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Mind Bomb-2 Is an E3 Ligase That Ubiquitinates the N-Methyl-d-aspartate Receptor NR2B Subunit in a Phosphorylation-dependent Manner

2007; Elsevier BV; Volume: 283; Issue: 1 Linguagem: Inglês

10.1074/jbc.m705580200

1083-351X

Rachel Jurd, Claire Thornton, Jun Wang, Ken Luong, Khanhky Phamluong, Viktor Kharazia, Stuart L. Gibb, Dorit Ron,

Cancer-related Molecular Pathways

The N-methyl-d-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination. The N-methyl-d-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination. The N-methyl-d-aspartate receptors (NMDARs) 4The abbreviations used are:NMDARN-methyl-d-aspartate receptorFynCAconstitutively active Fyn kinaseHEKhuman embryonic kidneyLTPlong-term potentiationMib2Mind bomb-2PSDpostsynaptic densityUPSubiquitin-proteasome systemHAhemagglutininGFPgreen fluorescent proteinPIphosphatidylinositolaaamino acidsMBPmaltose-binding protein are glutamate-gated ion channels that are important in synaptic plasticity events in the mammalian brain, and are comprised of an obligatory NR1 subunit and modulatory NR2 (A-D) or NR3 subunits (1Cull-Candy S. Brickley S. Farrant M. Curr. Opin. Neurobiol. 2001; 11: 327-335Crossref PubMed Scopus (1414) Google Scholar). While the NR1 subunit has a short intracellular tail, the NR2 subunits have large intracellular C-terminal tails that directly interact with proteins that play essential roles in the regulation of NMDAR function (2Wenthold R.J. Prybylowski K. Standley S. Sans N. Petralia R.S. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 335-358Crossref PubMed Scopus (293) Google Scholar, 3Sheng M. Kim M.J. Science. 2002; 298: 776-780Crossref PubMed Scopus (597) Google Scholar). N-methyl-d-aspartate receptor constitutively active Fyn kinase human embryonic kidney long-term potentiation Mind bomb-2 postsynaptic density ubiquitin-proteasome system hemagglutinin green fluorescent protein phosphatidylinositol amino acids maltose-binding protein The intracellular domains of NMDAR subunits are phosphorylated by a number of protein kinases. For example, Src-family protein-tyrosine kinases (PTKs), such as Src and Fyn, phosphorylate specific tyrosine sites within the NR2A and NR2B intracellular tails (4Suzuki T. Okumura-Noji K. Biochem. Biophys. Res. Commun. 1995; 216: 582-588Crossref PubMed Scopus (142) Google Scholar, 5Yaka R. He D.Y. Phamluong K. Ron D. J. Biol. Chem. 2003; 278: 9630-9638Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 6Nakazawa T. Komai S. Tezuka T. Hisatsune C. Umemori H. Semba K. Mishina M. Manabe T. Yamamoto T. J. Biol. Chem. 2001; 276: 693-699Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 7Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar), and this positively modulates channel function (5Yaka R. He D.Y. Phamluong K. Ron D. J. Biol. Chem. 2003; 278: 9630-9638Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 7Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar, 8Wang Y.T. Salter M.W. Nature. 1994; 369: 233-235Crossref PubMed Scopus (598) Google Scholar, 9Kohr G. Seeburg P.H. J. Physiol. (Lond). 1996; 492: 445-452Crossref Scopus (279) Google Scholar, 10Dunah A.W. Standaert D.G. J. Neurosci. 2001; 21: 5546-5558Crossref PubMed Google Scholar, 11Yaka R. Thornton C. Vagts A.J. Phamluong K. Bonci A. Ron D. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5710-5715Crossref PubMed Scopus (162) Google Scholar, 12Hallett P.J. Spoelgen R. Hyman B.T. Standaert D.G. Dunah A.W. J. Neurosci. 2006; 26: 4690-4700Crossref PubMed Scopus (173) Google Scholar). Tyrosine phosphorylation of the NR2 subunits of the NMDAR is enhanced under a number of synaptic plasticity-related events, including long-term potentiation (LTP) (13Rosenblum K. Dudai Y. Richter-Levin G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10457-10460Crossref PubMed Scopus (161) Google Scholar, 14Lu Y.M. Roder J.C. Davidow J. Salter M.W. Science. 1998; 279: 1363-1367Crossref PubMed Scopus (277) Google Scholar, 15Rostas 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: 10452-10456Crossref PubMed Scopus (214) Google Scholar), fear-related learning (16Nakazawa T. Komai S. Watabe A.M. Kiyama Y. Fukaya M. Arima-Yoshida F. Horai R. Sudo K. Ebine K. Delawary M. Goto J. Umemori H. Tezuka T. Iwakura Y. Watanabe M. Yamamoto T. Manabe T. EMBO J. 2006; 25: 2867-2877Crossref PubMed Scopus (125) Google Scholar), exposure to alcohol (17Miyakawa T. Yagi T. Kitazawa H. Yasuda M. Kawai N. Tsuboi K. Niki H. Science. 1997; 278: 698-701Crossref PubMed Scopus (267) Google Scholar, 18Yaka R. Phamluong K. Ron D. J. Neurosci. 2003; 23: 3623-3632Crossref PubMed Google Scholar, 19Wang J. Carnicella S. Phamluong K. Jeanblanc J. Ronesi J.A. Chaudhri N. Janak P.H. Lovinger D.M. Ron D. J. Neurosci. 2007; 27: 3593-3602Crossref PubMed Scopus (135) Google Scholar), and ischemia (20Takagi N. Sasakawa K. Besshoh S. Miyake-Takagi K. Takeo S. J. Neurochem. 2003; 84: 67-76Crossref PubMed Scopus (49) Google Scholar). Tyrosine phosphorylation also positively regulates the trafficking of NMDARs from intracellular compartments to the postsynaptic density (PSD) (10Dunah A.W. Standaert D.G. J. Neurosci. 2001; 21: 5546-5558Crossref PubMed Google Scholar, 12Hallett P.J. Spoelgen R. Hyman B.T. Standaert D.G. Dunah A.W. J. Neurosci. 2006; 26: 4690-4700Crossref PubMed Scopus (173) Google Scholar), the stability of NMDARs at the synaptic membrane (21Thornton C. Yaka R. Dinh S. Ron D. J. Biol. Chem. 2003; 278: 23823-23829Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 22Prybylowski K. Chang K. Sans N. Kan L. Vicini S. Wenthold R.J. Neuron. 2005; 47: 845-857Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar) and controls the association of the NMDAR with spectrin (23Wechsler A. Teichberg V.I. EMBO J. 1998; 17: 3931-3939Crossref PubMed Scopus (168) Google Scholar). Tyrosine phosphorylation can also influence which proteins bind to the receptor. For example, phosphatidylinositol 3-kinase (PI 3-kinase), and phospholipase C-γ (PLC-γ) bind to NR2 subunits in a tyrosine phosphorylation-dependent manner (24Gurd J.W. Bissoon N. J. Neurochem. 1997; 69: 623-630Crossref PubMed Scopus (50) Google Scholar, 25Hisatsune C. Umemori H. Mishina M. Yamamoto T. Genes Cells. 1999; 4: 657-666Crossref PubMed Scopus (77) Google Scholar), and the increased association of PI 3-kinase with phosphorylated NMDARs has been suggested to contribute to altered signaling in the hippocampus after ischemia (20Takagi N. Sasakawa K. Besshoh S. Miyake-Takagi K. Takeo S. J. Neurochem. 2003; 84: 67-76Crossref PubMed Scopus (49) Google Scholar). Thus, tyrosine phosphorylation plays an essential role in both the regulation of NMDAR channel activity and NMDAR-mediated downstream signaling cascades. In addition to phosphorylation, ubiquitination is another post-translational modification that plays an important role in regulating neuronal function. Ubiquitin is a 76-amino acid protein that covalently attaches to substrates in an ATP-dependent enzymatic reaction that requires the concerted efforts of E1, E2, and E3 ubiquitin ligases (26Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2944) Google Scholar). E3 ubiquitin ligases are the critical components that generate specificity in the reaction via substrate recognition. While many cellular processes are known to involve ubiquitination, its involvement in neuronal synaptic plasticity has only recently been investigated. Hippocampal LTP requires a balance of both protein synthesis and ubiquitin-dependent degradation (27Fonseca R. Vabulas R.M. Hartl F.U. Bonhoeffer T. Nagerl U.V. Neuron. 2006; 52: 239-245Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), and the stability of multiple proteins at the PSD is regulated by the ubiquitin-proteasome system (UPS) (28Ehlers M.D. Nat. Neurosci. 2003; 6: 231-242Crossref PubMed Scopus (834) Google Scholar, 29Patrick G.N. Bingol B. Weld H.A. Schuman E.M. Curr. Biol. 2003; 13: 2073-2081Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 30Colledge M. Snyder E.M. Crozier R.A. Soderling J.A. Jin Y. Langeberg L.K. Lu H. Bear M.F. Scott J.D. Neuron. 2003; 40: 595-607Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar). Thus, both phosphorylation and ubiquitination are likely to be important regulators of NMDAR function. Here we report the identification of the E3 ubiquitin ligase Mind bomb-2 (Mib2) as a novel NR2B subunit-interacting protein whose interaction with and ubiquitination of the NR2B subunit is enhanced in a Fyn phosphorylation-dependent manner. This leads to the down-regulation of NMDAR activity, suggesting a role for both phosphorylation and ubiquitination in NMDAR modulation. Antibodies and Reagents—Affinity-purified rabbit polyclonal sera directed against a peptide sequence of mouse Mib2 (amino acids (aa) 429-443) was generated by New England Peptide (Gardner, MA) (Supplemental Fig. S1). Anti-Mib2 (mouse) antibodies were from Abnova (Taipei, Taiwan). Anti-NR2B (rabbit) antibodies were from Covance (Berkeley, CA). Anti-NR1 (mouse) and MAP2(a,b) and MAP2(a-c) (mouse) antibodies were from Chemicon (Temecula, CA). Anti-hemagglutinin (HA, rat) and alkaline phosphatase-conjugated digoxigenin antibodies were from Roche Applied Science (Indianapolis, IN), and anti-α-actinin (mouse) antibodies from Sigma. Anti-phosphotyrosine (mouse) and anti-Myc (mouse) antibodies were from Upstate (Lake Placid, NY) and anti-PSD-95 (mouse) antibodies were from Affinity Bioreagents (Golden, CO). Anti-NR2B (goat), NR1 (goat), actin (goat), HA (mouse), Fyn (rabbit and mouse), normal IgG and all horseradish peroxidase-labeled secondary antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). Forskolin and MG-132 were purchased from Calbiochem, APV (2-amino-5-phosphonovaleric acid) was from Tocris (St. Louis, MO), and ketamine HCl was purchased from Henry Schein Inc (Palatine, IL). Construction of Yeast Plasmids—The sequence encoding the cytoplasmic tail of human NR2B (ctNR2B; aa 839-1482) was amplified from cDNA derived from L(-tk) cells expressing human NR1 and NR2B (Merck, Sharp and Dohme, Harlow, UK), and subcloned into the multiple cloning site I (MCS-I) of the pBridge yeast expression plasmid (Clontech, Mountain View). Full-length human Fyn cDNA was amplified from Marathon Race-Ready human brain cDNA (Clontech) and subcloned into pGEM-T (Promega, Madison, WI). This construct (Fyn WT) was used as a template for site-directed mutagenesis of the tyrosine residue 531 to phenylalanine (Y531F), generating a constitutively active form of Fyn (FynCA). After verifying the point mutation by sequencing, FynCA was subcloned into the MCS-II of pBridge-ctNR2B. MCS-I of pBridge is under the control of a CMV promoter, whereas MCS-II is under the control of a conditional methionine promoter. Thus, ctNR2B is constitutively expressed from the pBridge-ctNR2B-FynCA construct, whereas FynCA expression is repressed in the presence of methionine in the growth media. Yeast Three-hybrid Screening—An adult rat brain cDNA library in pACT2 (Clontech; “prey”) was screened using the pBridge-ctNR2B-FynCA plasmid for constitutive expression of the cytoplasmic tail of NR2B as “bait” and inducible expression of FynCA as the third component in the screen. The pBridge-ctNR2B-FynCA construct and cDNA library were sequentially co-transformed into AH109 yeast (Clontech) using standard techniques (31Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar). Selection was performed using minimal media plus/minus methionine and colonies that were β-galactosidase positive in the absence of methionine (FynCA expressed) but β-galactosidase negative in the presence of methionine (FynCA repressed) were identified. The β-galactosidase assay was repeated on restreaked positive clones to confirm the initial result. Plasmid DNA from positive yeast colonies was isolated and sequenced. Expression Constructs—Full-length human Mib2 cDNA (IMAGE clone 4341220) was purchased from ATCC (Manassas, VA) and subcloned into phCMV2 vector (Gene Therapy Systems; San Diego, CA) such that the 110-kDa Mib2 protein was fused in-frame with the N-terminal HA epitope. Similar constructs were made to express regions containing the zinc finger domain (aa 1-180), the ankyrin repeats (aa 504-855), and the RING finger domains (aa 857-1000) of Mib2. Each construct was verified by Western blot analysis using HA antibodies to detect the fusion protein after transient transfection of the cDNAs into HEK293 cells. The following cDNAs were kind gifts: GFP-NR2B, Robert Wenthold (National Institutes of Health, Bethesda, MD); NR2B and enhanced-GFP (eGFP), David Lovinger (National Institutes of Health, Bethesda, MD); NR1-1a, Michael Hollman (Ruhr University, Bochum, Germany); Myc-ubiquitin, Jennifer Johnston (Elan, South San Francisco, CA); maltose-binding protein (MBP), MBP-NR1 and MBP-NR2B constructs, Vivian Teichberg (Weizman Institute, Rehovot, Israel). MBP-NR2B was used as a template for site-directed mutagenesis of tyrosine residues 1252, 1336, and 1472 to aspartic acids (Y1252D, Y1336D, Y1472D), generating a phosphomimic mutant of NR2B that was verified by sequencing. Recombinant Proteins—MBP-tagged proteins were expressed in Escherichia coli, affinity-purified, and immobilized on amylose resin according to the manufacturer's protocol (New England Biolabs, Ipswich, MA). In Vitro Translation—[35S]methionine-labeled Mib2 was generated in rabbit reticulocyte lysates (TnT in vitro translation kit; Promega) using full-length Mib2 cDNA. In vitro translated products were analyzed by SDS-PAGE and fluorography. In Vitro Binding Assays—The assay was performed as previously described (32Thornton C. Tang K.C. Phamluong K. Luong K. Vagts A. Nikanjam D. Yaka R. Ron D. J. Biol. Chem. 2004; 279: 31357-31364Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Results were visualized by autoradiography using the Typhoon PhosphorImager (Amersham Biosciences) and quantified by NIH Image 1.61. Purity of the recombinant proteins was verified by Coomassie staining. Cell Culture—Human embryonic kidney (HEK293) cells were cultured on 10-cm plates in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT) and GIBCO™ MEM Non-Essential Amino Acids (Invitrogen). When cells reached 70% confluency, they were transiently transfected with cDNA constructs (up to 6 μg total) using Lipofectamine Plus (Invitrogen), in accordance with the manufacturer's instructions. The media was changed 24 h after transfection, and cells harvested 48 h after transfection. For the preparation of primary hippocampal neurons, newborn rats (P0) were decapitated, and hippocampi were dissected bilaterally. Cells were dissociated by enzyme digestion with papain (Worthington, Lakewood, NJ) followed by brief mechanical trituration and plated on 2-chamber CC2 glass chamber slides (Nalge Nunc, Naperville, IL). Cells were plated (∼1 × 105 cells/chamber) and maintained in Neurobasal medium supplemented with B27, penicillin, streptomycin, and Glutamax-1 (all from Invitrogen), and maintained in culture for up to 21 days. Preparation of Cell Homogenates—HEK293 cells were harvested in PBS (1.5 mm KH2PO4, 8 mm Na2HPO4, 2.7 mm KCl, 137 mm NaCl), spun at 2000 rpm for 2 min, washed twice with phosphate-buffered saline, and resuspended in lysis buffer (50 mm Tris-HCl, pH 7.4, 10 mm EGTA, 10 mm EDTA, 320 mm sucrose, 1% deoxycholate), 1% SDS, and protease (Roche Applied Science) and phosphatase inhibitors (Sigma) per the manufacturer's instructions. Samples were sonicated briefly, and lysis was allowed to proceed for 30 min on ice. Following protein concentration determination (using a BCA kit, per manufacturer's instructions; Pierce), samples were diluted to 2 mg/ml with lysis buffer before proceeding with immunoprecipitation assays. Rat hippocampal slices and homogenates were prepared as previously described (5Yaka R. He D.Y. Phamluong K. Ron D. J. Biol. Chem. 2003; 278: 9630-9638Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Immunoprecipitation Assays—Immunoprecipitation was performed with 5 μg of the appropriate antibodies and 500 μg of cell homogenate as previously described (5Yaka R. He D.Y. Phamluong K. Ron D. J. Biol. Chem. 2003; 278: 9630-9638Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Samples were resolved by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in milk solution (5% milk, PBS, 0.05% Tween-20) except for the detection of phosphorylated proteins when a bovine serum albumin (BSA) solution (5% BSA, 200 mm Tris-HCl, pH 7.4, 0.9% NaCl, 0.1% Tween-20) was used. Membranes were incubated with the specific primary antibody, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was detected by enhanced chemiluminescence ECL (GE Healthcare, Buckinghamshire, UK) and processed using the Typhoon PhosphorImager (Amersham Biosciences). Results were quantified by NIH Image 1.61. Ubiquitination Assays—Cells were transiently transfected with cDNA constructs and were treated with the proteasome inhibitor MG-132 (42 μm) 3 h prior to homogenization. Cell homogenates and immunoprecipitations were prepared as described above, except that 42 μm MG-132 and 5 mmN-ethylmaledimide (Sigma) were added to all buffers. In addition, samples were diluted in lysis buffer containing 2% SDS to dissociate protein-protein interactions. In Situ Hybridization—A fragment of the mouse Mib2 gene (aa 263-428) was generated by PCR using adult mouse brain cDNA (Clontech) as a template. The PCR product was subcloned into pGEM-T (Promega) and used as a template to generate sense and antisense digoxygenin-labeled riboprobes using the Digoxygenin RNA labeling kit (Roche Applied Science), as per the manufacturer's instructions. This sequence contained no significant homology to other known cDNAs, as determined by BLAST analysis. Hybridization was performed on 16-20 μm-thick sagittal cryostat sections as previously described (33Ashique A.M. Kharazia V. Yaka R. Phamluong K. Peterson A.S. Ron D. Brain Res. 2006; 1069: 31-38Crossref PubMed Scopus (31) Google Scholar). Digoxigenin antibodies conjugated to alkaline phosphatase were used at a concentration of 1:2,000, and the detection of alkaline phosphatase was performed with NBT/BCIP substrate according to the manufacturer's instructions (Roche Applied Science). Immunocytochemistry—Rats were deeply anesthetized with an overdose of Euthasol® (Virbac, Forth Worth, TX) and perfused with 0.9% NaCl for 2 min, followed by 4% paraformaldehyde (PFA) for 10 min. Brains were removed, post-fixed in 4% PFA for 2 h, and sectioned (40 μm) using a vibratome. Free-floating sagittal sections were immunostained as previously described (33Ashique A.M. Kharazia V. Yaka R. Phamluong K. Peterson A.S. Ron D. Brain Res. 2006; 1069: 31-38Crossref PubMed Scopus (31) Google Scholar), using primary antibodies at the following concentrations: anti-Mib2 (rabbit; 1:500), anti-MAP2(a,b) (mouse; 1:500) and anti-NR1 (mouse; 1:500). Secondary antibodies were Cy3-labeled donkey anti-rabbit and FITC-labeled anti-mouse (1:250; Jackson ImmunoResearch, WestGrove, PA). Primary hippocampal neurons were fixed in 4% PFA, followed by incubation in 3% normal donkey serum (Jackson ImmunoResearch) in PBS containing 0.1% Triton X-100 for 2 h at room temperature. Fixed neurons were incubated (overnight, 4 °C) in primary antibodies against Mib2 (1:250), MAP(a-c) (1:250), PSD-95 (1:100), or α-actinin (1:500,000) diluted in PBS/0.1% Triton X-100. Alexa Fluor-594 labeled donkey anti-rabbit and Alexa Fluor-488 labeled donkey anti-mouse secondary antibodies (Molecular Probes, Eugene, OR) were incubated for 2 h at room temperature, followed by washing 3× in phosphate-buffered saline/0.1% Triton X-100 and coverslipped using Vectashield mounting medium (Vector laboratories, Burlingame, CA). Images were acquired using the LSM 510 confocal microscope (Zeiss, Thornwood, NY). Electrophysiology—HEK293 cells were transfected with cDNA constructs as described above, except that APV (500 μm) and ketamine (0.7 mm) were added to the media to prevent glutamate-induced excitatoxicity. 24 h after transfection, cells were reseeded at a density of 1 × 105 cells in 35-mm plates coated with 1% gelatin. 24-48 h after replating, one dish was transferred to the chamber of an upright microscope (BX50WI, Olympus, Tokyo, Japan) equipped with epifluorescent optics. Cells were continuously perfused with external solution that contained (in mm): 145 NaCl, 5.4 KCl, 1.8 BaCl2, 15 HEPES, 10 glucose, 0.05 glycine, and 0.2 sodium orthovanadate (pH 7.3). When testing the effect of MG-132 (10 μm), we added it to the culture media 2-3 h before transferring cells to the recording chamber, and MG-132 (1 μm) was also present in the recording solution. Transfected cells were identified by the fluorescence of eGFP that was co-transfected with the cDNA constructs. NMDA was dissolved into external solution daily at a concentration of 1 mm. Fast switching to NMDA-containing external solution was achieved by piezoelectric control of the lateral movement of a three-barrel square glass application pipette (SF-77B, Warner Instrument, CT). NMDA-elicited currents were measured at -60 mV in the whole cell voltage-clamp mode using Multiclamp 700A (Molecular Device, Union City, CA), as described previously (34Bradley J. Carter S.R. Rao V.R. Wang J. Finkbeiner S. J. Neurosci. 2006; 26: 1065-1076Crossref PubMed Scopus (57) Google Scholar). Recording electrodes (2-3 MΩ) were filled with internal solution containing (in mm): 121 cesium methanesulfate, 10 CsCl, 15 HEPES, 0.6 EGTA, 4 MgATP, 0.3 NaGTP, and 7 Na2CrPO4 (pH 7.25) with an osmolarity of 275 mOsm. Whole cell currents were low-pass filtered at 1 kHz and digitized at 2 kHz by Multicamp 700A software and pClamp 9 (Molecular Device, Union City, CA). Cell membrane capacitance was measured and compensated automatically. Series resistance was 50% compensated. Statistical Analysis—All data are expressed as mean ± S.E. Electrophysiological results were analyzed by Student's t test. Biochemical results were analyzed using the one-sample t test or one-way analysis of variance, followed by post-hoc tests using Newman-Keuls method. Significance for all tests was set at p < 0.05. Mib2 Interacts with the NR2B Cytoplasmic Tail in a Fyn-dependent Manner in Yeast—Because tyrosine phosphorylation of the NMDAR is a critical mechanism regulating channel activity and signal transduction (7Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (646) Google Scholar), we designed a screen to identify proteins that interact with the NMDAR in a tyrosine phosphorylation-dependent manner. This yeast three-hybrid screen (35Fuller K.J. Morse M.A. White J.H. Dowell S.J. Sims M.J. BioTechniques. 1998; 25 (90–82): 85-88Crossref PubMed Scopus (24) Google Scholar) consisted of a bait plasmid containing the cytoplasmic tail of NR2B (ctNR2B), prey proteins expressed from a rat brain cDNA library, and a constitutively active form of Fyn (FynY531F; FynCA (36Harrison S.C. Cell. 2003; 112: 737-740Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar)) whose expression was under the control of a conditional methionine promoter. In the presence of methionine in the growth media, FynCA expression was repressed. In the absence of methionine, FynCA expression was induced, and tyrosine phosphorylation of ctNR2B was observed (data not shown). Approximately 5 × 105 independent transformant clones were screened in the absence and presence of methionine in the growth media. This resulted in the isolation of 32 colonies that were true three-hybrid interactants (i.e. interacted with ctNR2B only when FynCA was expressed; Fig. 1). Three of these colonies contained plasmids encoding the RING finger domain of Mib2, a protein that has previously been reported to have E3 ubiquitin ligase activity (37Koo B.K. Yoon K.J. Yoo K.W. Lim H.S. Song R. So J.H. Kim C.H. Kong Y.Y. J. Biol. Chem. 2005; 280: 22335-22342Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Because of the recently identified role of the UPS in activity-dependent regulation of excitatory synaptic proteins (28Ehlers M.D. Nat. Neurosci. 2003; 6: 231-242Crossref PubMed Scopus (834) Google Scholar, 29Patrick G.N. Bingol B. Weld H.A. Schuman E.M. Curr. Biol. 2003; 13: 2073-2081Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 30Colledge M. Snyder E.M. Crozier R.A. Soderling J.A. Jin Y. Langeberg L.K. Lu H. Bear M.F. Scott J.D. Neuron. 2003; 40: 595-607Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar), we were interested in further characterizing whether Mib2 was a phosphorylation-dependent NR2B-interacting protein. Mib2 Interacts with the NR2B Subunit in a Phosphorylation-dependent Manner—Having identified the RING finger domain of Mib2 as a phosphorylation-dependent interactant with the cytoplasmic tail of NR2B in a yeast system, we next set out to determine whether this interaction occurs in mammalian cells. The RING finger domain of Mib2 was expressed as a HA-tagged protein together with GFP-tagged NR2B subunit in HEK293 cells, and the interaction between NR2B and this Mib2 domain was determined by immunoprecipitation. As shown in Fig. 2A (lane 1), anti-NR2B antibodies co-immunoprecipitated the RING finger domain of Mib2, indicating that the two proteins exist as a complex. IgG antibodies did not immunoprecipitate either NR2B or the RING finger domain, demonstrating the specificity of the observed interaction (Fig. 2A, lane 2). Next, we determined whether the interaction between Mib2 and the NR2B subunit is regulated by Fyn phosphorylation. To do so, FynCA was transfected into HEK293 cells in addition to Mib2 and NR2B. Expression of FynCA in HEK293 cells resulted in robust tyrosine phosphorylation of the NR2B subunit (Fig. 2A, lane 4 versus lane 1, top panel). The association of the RING finger domain of Mib2 with NR2B showed a small, but significant, enhancement in the presence of FynCA (Fig. 2A, lane 4 versus lane 1, bottom panel, and Fig. 2B). However, transfection of full-length Mib2 into the same mammalian system resulted in a marked 2-fold increase in association in the presence of Fyn kinase (Fig. 2, C and D), suggesting that additional domains contained in the full-length protein contribute to its binding and regulated interaction with the NR2B subunit. Taken together, we show that the interaction between Mib2 and NR2B is significantly enhanced in a Fyn phosphorylation-dependent manner in mammalian cells. To assess whether the interaction between NR2B and Mib2 is specific, we determined if Mib2 interacts with the NR1 obligatory subunit of the NMDAR. To do so, we expressed Mib2 together with NR1 in HEK293 cells. Anti-NR1 antibodies immunoprecipitated NR1, but failed to co-immunoprecipitate Mib2 (Fig. 2E), suggesting that Mib2 interacts specifically with NR2B, but not the NR1 subunit of the NMDAR. Mib2 Interacts Directly with the Cytoplasmic Tail of NR2B—We next set out to determine if the interaction that we observed in mammalian cells was occurring through a direct protein-protein interaction between Mib2 and NR2B. Proteins encoding the C-terminal regions of the NR1 and NR2B subunit tagged with MBP were expressed in E. coli and incubated with in vitro translated radiolabeled Mib2 (35S-Mib2). As shown in Fig. 3A, 35S-Mib2 binds to MBP-NR2B (lane 3) but not to MBP-NR1 (lane 2) or the MBP tag alone (lane 1). Furthermore, 35S-Mib2 bound strongly to a phosphomimic mutant form of MBP-NR2B (MBP-NR2B-mut) in which the three main sites of tyrosine phosphorylation by Src family kinases (Tyr1252, Tyr1336, Tyr1472) have been mutated to aspartic acids to mimic constitutive phosphorylation (Fig. 3A, lanes 4 versus 3). 35S-Mib2 was also observed to bind significantly more strongly to the distal portio