src contains a binding site for H-Ras (Fig. 2a, lane 6). To confirm the identification of the binding site, a competition assay was carried out in which H-Ras-GST-agarose was preincubated with either control (in vitro translated empty plasmid) or non-radiolabeled in vitro translated kinase domain (KD; Fig. 2b). Radiolabeled kinase domain (35S-labeled KD) was then added to the reaction, and the incubation was allowed to proceed. Assuming that the kinase domain is indeed the binding site for H-Ras, fewer binding sites would be available for 35S-labeled KD binding after preincubation with unlabeled KD. Indeed, there was a decrease in 35S-labeled KD detected after H-Ras-GST-agarose preincubation with unlabeled KD (lane 2) when compared with H-Ras-GST-agarose preincubation with control (lane 1). Therefore, the Src-H-Ras interaction was mediated at least in part through binding between H-Ras and the kinase domain of Src. H-Ras Inhibits Src Kinase Activity and Subsequent NR2A Phosphorylation—The activation state of Src is affected by its structure. Phosphorylation of the C-terminal tyrosine of Src (Tyr-529) by an upstream kinase, Csk, results in an intramolecular interaction between the phosphorylated residue and the Src-SH2 domain, inactivating Src (36Xu W. Doshi A. Lei M. Eck M.J. Harrison S.C. Mol. Cell. 1999; 3: 629-638Google Scholar, 37Nada S. Okada M. MacAuley A. Cooper J.A. Nakagawa H. Nature. 1991; 351: 69-72Google Scholar, 38Brown M.T. Cooper J.A. Biochim. Biophys. Acta. 1996; 1287: 121-149Google Scholar). Dephosphorylation or binding by other molecules displaces this folding, allowing Src autophosphorylation of Tyr-418 and activation (for review, see Refs. 39Thomas S.M. Brugge J.S. Annu. Rev. Cell Dev. Biol. 1997; 13: 513-609Google Scholar and 40Ma Y.C. Huang J. Ali S. Lowry W. Huang X.Y. Cell. 2000; 102: 635-646Google Scholar). Because H-Ras interacts via the kinase domain of Src (Fig. 2, a and b), we tested whether the binding of H-Ras to Src affected Src kinase activity. L(-tk) mouse fibroblast cells were transiently transfected with combinations of SrcWT and H-RasWT cDNA. Immunoprecipitation using anti-Src-agarose confirmed that in these cells, Src co-immunoprecipitated with H-Ras (Fig. 3a, lane 2). Therefore Src activity was measured in the presence of H-Ras using anti-Src[pY418], an antibody that recognizes active autophosphorylated Src. In cells transfected with SrcWT alone, anti-Src[pY418] detected a robust signal corresponding to Src at 60 kDa (Fig. 3b, top panel, lane 1), suggesting that transfected Src was active. Cotransfection of H-RasWT with SrcWT resulted in a sharp decrease in active Src (Fig. 3b, top panel, lane 2). Our results show that H-Ras significantly inhibits the ability of Src to autophosphorylate Tyr-418, implying that Src kinase activity is abrogated by the binding of H-Ras to the Src kinase domain. Because NR2A is a substrate for tyrosine phosphorylation by Src (6Kohr G. Seeburg P.H. J. Physiol. 1996; 492: 445-452Google Scholar), we analyzed the effect of H-Ras overexpression and the consequent inhibition of Src activity on the tyrosine phosphorylation state of NR2A. To do so we used L(-tk) cells stably transfected with NR1 and NR2A and transiently transfected them with combinations of SrcWT and H-RasWT cDNA. In addition, we generated a polyclonal rabbit antibody against tyrosine 1325-phosphorylated NR2A (41Yang M. Leonard J.P. J. Neurochem. 2001; 77: 580-588Google Scholar) and tested for the effects of H-Ras overexpression on basal level phosphorylation of NR2A. As predicted, there was a decrease in phosphorylated NR2A in SrcWT/H-RasWT-transfected cells compared with those transfected with Src alone (Fig. 3c). Thus, co-transfection with H-RasWT decreased Src kinase activity, consequently inhibiting Src-mediated NR2A phosphorylation. Active Tat-H-Ras Transduces into Cultured Cells and Brain and Inhibits Endogenous Src Kinase Activity—To determine whether H-Ras modulates endogenous Src kinase activity and NR2A-mediated channel function, we used the Tat fusion protein transduction system (42Nagahara H. Vocero-Akbani A.M. Snyder E.L. Ho A. Latham D.G. Lissy N.A. Becker-Hapak M. Ezhevsky S.A. Dowdy S.F. Nat. Med. 1998; 4: 1449-1452Google Scholar) to elevate H-Ras protein levels in brain slices. We generated a Tat-H-Ras construct and expressed and purified the fusion protein from E. coli as described previously (33He D.Y. Vagts A.J. Yaka R. Ron D. Mol. Pharmacol. 2002; 62: 272-280Google Scholar). First, we used immunofluorescence and confocal microscopy to confirm that the fusion protein was capable of entering cells. Control and Tat-H-Ras (1 μm)-transduced L(-tk) cells were fixed and analyzed using an antibody against an HA tag engineered into the Tat fusion protein (Fig. 4a). There was a clear antibody signal detected in cells treated with Tat-H-Ras (right panel) but not in control (left panel). Because the images shown represent the middle sections of a projected Z series, Tat-H-Ras has been successfully transduced through the cell membrane. Next, we tested Tat-H-Ras transduction in brain slices. Coronal rat whole brain slices were incubated in the absence or presence of Tat-H-Ras, homogenized, and fractionated to P2 (crude synaptosomes) and S2 (cell cytosol, light membranes) fractions. The integrity of the fractions was confirmed using an antibody against PSD-95 (data not shown), a protein enriched in the PSD (43Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Google Scholar), and Tat-H-Ras was detected in the P2 pellet fraction (Fig. 4b, lane 3) correlating with previous data showing that H-Ras is localized to the membrane (44Hancock J.F. Paterson H. Marshall C.J. Cell. 1990; 63: 133-139Google Scholar, 45Prior I.A. Harding A. Yan J. Sluimer J. Parton R.G. Hancock J.F. Nat. Cell Biol. 2001; 3: 368-375Google Scholar). To determine whether transduced Tat-H-Ras was functional, we utilized the Raf-1 binding assay (Fig. 4b). Initiation of the mitogen-activated protein kinase pathway requires H-Ras to be active (i.e. GTP-bound) before it can bind to and activate Raf-1, a kinase downstream of H-Ras (46Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Google Scholar). Thus, the activation state of Tat-H-Ras can be determined by its binding to the H-Ras binding domain on Raf-1. We found that transduced Tat-H-Ras did indeed bind to Raf-1 H-Ras binding domain (Fig. 4b, lane 3), suggesting that Tat-H-Ras was successfully transduced into brain, activated, and correctly compartmentalized. Because transfection of H-Ras caused a decrease in Src kinase activity in L(-tk) cells (Fig. 3a), we examined Src kinase activity in Tat-H-Ras-treated brain slices using anti-Src[pY418] antibodies. The pY418 signal was reduced in slices treated with Tat-H-Ras (Fig. 4c, top panel, lanes 1 and 3), implying that transduction of Tat-H-Ras decreases Src kinase activity through in vivo interaction with the Src kinase domain. Tat-H-Ras Decreases NR2A Retention and NR2A Phosphorylation in Synaptic Membranes—Interestingly, we found that the level of NR2A in the crude synaptosomal membrane fraction (P2) was decreased after transduction of Tat-H-Ras (Fig. 5a, top panel, lanes 1 and 3). In addition, there was a concomitant decrease in phosphorylated NR2A on Tat-H-Ras treatment (Fig. 5a, middle panel, lanes 1 and 3), consistent with the observations made for NR2A in L(-tk) cells (Fig. 3c). Because Src is capable of phosphorylating NR2B as well as NR2A in vitro (47Hisatsune C. Umemori H. Mishina M. Yamamoto T. Genes Cells. 1999; 4: 657-666Google Scholar) and because inhibition of Src-mediated phosphorylation by H-Ras decreased NR2A in the synaptic membrane, we examined whether H-Ras affects retention of NR2B in the synaptic membrane. There was no change in NR2B levels in the synaptic membrane after Tat-H-Ras treatment (Fig. 5b), suggesting that Src-mediated tyrosine phosphorylation plays a major role in specifically retaining NR2A-containing NMDA receptors in the membrane. Retention of NR2A in the Membrane Is Mediated by a Src-specific Mechanism—To confirm that the observed membrane depletion of NR2A was mediated via a Src tyrosine kinase mechanism, we transduced Tat-H-Ras into brain slices from Src –/– and Fyn –/– mice. Tat-H-Ras-transd
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