Regulation of the Neuron-specific Ras GTPase-activating Protein, synGAP, by Ca2+/Calmodulin-dependent Protein Kinase II
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m314109200
ISSN1083-351X
AutoresJeong Su Oh, Pasquale Manzerra, Mary B. Kennedy,
Tópico(s)Ion channel regulation and function
ResumosynGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-d-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca2+/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca2+/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ± 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-d-aspartate. synGAP is a neuron-specific Ras GTPase-activating protein found in high concentration in the postsynaptic density fraction from mammalian forebrain. Proteins in the postsynaptic density, including synGAP, are part of a signaling complex attached to the cytoplasmic tail of the N-methyl-d-aspartate-type glutamate receptor. synGAP can be phosphorylated by a second prominent component of the complex, Ca2+/calmodulin-dependent protein kinase II. Here we show that phosphorylation of synGAP by Ca2+/calmodulin-dependent protein kinase II increases its Ras GTPase-activating activity by 70-95%. We identify four major sites of phosphorylation, serines 1123, 1058, 750/751/756, and 764/765. These sites together with other minor phosphorylation sites in the carboxyl tail of synGAP control stimulation of GTPase-activating activity. When three of these sites and four other serines in the carboxyl tail are mutated, stimulation of GAP activity after phosphorylation is reduced to 21 ± 5% compared with 70-95% for the wild type protein. We used phosphosite-specific antibodies to show that, as predicted, phosphorylation of serines 765 and 1123 is increased in cultured cortical neurons after exposure of the neurons to the agonist N-methyl-d-aspartate. Storage of information in the brain is mediated in part by changes in the strength of synaptic connections between neurons initiated by specific patterns of electrical activity (1Sjostrom P.J. Nelson S.B. Curr. Opin. Neurobiol. 2002; 12: 305-314Crossref PubMed Scopus (179) Google Scholar). These changes involve complex regulatory pathways that are controlled by the pattern of influx of Ca2+ ion through N-methyl-d-aspartate (NMDA) 1The abbreviations used are: NMDA, N-methyl-d-aspartate; CaMKII, calmodulin-dependent protein kinase II; PSD, postsynaptic density; GST, glutathione S-transferase; GAP, GTPase-activating protein; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DIV, days in vitro; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry; HCSS, HEPES-control salt solution; ctm, carboxyl-terminal synGAP mutant. 1The abbreviations used are: NMDA, N-methyl-d-aspartate; CaMKII, calmodulin-dependent protein kinase II; PSD, postsynaptic density; GST, glutathione S-transferase; GAP, GTPase-activating protein; HPLC, high performance liquid chromatography; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; DIV, days in vitro; DTT, dithiothreitol; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MS/MS, tandem mass spectrometry; HCSS, HEPES-control salt solution; ctm, carboxyl-terminal synGAP mutant.-type glutamate receptors (NMDA receptors) at postsynaptic sites. Much present research concerns the nature of the relevant biochemical pathways and the mechanisms of Ca2+ control. One set of regulatory proteins associates tightly with the cytosolic portion of the NMDA receptor (2Sheng M. Sala C. Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1029) Google Scholar, 3Kennedy M.B. Science. 2000; 290: 750-754Crossref PubMed Scopus (647) Google Scholar). These include Ca2+/calmodulin-dependent protein kinase II (CaMKII) (4Leonard A.S. Lim I.A. Hemsworth D.E. Horne M.C. Hell J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3239-3244Crossref PubMed Scopus (323) Google Scholar, 5Bayer K.U. De Koninck P. Leonard A.S. Hell J.W. Schulman H. Nature. 2001; 411: 801-805Crossref PubMed Scopus (552) Google Scholar), which is activated by Ca2+ flux through the receptor, and several proteins that are held near the receptor by the scaffold protein PSD-95 (6Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (997) Google Scholar, 7Kornau H.-C. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1611) Google Scholar). A complex containing many of these proteins can be isolated from brain homogenates and is called the postsynaptic density (PSD) fraction (8Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). synGAP was identified as a prominent 140-kDa protein in the PSD fraction (9Chen H.-J. Kennedy M.B. Abstr. Soc. Neurosci. 1997; 23 (abstr.)1466Google Scholar, 10Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) and as a protein that interacts with PSD-95 in a yeast two-hybrid screen (11Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Its message is detected only in brain (10Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar, 11Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). It is expressed only in neurons, including most excitatory neurons and a subset of inhibitory neurons (12Zhang W. Vazquez L. Apperson M.L. Kennedy M.B. J. Neurosci. 1999; 19: 96-108Crossref PubMed Google Scholar), where it is highly localized to the postsynaptic density (10Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). It contains a PH domain, a C2 domain, and a Ras GAP domain that are 23%, 33%, and 47% similar, respectively, to those of the prototype Ras GAP protein p120 Ras GAP (13McCormick F. Martin G.A. Clark R. Bollag G. Polakis P. Cold Spring Harbor Symp. Quant. Biol. 1991; 56: 237-241Crossref PubMed Scopus (19) Google Scholar). In brain homogenates, synGAP is tightly bound to the particulate fraction and full-length synGAP has not yet been purified in soluble form; however, the GAP domain, expressed as a GST fusion protein in Escherichia coli, has been shown to stimulate hydrolysis of bound GTP by Ras (11Kim J.H. Liao D. Lau L.-F. Huganir R.L. Neuron. 1998; 20: 683-691Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). synGAP plays a crucial role in early development of the brain and in control of synaptic plasticity in the adult brain, as demonstrated by the phenotypes of mouse synGAP mutants (14Komiyama N.H. Watabe A.M. Carlisle H.J. Porter K. Charlesworth P. Monti J. Strathdee D.J. O'Carroll C.M. Martin S.J. Morris R.G. O'Dell T.J. Grant S.G. J. Neurosci. 2002; 22: 9721-9732Crossref PubMed Google Scholar, 15Kim J.H. Lee H.K. Takamiya K. Huganir R.L. J. Neurosci. 2003; 23: 1119-1124Crossref PubMed Google Scholar). Newborn mice with a deletion of synGAP die a few days after birth; whereas adult mice heterozygous for the deletion have altered synaptic plasticity (14Komiyama N.H. Watabe A.M. Carlisle H.J. Porter K. Charlesworth P. Monti J. Strathdee D.J. O'Carroll C.M. Martin S.J. Morris R.G. O'Dell T.J. Grant S.G. J. Neurosci. 2002; 22: 9721-9732Crossref PubMed Google Scholar). In our first report of the characterization of synGAP, we showed that it is a prominent substrate for CaMKII in the postsynaptic density fraction and presented evidence that phosphorylation by CaMKII reduced its GAP activity ∼50% (10Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). We later reported that this inhibition was an artifact caused by the combined presence of residual ATP from the phosphorylation reaction and the phosphatase inhibitor pyrophosphate in our Ras GAP assay (16Oh J.S. Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 2002; 33: 151Abstract Full Text Full Text PDF Scopus (6) Google Scholar). Here we have identified four of the principal phosphorylation sites for CaMKII in synGAP and re-examined the regulatory effect of their phosphorylation. We report that phosphorylation of these sites by CaMKII produces a 70-95% increase in the Ras GAP activity of synGAP. Because CaMKII is a major target of calcium influx through the NMDA receptor, this finding suggests that one result of activation of NMDA receptors at a synapse may be an increase in the rate of inactivation of Ras at that synapse. This mechanism might provide a means by which NMDA receptor activation modulates the action of receptor tyrosine kinases at excitatory synapses. Materials—Acetonitrile, UV/HPLC grade, was purchased from EM Science (Gibbstown, NJ); HPLC/Spectra grade trifluoroacetic acid from Pierce (Rockford, IL); iodoacetamide from Sigma (St. Louis, MO); [γ-32P]ATP and [α-32P]GTP from ICN Pharmaceuticals Inc. (Irvine, CA); modified sequencing grade trypsin from Promega (Madison, WI); C18 reversed-phase HPLC columns (4.6 × 250 mm) from Vydac (Hesperia, CA); cellulose-coated TLC sheets (20 × 20 mm) from EM Science; glutathione-agarose from Sigma; and PhosphorImager screens and scanner from Amersham Biosciences (Piscataway, NJ). Calmodulin was purchased from Calbiochem (San Diego, CA). CaMKII was purified from rat forebrain as previously described (17Miller S.G. Kennedy M.B. J. Biol. Chem. 1985; 260: 9039-9046Abstract Full Text PDF PubMed Google Scholar). Preparation of Postsynaptic Density Fraction from Rat Brain—The crude PSD fraction was prepared as described previously (6Cho K.-O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (997) Google Scholar) by a modification of the method of Carlin et al. (18Carlin R.K. Grab D.J. Cohen R.S. Siekevitz P. J. Cell Biol. 1980; 86: 831-843Crossref PubMed Scopus (597) Google Scholar). Expression and Purification of GST Fusion Proteins Containing Portions of synGAP—A vector for expression of a fusion protein containing a portion of the carboxyl tail of synGAP (residues 946-1167) fused to the carboxyl terminus of glutathione S-transferase (GST-ctsynGAP) was constructed in the pGEX plasmid, according to the manufacturer's instructions (Amersham Biosciences). The fusion protein contained the sequence of synGAP encoded by positions 2836-3501 in the synGAP cDNA (accession number AF048976). E. coli cells transformed with the plasmids were grown in cultures containing 50 μg/ml ampicillin at 37 °C. At mid-log phase, expression was induced by addition of 0.1 mm isopropyl-1-thio-β-d-galactopyranoside and continued until late log phase. Cells were harvested and frozen at -80 °C. Cells were lysed by sonication in 137 mm NaCl, 2.7 mm KCl, 4.3 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.3 (PBS), containing 1% Triton X-100, proteinase inhibitor mixture (Roche Applied Science, Mannheim, Germany), 0.1 mm PMSF, 0.5 mm dithiothreitol (DTT). After centrifugation at 15,000 × g for 10 min, lysate supernatants were incubated at room temperature for 2 h, or at 4 °C overnight with glutathione-agarose beads. In some cases, to recover more fusion protein, the pellets were also resuspended in PBS plus 1% n-lauroyl sarcosine, 1% Triton X-100, and 0.1 mm PMSF. The suspension was sonicated and subjected to centrifugation at 15,000 × g. The supernatant from this spin was pooled with the lysate supernatants and incubated with glutathione-agarose beads for 2 h at 4 °C. The bead suspension was transferred to a column, and the beads were washed with PBS. GST fusion proteins were eluted in 50 mm Tris (pH 8.0), 20 mm reduced glutathione, and 0.1 mm PMSF at 4 °C. The protein concentration was determined by a modification of the method of Lowry (19Peterson G.L. Methods Enzymol. 1983; 91: 95-119Crossref PubMed Scopus (1136) Google Scholar) and stored at -80 °C. Expression of synGAP in Insect Cells—The entire sequence encoding synGAP was inserted into plasmid pVL1392 (BD Pharmingen, San Diego, CA) at EcoR1 and BamH1 restriction sites, which added a FLAG tag to the amino terminus. The recombinant FLAG-tagged synGAP (rsynGAP) was expressed in Hi-5 insect cells by the Caltech Protein Expression Laboratory. Cells were harvested by centrifugation, and the cell pellets were frozen at -80 °C. Pellets were resuspended in 20 mm Tris-Cl (pH 8.0), 2 mm EDTA, 2 mm DTT, 0.1 mm PMSF, 0.5% Triton X-100, 1 μg/ml deoxyribonuclease I, and protease inhibitor mixture (Roche Applied Science) and lysed by homogenization at 4 °C. Nuclei were removed by centrifugation at 100 × g, and membranes were harvested at 100,000 × g. Like endogenous synGAP, rsynGAP is tightly bound to membranes. All attempts to extract it resulted in loss of GAP activity. Membrane fractions from control insect cells in which synGAP was not present had no detectable GAP activity (data not shown); thus, we are able to reliably measure synGAP activity in the membrane fractions. Recombinant synGAP in the membranes was detected by immunoblotting with an anti-FLAG antibody (Sigma). Phosphorylation of synGAP and GST-ctsynGAP by CaMKII—Phosphorylation by purified CaMKII was carried out in a reaction mix containing 50 mm Tris-HCl (pH 8.0), 10 mm MgCl2, 0.7 mm CaCl2, 0.4 mm EGTA, 30 μm [γ-32P]ATP (1000-3000 cpm/pmol) or 30 μm ATP, 10 μg/ml calmodulin, 10 mm DTT, 3 μg of purified rat brain CaMKII and Hi-5 cell membranes containing 1.5-2 μg of rsynGAP, or 45 ng of purified CaMKII and 3 μg of GST-ctsynGAP. Phosphorylation was initiated by addition of CaMKII and ATP to a 30-μl reaction mix prewarmed to 30 °C for 2 min. The reaction was carried out for 2 min, or as indicated, and stopped by the addition of SDS-PAGE sample buffer. The mixture was placed in a boiling water bath for 3 min then samples were fractionated by SDS-PAGE on 10% gels for GST-ctsynGAP, and 7.5% gels for rsynGAP. The gels were dried and exposed to x-ray film to identify phosphorylated proteins. To quantify the amount of phosphate incorporated, the level of 32P in the bands was determined with a Storm PhosphorImager (Amersham Biosciences). The relative densities measured by the Imager were converted to counts per minute by comparison to signals from standard amounts of [32P]phosphate spotted onto filter paper and imaged at the same time. Determination of Stoichiometry of Phosphorylation of synGAP by CaMKII—The moles of synGAP per milligram of protein in either the postsynaptic density or the membranes of Hi-5 insect cells was determined from immunoblots containing increasing amounts of protein from each sample, labeled with affinity-purified primary antibodies specific for synGAP (Affinity Bioreagents, Golden, CO), and secondary antibodies conjugated to Alexa fluor-488 (Molecular Probes, Eugene, OR). The labeling was quantified with the use of the Storm system and compared with the labeling of standard amounts of GST-ctsynGAP protein, which contains the epitopes of synGAP used to prepare the primary antibody. We then calculated the nanomoles of phosphate incorporated into the synGAP band (determined as described above) per nanomoles of total synGAP. Trypsinization of Phosphorylated GST Fusion Proteins and Recombinant synGAP—Proteins were phosphorylated as described above with the following modifications. The reaction was conducted in a volume of 500 μl containing 80-120 μg/ml GST fusion protein or 300-400 μg/ml Hi-5 membrane protein containing recombinant synGAP. The reaction mixture was preincubated at 30 °C for 5 min, and the reaction was initiated by addition of 2-4 μg/ml CaMKII for phosphorylation of GST fusion protein or 10-20 μg/ml CaMKII for phosphorylation of membrane-bound recombinant synGAP in the presence of 30 μm [γ-32P]ATP (1500-3000 cpm/pmol). The reaction was continued for 5, 15, or 30 min at 30 °C and stopped by addition of 3× SDS sample buffer. The mixture was placed in a boiling water bath for 3 min then fractionated by SDS-PAGE. The gel was stained with Coomassie Blue R-250, and the appropriate band of GST fusion protein or recombinant synGAP was identified by comparison to molecular weight markers. Bands were excised, chopped into small pieces, and transferred to 1.5-ml Eppendorf tubes. The pieces were incubated for 30 min at 37 °C in 2 mm tris-(2-carboxyethyl)phosphine hydrochloride, 50% acetonitrile, 0.5 m ammonium bicarbonate (pH > 8) to reduce the protein and destain the gel piece. The protein was then alkylated by transferring the gel piece to 25 mm iodoacetamide, 50% acetonitrile, 25 mm ammonium bicarbonate and incubating at room temperature in the dark for 20 min. Gel pieces were rinsed with 50 mm ammonium bicarbonate. Proteins were then trypsinized in the gel as previously described (20Hellman U. Wernstedt C. Gonez J. Heldin C.-H. Analyt. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (684) Google Scholar). HPLC Fractionation of Phosphopeptides—Trypsinized phosphopeptides were fractionated by HPLC on a C18 reversed-phase column developed at 1 ml/min with a gradient of 0-30% acetonitrile in 0.1% trifluoroacetic acid. Radioactivity in each 0.5-ml fraction was measured in a Beckman LS 7800 scintillation counter by detection of Cerenkov radiation. Mass Spectrometry and Sequencing of Phosphopeptides—Mass spectrometry was conducted by the Protein and Peptide Microanalytical Laboratory at Caltech with a PerSeptive Biosystems/Vestec Lasertech II reflector for matrix-assisted, laser desorption ionization, time-of-flight (MALDI-TOF) mass spectrometry. Data were collected in both linear and reflector modes. Serine phosphopeptides are reliably identified by appearance of a new peptide in reflector mode that is reduced in mass by ∼98 atomic mass units from the parent because of cleavage of a phosphate group from the parent. The identity of some of the phosphopeptides that we detected was confirmed by amino acid sequencing by Tandem mass spectrometry (MS/MS). Identification of Tryptic Peptides with Peptidesort Software—The Peptidesort software package (GCG, Accyleris, Inc.) permits identification of a peptide from its molecular mass and its relative retention time during HPLC. We used the program to predict all possible tryptic peptides, sorted by retention time and molecular mass, from the amino acid sequence of synGAP. We compared the retention times from HPLC and the molecular masses of each phosphopeptide that we detected by mass spectrometry with those predicted by the program. Mass spectrometry measures the mass of the peptide fragment plus a proton, whereas the program predicts the mass of the corresponding hydrolysis product. Therefore, for comparison, we subtracted 17 atomic mass units from the mass of each peptide predicted by the program. After this correction, unless noted, the differences between masses predicted by the program, and those of peptides determined by mass spectrometry and reported in Tables I and II (see below), were less than 1 atomic mass unit.Table IIdentification of phosphopeptides in HPLC fractions shown in Fig. 4HPLC fractionsIdentified sitesPeptide mass (MALDI-TOF)Predicted peptide massbThe mass of peptides predicted from tryptic hydrolysis in Peptidesort software were corrected by subtracting 17 atomic mass units to match the M + H+ mass measured by MALDI-TOF.Residues in synGAPLinearReflectedaMass of peptide after neutral loss of phosphate in reflector mode.atomic mass units77cIdentity of phosphopeptide was confirmed by sequencing by MS/MS as described under "Experimental Procedures."S10581678.781581.721580.871056-107095cIdentity of phosphopeptide was confirmed by sequencing by MS/MS as described under "Experimental Procedures."S10992054.101956.321957.101097-1120103cIdentity of phosphopeptide was confirmed by sequencing by MS/MS as described under "Experimental Procedures."S11232045.281947.831948.101121-1138202S1058 or 7 Ss, 3 Ts3770.063672.463674.06 or 3673.901056-1090 or 970-1004a Mass of peptide after neutral loss of phosphate in reflector mode.b The mass of peptides predicted from tryptic hydrolysis in Peptidesort software were corrected by subtracting 17 atomic mass units to match the M + H+ mass measured by MALDI-TOF.c Identity of phosphopeptide was confirmed by sequencing by MS/MS as described under "Experimental Procedures." Open table in a new tab Table IIIdentification of phosphopeptides in HPLC fractions shown in Fig. 6HPLC fractionsIdentified sitesPeptide mass (MALDI-TOF)Predicted peptide massbThe mass of peptides predicted from tryptic hydrolysis in Peptidesort software were corrected by subtracting 17 atomic mass units to match the M + H+ mass measured by MALDI-TOF.Residues in synGAPLinearReflectedaMass of peptide after neutral loss of phosphate in reflector mode.atomic mass units79S1093/10951023.6926.3926.11089-109688S764/7651161.61064.21064.2761-770214S750/751/7561676.81579.31579.7747-760a Mass of peptide after neutral loss of phosphate in reflector mode.b The mass of peptides predicted from tryptic hydrolysis in Peptidesort software were corrected by subtracting 17 atomic mass units to match the M + H+ mass measured by MALDI-TOF. Open table in a new tab Assay of Ras GTPase-activating Activity—GAP assays of 20-35 μg of PSD and 5-15 μg of recombinant synGAP were performed after phosphorylation as described above, in the absence or presence of 0.3 mm free Ca2+ and 10 μg/ml calmodulin for 2 min at 30 °C, but in a final volume of 30 μl. In some reactions, antibody 6G6 against PSD-95 or inhibiting antibodies 4A11 and 6E9 (21Molloy S.S. Kennedy M.B. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4756-4760Crossref PubMed Scopus (100) Google Scholar) against CaMKII (20 μg each of IgG partially purified from Ascites fluid by precipitation with 50% ammonium sulfate) were included in the reaction. The phosphorylation reactions were stopped by addition of 60 μl of a mixture to bring the solution to 0.66 mm EGTA, 11.7 μm okadaic acid, 22 mm Tris-Cl (pH 7.5), 9.3 mm MgCl2, 111 mm NaCl, and 2.2 mm DTT. The GAP assay was then initiated by addition of 10 μl containing 2 pmol of [α-32P]GTP-bound GST-Ras fusion protein (10Chen H.-J. Rojas-Soto M. Oguni A. Kennedy M.B. Neuron. 1998; 20: 895-904Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) for a total volume of 100 μl of GAP reaction mixture (22Bollag G. McCormick F. Methods Enzymol. 1995; 255: 161-170Crossref PubMed Scopus (40) Google Scholar). The reactions were carried out at 30 °C for 15 min, stopped by addition of 600 μl of ice-cold 5% glutathione-agarose beads and 80 μl of ice-cold 50 mm EDTA, and then incubated at 4 °C for 45 min on an end-over-end mixer. Beads were washed three times (23Smith D.B. Corcoran L.M. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. K. S. Current Protocols in Molecular Biology. Vol. 2. Wiley Interscience, New York1995: 1671-1677Google Scholar), nucleotides were dissociated from the column-bound GST-Ras, and [α-32P]GTP and [α-32P]GDP were separated by thin layer chromatography (22Bollag G. McCormick F. Methods Enzymol. 1995; 255: 161-170Crossref PubMed Scopus (40) Google Scholar). The separated nucleotides were visualized by PhosphorImager analysis (Amersham Biosciences) and quantified with ImageQuaNT software to determine the percent GDP generated in the assay (GDP/[GDP + GTP] × 100), a measure of Ras GTPase activity. Site-directed Mutagenesis of Recombinant synGAP—We prepared various mutant constructs to study the effect of mutation of the identified phosphorylation sites to alanine (serines 764, 765, 1058, 1062, 1064, 1093, 1095, and 1123). Mutagenic oligonucleotides (18-to 25-mer) that contained alanine instead of the identified serine were synthesized at the Caltech Oligonucleotide Synthesis Laboratory. The oligonucleotides were phosphorylated at the 5′ end by T4 kinase then annealed to the denatured synGAP plasmid (pVL1392) at room temperature for 30 min. The oligonucleotides were extended with T4 DNA polymerase and T4 DNA ligase in vitro to generate a hemi-methylated, double-stranded DNA molecule. A restriction digestion was performed with Dpn-1 to eliminate non-mutant plasmid DNA (those with two methylated strands). The DNA molecules were then transformed into the E. coli mutS strain (deficient in the methylation-specific repair system), and colonies were screened by DNA sequencing for plasmids containing the alanine mutations (24Kramer B. Kramer W. Fritz H.J. Cell. 1984; 38: 879-887Abstract Full Text PDF PubMed Scopus (338) Google Scholar). Plasmids containing the desired mutations were transformed into E. coli DH5α for propagation, and mutations were confirmed by DNA sequencing. Production of Phosphosite-specific Antibodies—Synthetic peptides with the sequence ITKQH-S(PO3)-QTPSTC (P-S1123) and RGLNSS(PO3)-MDMARC (P-S765) were purchased from SynPep (Dublin, CA). Purified peptides were conjugated via succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate to keyhole limpet hemocyanin. Rabbit antisera against the conjugated P-S1123 and P-S765 were raised by CoCalico Biologicals (Reamstown, PA) and Sigma Genosis (The Woodlands, TX), respectively. Specificity and optimum dilution were determined for each bleed by immunoblotting against 15-20 μg of PSD fraction phosphorylated by endogenous CaMKII, and of equivalent nonphosphorylated PSD fraction. Antibodies from sera that contained a high titer specific for phospho-synGAP were purified by peptide affinity chromatography. Five milligrams of P-S1123 or P-S765 peptide was conjugated to 5 ml of Sulfolink coupling resin (Pierce, Rockford, IL) according to the manufacturer's instructions. The coupled resin was then mixed for 1 h with a blocker consisting of 0.1 m cysteine in TE85 (50 mm Tris, pH 8.5, 5 mm EDTA), then washed twice with 5 volumes each of TE85, TE85, 1 m NaCl, and finally with 50 mm Tris, pH 7.5, 5 mm EDTA (TE75). Before each use, the resin was blocked for 1 h with 3 volumes of TE75, 20% non-immune rabbit serum, washed with TE75, G elution buffer (Pierce), and finally re-equilibrated with TE75 buffer. IgG from ∼25 ml of serum was concentrated by precipitation in 50% ammonium sulfate, 0.1 m Tris-Cl, pH 7.5. The pellet was redissolved in 50 ml of TE75 and dialyzed against two changes of the same buffer. The dialyzed protein was stirred with the peptide resin for 2 h, poured into a column, and washed with 10 column volumes of TE75. Bound IgG was eluted in Gentle Elution Buffer (Pierce), collecting 1-ml fractions into tubes containing 0.1 ml of 1 m Tris-Cl, pH 7.5. Protein was detected in each fraction by absorbance at 280 nm. Fractions containing high amounts of protein were further characterized by immunoblotting against both phosphorylated and nonphosphorylated PSD protein. Fractions with the highest concentration of phosphosite-specific antibodies were pooled. Preparation of Cortical Neuronal Cultures—Cultures of cortical neurons with less than 1% astrocytes were prepared from fetal mice (15-16 days gestation) as previously described (25Rose K. Goldberg M. Choi D.W. Tyson C. Frazier J. In Vitro Biological Methods. Methods in Toxicology. Academic Press, San Diego1993: 46-60Google Scholar) in 24-well plates coated with 50 ng/ml poly-d-lysine (Sigma) and 2 ng/ml laminin (BD Biosciences) and Neurobasal medium (Invitrogen), B27 supplement (Invitrogen), 0.5 mm Glutamax I (Invitrogen), 25 μm glutamate, and 25 μmβ-mercaptoethanol. After 3 days in vitro (DIV), cytosine arabinoside (Sigma) was added (10 μm) to halt the growth of non-neuronal cells. Cells were used at 13-14 DIV. Cell Treatment and Protein Extraction —Cell cultures (13-14 days in vitro (DIV)) were washed three times in HEPES-control salt solution (HCSS) containing (in mm): 120 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 10 NaOH, 20 HEPES, and 5.5 glucose, pH 7.4, and then half were exposed to 25 μm N-methyl-d-aspartic acid (NMDA) dissolved in HCSS or to an equal amount of additional HCSS for 15 s (2-4 wells for each condition). After treatment, cells were washed quickly with ice-cold PBS and extracted with lysis buffer (1% SDS, 20 mm Tris-Cl, pH 7.5, 10 mm EGTA, 40 mm β-glycerophosphate, 2.5 mm MgCl2, 2 mm orthovanadate, and complete mini protease inhibitor mixture (Roche Applied Science)). Extracts were heated at 90 °C for 5 min, and insoluble material was removed by centrifugation at 14,000 × g for 30 min. Protein concentrations were determined by the bicinchoninic acid method (Pierce) using bovine serum albumin as standard. Western Blotting—To determine synGAP phosphorylation, 5 μg of protein samples was dissolved in SDS-PAGE sample buffer, heated at 90 °C for 5 min, fractionated by SDS-PAGE on 8% gels, and transferred to nitrocellulose membranes (Schleicher & Schuell) in transfer buffer containing 50 mm Tris, 380 mm glycine, 0.1% SDS, and 20% methanol. Membranes were blocked with 5% milk in TBS-T buffer (20 mm Tris, 150 mm NaCl, 0.05% Tween 20) and were
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