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• W2023928079 abstract "The COOH-terminal domain of the NR2D subunit of the NMDA receptor contains proline-rich regions that show striking homology to sequences known to bind to Src homology 3 (SH3) domains. To determine whether the proline-rich region of the NR2D subunit interacts with specific SH3 domains, in vitro SH3 domain binding assays were performed. A proline-rich fragment of the NR2D subunit (2D866–1064) bound to the Abl SH3 domain but not to the SH3 domains from Src, Fyn, Grb2, GAP, or phospholipase C-γ (PLCγ). Co-immunoprecipitation of NR2D with Abl suggests stable association of NR2D and Abl in transfected cells. The SH3 domain plays an important role in the negative regulation of Abl kinase activity. To determine whether the interaction of NR2D with the Abl SH3 domain alters Abl kinase activity, Abl was expressed alone or with NR2D in 293T cells. Autophosphorylation of Abl was readily observed when Abl was expressed alone. However, co-expression of Abl with 2D866–1064 or full-length NR2D inhibited autophosphorylation. 2D866–1064did not inhibit ΔSH3 Abl, indicating a requirement for the Abl SH3 domain in the inhibitory effect. Similarly, 2D866–1064 did not inhibit the catalytic activity of Abl-PP, which contains two point mutations in the SH2-kinase linker domain that release the negative kinase regulation by the SH3 domain. In contrast, the full-length NR2D subunit partially inhibited the autokinase activity of both ΔSH3 Abl and Abl-PP, suggesting that NR2D and Abl may interact at multiple sites. Taken together, the data in this report provide the first evidence for a novel inhibitory interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase. The COOH-terminal domain of the NR2D subunit of the NMDA receptor contains proline-rich regions that show striking homology to sequences known to bind to Src homology 3 (SH3) domains. To determine whether the proline-rich region of the NR2D subunit interacts with specific SH3 domains, in vitro SH3 domain binding assays were performed. A proline-rich fragment of the NR2D subunit (2D866–1064) bound to the Abl SH3 domain but not to the SH3 domains from Src, Fyn, Grb2, GAP, or phospholipase C-γ (PLCγ). Co-immunoprecipitation of NR2D with Abl suggests stable association of NR2D and Abl in transfected cells. The SH3 domain plays an important role in the negative regulation of Abl kinase activity. To determine whether the interaction of NR2D with the Abl SH3 domain alters Abl kinase activity, Abl was expressed alone or with NR2D in 293T cells. Autophosphorylation of Abl was readily observed when Abl was expressed alone. However, co-expression of Abl with 2D866–1064 or full-length NR2D inhibited autophosphorylation. 2D866–1064did not inhibit ΔSH3 Abl, indicating a requirement for the Abl SH3 domain in the inhibitory effect. Similarly, 2D866–1064 did not inhibit the catalytic activity of Abl-PP, which contains two point mutations in the SH2-kinase linker domain that release the negative kinase regulation by the SH3 domain. In contrast, the full-length NR2D subunit partially inhibited the autokinase activity of both ΔSH3 Abl and Abl-PP, suggesting that NR2D and Abl may interact at multiple sites. Taken together, the data in this report provide the first evidence for a novel inhibitory interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase. N-methyl-d-aspartic acid Src homology 3 glutathione S-transferase phospholipase polymerase chain reaction polyacrylamide gel electrophoresis wild-type NMDA1 receptors mediate synaptic transmission and neural plasticity at many sites in the mammalian CNS (1.Bear M.F. Malenka R.C. Curr. Opin. Neurobiol. 1994; 4: 389-399Crossref PubMed Scopus (1045) Google Scholar, 2.Monaghan D.T. Bridges R.J. Cotman C.W. Annu. Rev. Pharmacol. Toxicol. 1989; 29: 365-402Crossref PubMed Scopus (2014) Google Scholar). NMDA receptors are also involved in epileptiform activity and neuronal cell death in a number of experimental and pathological conditions (3.Lipton S.A. Rosenberg P.A. N. Engl. J. Med. 1994; 330: 613-622Crossref PubMed Scopus (2550) Google Scholar). Two NMDA receptor subunit families (NR1a-h and NR2A-D) have been identified; alternative splicing of a single NR1 gene generates eight isoforms with distinct functional properties, while heterogeneity within the NR2 subunit family results from expression of four closely related genes (for review see, Refs. 4.Mori H. Mishina M. Neuropharmacology. 1995; 34: 1219-1237Crossref PubMed Scopus (588) Google Scholar and5.Yamakura T. Shimoji K. Prog. Neurobiol. 1999; 59: 279-298Crossref PubMed Scopus (268) Google Scholar). Native NMDA receptors are believed to be oligomeric complexes formed from combinations of NR1 and NR2 subunits, with the NR2 subunit imparting distinct functional and pharmacological properties (6.Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2897) Google Scholar, 7.Kutsuwada T. Kashiwabuchi H. Mori H. Sakimura K. Kushiya E. Araki K. Meguro H. Masaki H. Kumanishi T. Arakawa M. Mishina M. Nature. 1992; 358: 36-41Crossref PubMed Scopus (1226) Google Scholar, 8.Laurie D.J. Seeburg P.H. Eur. J. Pharmacol. 1994; 268: 335-345Crossref PubMed Scopus (383) Google Scholar, 9.Buller A.L. Monaghan D.T. Eur. J. Pharmacol. 1997; 320: 87-94Crossref PubMed Scopus (75) Google Scholar, 10.Buller A.L. Larson H.C. Schneider B.E. Beaton J.A. Morrisett R.A. Monaghan D.T. J. Neurosci. 1994; 14: 5471-5484Crossref PubMed Google Scholar). The four NR2 subunits show distinct patterns of expression in brain that parallel the distribution of native receptor subtypes (10.Buller A.L. Larson H.C. Schneider B.E. Beaton J.A. Morrisett R.A. Monaghan D.T. J. Neurosci. 1994; 14: 5471-5484Crossref PubMed Google Scholar). NR2D subunit expression is developmentally regulated, with widespread distribution in embryonic and neonatal diencephalon, brainstem, and spinal cord (11.Wenzel A. Villa M. Mohler H. Benke D. J. Neurochem. 1996; 66: 1240-1248Crossref PubMed Scopus (128) Google Scholar, 12.Dunah A.W. Yasuda R.P. Wang Y. Luo J. Davila-Garcia M. Gbadegesin M. Vicini S. Wolfe B.B. J. Neurochem. 1996; 67: 2335-2345Crossref PubMed Scopus (122) Google Scholar) and lower levels of expression in adult brain (10.Buller A.L. Larson H.C. Schneider B.E. Beaton J.A. Morrisett R.A. Monaghan D.T. J. Neurosci. 1994; 14: 5471-5484Crossref PubMed Google Scholar,13.Watanabe M. Inoue Y. Sakimura K. Mishina M. Neuroreport. 1992; 3: 1138-1140Crossref PubMed Scopus (617) Google Scholar). Recent evidence has demonstrated that the NMDA receptor interacts with a number of cellular signaling proteins that may modulate the structure, function, and localization of the receptor (14.Wechsler A. Teichberg V.I. EMBO J. 1998; 17: 3931-3939Crossref PubMed Scopus (168) Google Scholar, 15.Wang Y.T. Salter M.W. Nature. 1994; 369: 233-235Crossref PubMed Scopus (598) Google Scholar, 16.Kohr G. Seeburg P.H. J. Physiol. (Lond.). 1996; 492: 445-452Crossref Scopus (279) Google Scholar, 17.Kornau H. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 18.Tezuka T. Umemori H. Akiyama T. Nakanishi S. Yamamoto T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 435-440Crossref PubMed Scopus (334) Google Scholar). Intracellular signaling proteins, including some nonreceptor protein-tyrosine kinases, frequently contain modular domains that mediate interactions with effector and regulatory proteins. Src homology 3 (SH3) domains mediate protein-protein interactions with proline-rich target sequences that adopt a left-handed helical conformation (polyproline type II helix (19.Feng S. Chen J.K., Yu, H. Simon J.A. Schreiber S.L. Science. 1994; 266: 1241-1246Crossref PubMed Scopus (747) Google Scholar, 20.Yu H. Chen J.K. Feng S. Dalgarno D.C. Brauer A.W. Schreiber S.L. Cell. 1994; 76: 933-945Abstract Full Text PDF PubMed Scopus (876) Google Scholar)). In the case of the tyrosine kinases Src and Abl, SH3 domains function in both negative regulation of kinase activity and substrate recruitment (21.Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-673Crossref PubMed Scopus (1444) Google Scholar, 22.Musacchio A. Gibson T. Lehto V. Saraste M. FEBS Lett. 1992; 307: 55-60Crossref PubMed Scopus (235) Google Scholar, 23.Bar-Sagi D. Rotin D. Batzer A. Mandiyan V. Schlessinger J. Cell. 1993; 74: 83-91Abstract Full Text PDF PubMed Scopus (308) Google Scholar, 24.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar). SH3 domains are also found in many other proteins involved in signal transduction and may be involved in cytoskeletal organization and the targeting of signaling molecules to specific subcellular locations (22.Musacchio A. Gibson T. Lehto V. Saraste M. FEBS Lett. 1992; 307: 55-60Crossref PubMed Scopus (235) Google Scholar,25.Gomperts S.N. Cell. 1996; 84: 659-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Analysis of rat NMDA receptor subunit amino acid sequences revealed a proline-rich region in the COOH-terminal portion of the NR2D subunit. This proline-rich region contains sequences that show striking homology to motifs known to bind to SH3 domains (19.Feng S. Chen J.K., Yu, H. Simon J.A. Schreiber S.L. Science. 1994; 266: 1241-1246Crossref PubMed Scopus (747) Google Scholar, 26.Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar, 27.Rickles R. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar, 28.Rickles R.J. Botfield M.C. Zhou X.M. Henry P.A. Brugge J.S. Zoller M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10909-10913Crossref PubMed Scopus (175) Google Scholar). In the present report, we tested the hypothesis that the proline-rich region of the NR2D subunit interacts with SH3 domains. Our results reveal a selective interaction of the proline-rich region of the NR2D subunit with the SH3 domain of the Abl tyrosine kinase. Co-expression of NR2D with Abl in a model system results in suppression of kinase activity, suggesting a biologically significant interaction between these proteins. The anti-Abl monoclonal antibody K-12 and the antiphosphotyrosine antibodies PY20 and PY99 were obtained from Santa Cruz Biotechnology. The anti-Abl antibody 8e9 has been described (29.McWhirter J.R. Wang J.Y.J. Mol. Cell. Biol. 1991; 11: 1553-1565Crossref PubMed Google Scholar). The M2 anti-FLAG antibody and M2 anti-FLAG antibody resin were obtained from Kodak Scientific Imaging Systems or Sigma. Appropriate alkaline phosphatase-conjugated secondary antibodies were from Southern Biotechnology Associates. NR1a cDNA was generously provided by Dr. Shigetada Nakanishi (Kyoto University Faculty of Medicine, Kyoto, Japan). NR2D cDNA (6.Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2897) Google Scholar) was the gift of Dr. Peter Seeburg (University of Heidelberg, Heidelberg, Germany). GST fusion proteins containing the SH3 domains of Fyn and PLCγ and the full-length Grb2 protein were purchased from Santa Cruz Biotechnology. Glutathione-agarose and protein A-Sepharose were from Sigma. The coding sequence of the 8-amino acid FLAG epitope (DYKDDDDK) was added to a COOH-terminal fragment of the NR2D NMDA receptor subunit by PCR to generate the 2D866–1064 construct. This construct contains residues 866–1064 of the rat NR2D subunit, beginning immediately COOH-terminal to the M4 domain at Val866, extending through Gln1064, and terminating at the FLAG epitope. The nucleotide sequence of the PCR-amplified fragment was confirmed by automated DNA sequence analysis (Applied Biosystems). 2D866–1064 was subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) for expression in 293T cells. PCR was used to amplify the COOH-terminal coding region downstream from the unique SacI site. The reverse primer added the FLAG epitope immediately adjacent to the COOH-terminal Val, thereby removing the naturally occurring stop codon. The forward primer included the unique SacI site. The resulting PCR product was subcloned into NR2DpcDNA3 to replace the normal COOH-terminal region. The nucleotide sequence of the PCR-amplified fragment was confirmed by automated sequence analysis (Applied Biosystems). Abl and ΔSH3 Abl were subcloned into pcDNA3.1 (Invitrogen, Carlsbad CA). The constitutively active Abl linker mutant, Abl-PP (24.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar), was generated by two sequential rounds of mutagenesis in which prolines 242 and 249 were each mutated to alanine using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). Mutants were confirmed by automated DNA sequence analysis (Applied Biosystems). Calcium phosphate-mediated transfection of 293T cells was as described previously (30.Rogers J.A. Read R.D. Li J. Peters K.L. Smithgall T.E. J. Biol. Chem. 1996; 271: 17519-17525Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). 2–3 days after transfection, 293T cells were harvested by centrifugation and lysed. For preparation of total cellular lysates, cells were sonicated in SDS-PAGE sample buffer; for immunoprecipitation from cells expressing 2D866–1064, cells were sonicated in 50 mm Tris-HCl, pH 7.4, 50 mm NaCl, 1 mm EDTA, 1 mmMgCl2, 0.1% Triton X-100 supplemented with 25 μg/ml aprotinin, 50 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, and 50 μm Na2MoO4. For immunoprecipitation from cells expressing full-length NR2D, cells were sonicated in 50 mm Hepes, pH 7.4, 0.5 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 0.1% deoxycholate supplemented with 25 μg/ml aprotinin, 50 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mmNa3VO4, and 50 μmNa2MoO4. For immunoprecipitation, clarified cytosolic extracts were incubated with antibody and protein A-Sepharose for 1 h at 4 °C. Immune complexes were isolated by centrifugation and washed three times with radioimmune precipitation buffer. Final immunoprecipitates were heated in SDS sample buffer. For immunoblotting, cytosolic extracts or immunoprecipitated proteins were run on 4–15% gradient polyacrylamide gels and transferred to nitrocellulose. Membranes were incubated for 1 h with the appropriate primary antibody at 1 μg/ml in Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 1.5% bovine serum albumin. The membranes were then washed in TBS-T and probed with a secondary antibody-alkaline phosphatase conjugate in TBS-T containing bovine serum albumin. Following further TBS-T washes, immunoreactive proteins were visualized colorimetrically using the alkaline phosphatase substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. The coding sequence of the Abl SH3 domain was amplified by PCR using Bcr/Abl as a template and subcloned into the pGEX2T expression vector (Amersham Pharmacia Biotech). SH3 domains from Src and GAP were subcloned into pGEX2T as described previously (31.Hjermstad S.J. Briggs S.D. Smithgall T.E. Biochemistry. 1993; 32: 10519-10525Crossref PubMed Scopus (25) Google Scholar, 32.Briggs S.D. Bryant S.S. Jove R. Sanderson S.D. Smithgall T.E. J. Biol. Chem. 1995; 270: 14718-14724Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The resulting plasmids were used to express the SH3 domains as GST fusion proteins in bacteria. Fusion proteins were purified using glutathione-agarose beads as described previously (31.Hjermstad S.J. Briggs S.D. Smithgall T.E. Biochemistry. 1993; 32: 10519-10525Crossref PubMed Scopus (25) Google Scholar, 33.Hjermstad S. Peters K.L. Brigss S.D. Glazer R.I. Smithgall T.E. Oncogene. 1993; 8: 2283-2292PubMed Google Scholar). Combined lysates from 293T cells expressing 2D866–1064 were split into aliquots and incubated with the GST-SH3 fusions or GST alone (10 μg) immobilized on glutathione-agarose beads in a final volume of 1.0 ml of lysis buffer. Following incubation at 4 °C for 1 h, protein complexes were pelleted by centrifugation and washed three times with radioimmune precipitation buffer. Associated proteins were eluted by heating in SDS-PAGE sample buffer, and the presence of 2D866–1064 was determined by immunoblotting with anti-FLAG monoclonal antibody (M2, Santa Cruz Biotechnology). Analysis of deduced rat NMDA receptor subunit amino acid sequences revealed proline-rich sequences in the COOH-terminal portion of the NR2D subunit (Fig. 1). These sequences show striking similarities to those found in SH3 domain binding proteins (27.Rickles R. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar). To determine whether the proline-rich domain of the NR2D subunit can act as an SH3 domain binding protein, in vitroSH3 domain binding assays were performed. A fragment of the NR2D cDNA, encoding amino acids 866–1064, was amplified by PCR and tagged at its COOH terminus with the 8-amino acid FLAG epitope. The resulting construct, 2D866–1064, was expressed in 293T human embryonic kidney cells and expression was verified by immunoblot and immunoprecipitation using anti-FLAG antibodies (Fig.2). Lysates from 293T cells expressing 2D866–1064 were incubated with recombinant immobilized SH3 domain-GST fusion proteins, expressed and purified from E. coli. Following incubation and washing, bound 2D866–1064 was visualized by immunoblotting with anti-FLAG antibodies. As shown in Fig. 2, the proline-rich fragment of the NR2D subunit bound only to the Abl SH3 domain. 2D866–1064 did not bind to other SH3 domains tested, including those from phospholipase C-γ, Src, Fyn, GAP, or the full-length Grb2 protein. In addition, 2D866–1064 did not bind GST alone. Lysates from nontransfected control cells did not bind to any of the SH3 domains tested (data not shown).Figure 2The proline-rich region of the NR2D subunit binds to the Abl SH3 domain. Left, Expression of 2D866–1064 in 293T human embryonic kidney cells. Total cellular lysates (lysate) from 293T cells expressing the proline-rich region of the NR2D subunit, 2D866–1064, were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted using an antibody specific for the FLAG epitope. For immunoprecipitation (IP), soluble cellular extracts were incubated with the anti-FLAG antibody and protein G-Sepharose. Immunoprecipitates were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted using the anti-FLAG antibody. C, control cells; T, transfected cells. The position of 2D866–1064 is indicated by the arrowhead. Right, in vitro SH3 domain association assays. Lysates from 293T cells expressing 2D866–1064 were incubated with purified and immobilized GST-SH3 fusion proteins. 2D866–1064 bound to SH3 domains was eluted by heating in SDS-PAGE sample buffer and detected by immunoblotting with the anti-FLAG antibody. The SH3 domains tested are indicated. GST indicates the results from incubation of transfected 293T cell lysates with GST alone. The position of SH3 bound 2D866–1064 is indicated by the arrowhead. Note that shadow bands are visible on the Western blots for Abl SH3-GST, Src SH3-GST, GAP SH3-GST, and GST proteins. The Fyn SH3, PLCγ SH3, and Grb2 GST fusion proteins are covalently coupled to the solid support (Santa Cruz) and are not visible as shadow bands on Western blots.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The interaction of the Abl SH3 domain with the proline-rich region of NR2D in vitro suggests that these two proteins may associate in a living cell. Co-immunoprecipitation experiments were performed to determine whether full-length Abl and NR2D form a stable complex in co-transfected 293T human embryonic kidney cells. As shown in Fig.3, both NR2D (tagged with the FLAG epitope at its carboxyl terminus) and 2D866–1064co-immunoprecipitated with Abl in 293T cells expressing both proteins. NR2D was not seen in the anti-Abl immunoprecipitates from cells expressing either form of NR2D alone (see Fig. 3, center lanes). These data suggest that the presence of NR2D in the anti-Abl immunoprecipitates results from a specific and stable interaction between the full-length proteins. To investigate whether NR2D and Abl functionally interact in a model system, 293T cells were transiently transfected with Abl and NR2D cDNAs. Transfection of Abl cDNA in a mammalian overexpression system, such as 293T cells, results in constitutive Abl kinase activity (34.Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar, 35.Sawyers C.L. McLaughlin J. Goga A. Havlik M. Witte O. Cell. 1994; 77: 121-131Abstract Full Text PDF PubMed Scopus (250) Google Scholar). Lysates were prepared from cells co-expressing wild-type Abl (WT) and either the full-length NR2D subunit or 2D866–1064, the proline-rich fragment of the NR2D subunit, and immunoblotted with anti-phosphotyrosine antibodies. As shown in Fig. 4, overexpression of WT Abl in this system releases the negative regulation of tyrosine kinase activity as evidenced by autophosphorylation, in agreement with previous studies (34.Pendergast A.M. Muller A.J. Havlik M.H. Clark R. McCormick F. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5927-5931Crossref PubMed Scopus (123) Google Scholar, 35.Sawyers C.L. McLaughlin J. Goga A. Havlik M. Witte O. Cell. 1994; 77: 121-131Abstract Full Text PDF PubMed Scopus (250) Google Scholar). Surprisingly, co-expression of either full-length NR2D or 2D866–1064 with WT Abl resulted in inhibition of Abl autokinase activity as indicated on the phosphotyrosine blot. Control blots verify expression of Abl and NR2D proteins. As shown in Fig.5 B, inhibition of Abl autophosphorylation was dependent on the amount of NR2D or 2D866–1064 cDNA transfected, with nearly complete inhibition at the highest NR2D levels tested. Pretreatment of transfected cells with vanadate (3–300 μm) did not alter the inhibition of Abl by NR2D (data not shown), ruling out the possibility that NR2D works through an intermediate protein-tyrosine phosphatase.Figure 5Inhibition of Abl by NR2D is dependent on the amount of NR2D expressed. A, lysates from cells co-transfected with Abl and the indicated microgram amount of 2D866–1064 expression plasmid DNA (left panels) were immunoblotted with anti-phosphotyrosine antibodies. The position of autophosphorylated Abl is indicted by the arrowhead. Control levels of Abl (middle blots) and NR2D (bottom blots) proteins are indicated by arrowheads. Abl was immunoprecipitated from lysates of cells co-transfected with Abl and the indicated amount of NR2D (right panels) using the anti-Abl antibody, K-12. Immunoprecipitates were immunoblotted with antiphosphotyrosine (top blot) or anti-Abl (middle blot) antibodies. Arrowheads indicate the position of autophosphorylated Abl (top blot). Control levels of immunoprecipitated Abl (middle blot) or NR2D in cellular lysates (bottom blot) are indicated by thearrowheads. B, phosphotyrosine and anti-Abl immunoblots were scanned and immunoreactivity was quantitated using a Bio-Rad GS-710 calibrated imaging densitometer. The ratio of anti-phosphotyrosine immunoreactivity to anti-Abl immunoreactivity was determined to standardize tyrosine phosphorylation to levels of protein expression. Data are expressed as the percent control tyrosine phosphorylation (determined in the absence of NR2D). Shown are quantifications of experiments presented in A; these results are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because native NR2D exists as part of an oligomeric complex consisting minimally of NR2D and NR1 (36.Dunah A.W. Luo J. Wang Y.H. Yasuda R.P. Wolfe B.B. Mol. Pharmacol. 1998; 53: 429-437Crossref PubMed Scopus (104) Google Scholar), the NR2D subunit expressed in the absence of NR1 may not localize properly. Furthermore, it is possible that interactions with Abl in the absence of NR1 may not reflect the associations of the mature receptor with other proteins. To determine whether co-expression with the NR1 subunit alters the ability of NR2D to inhibit Abl autophosphorylation, 293T cells were co-transfected with NR1, NR2D, and Abl alone and in various combinations. Inhibition of Abl autokinase activity was observed in cells expressing Abl in combination with either NR2D alone or NR2D and NR1 but not in cells expressing Abl in combination with NR1 alone (Fig. 6). It is important to note that functional heteromeric receptors are observed in 293T cells 2(A. Qian, J. W. Johnson, and A. L. Buller, unpublished observations.co-expressing NR1 and NR2D subunits but not in cells expressing NR2 subunits alone (7.Kutsuwada T. Kashiwabuchi H. Mori H. Sakimura K. Kushiya E. Araki K. Meguro H. Masaki H. Kumanishi T. Arakawa M. Mishina M. Nature. 1992; 358: 36-41Crossref PubMed Scopus (1226) Google Scholar), suggesting that in the presence of the NR1 subunit, NR2D is inserted in the proper cellular location. Negative regulation of autokinase activity by the Abl SH3 domain has been reported (37.Franz W.M. Berger P. Wang J.Y.J. EMBO J. 1989; 8: 137-147Crossref PubMed Scopus (161) Google Scholar). To determine whether the Abl SH3 domain is critical for the inhibitory effect of NR2D on kinase activity, an Abl mutant lacking the SH3 domain (ΔSH3) was co-expressed with NR2D in 293T cells. 2D866–1064 did not inhibit autophosphorylation of ΔSH3 Abl (Fig. 7 A), indicating that inhibition by 2D866–1064 is dependent upon the Abl SH3 domain. In contrast to 2D866–1064, co-expression of the full-length NR2D subunit with ΔSH3 Abl produced partial inhibition of autokinase activity, suggesting the presence of additional sites of interaction between NR2D and Abl. Similar results were also obtained with Abl-PP, an Abl mutant containing two point mutations in the SH2-kinase linker region (Fig. 7 B). These mutations have been proposed to release negative regulation by the SH3 domain (24.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar). As seen with ΔSH3, 2D866–1064did not inhibit Abl-PP autokinase activity (Fig. 7 B), suggesting that the proper spatial relationship between 2D866–1064 and the Abl SH3 and kinase domains is required for the inhibitory effect. In contrast, the full-length NR2D subunit partially inhibited Abl-PP kinase activity, similar to that seen for ΔSH3 Abl kinase activity. One of the most striking features of the NR2D subunit amino acid sequence is the prevalence of proline residues in the COOH-terminal region of the molecule, immediately downstream from the M4 domain. This proline-rich region localizes to the cytoplasmic surface of the cell membrane (38.Hollmann M. Monaghan D.T. Wenthold R.J. The Ionotropic Glutamate Receptors. Humana Press, Totowa, NJ1997: 39-79Crossref Google Scholar), making it a potential target for interactions with regulatory molecules. The data in this report provide evidence of a novel interaction between the NR2D subunit of the NMDA receptor and the Abl tyrosine kinase that may involve the proline-rich region of NR2D and the SH3 domain of Abl. SH3 domain-dependent interactions of ion channels with nonreceptor tyrosine kinases have been reported in other systems. Src kinase inhibits hKv1.5 potassium channel currents in an SH3 domain-dependent manner (39.Holmes T.C. Fadool D.A. Ren R. Levitan I.B. Science. 1996; 274: 2089-2091Crossref PubMed Scopus (231) Google Scholar). Given the structural homology between the potassium channel and the NMDA receptor, it is interesting that the proline-rich domain of NR2D does not interact with the Src SH3 domain (see Fig. 2). However, the preferred Src SH3 domain binding motif (RPLPXXP (26.Ren R. Mayer B.J. Cicchetti P. Baltimore D. Science. 1993; 259: 1157-1161Crossref PubMed Scopus (1022) Google Scholar, 27.Rickles R. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar)) is not present in the NR2D subunit. The Abl SH3 domain shows a preference for the peptide sequenceXPXXPPPΦXP, where Φ is any hydrophobic residue (27.Rickles R. Botfield M.C. Weng Z. Taylor J.A. Green O.M. Brugge J.S. Zoller M.J. EMBO J. 1994; 13: 5598-5604Crossref PubMed Scopus (223) Google Scholar, 28.Rickles R.J. Botfield M.C. Zhou X.M. Henry P.A. Brugge J.S. Zoller M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10909-10913Crossref PubMed Scopus (175) Google Scholar). This consensus sequence is closely approximated in the NR2D subunit (PPAKPPPQP; see Fig. 1). Data presented here demonstrate that complete inhibition of Abl autophosphorylation by NR2D requires the Abl SH3 domain, which has been implicated in the negative regulation of Abl kinase activity. An inhibitory intramolecular association between the Abl SH3 domain and sequences connecting the SH2 and kinase domains (SH2-kinase linker) has been proposed (24.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar), based on an analogous interaction revealed in the x-ray crystal structures of the inactive form of c-Src and the closely related tyrosine kinase Hck (40.Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar, 41.Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). Mutations in the Src, Hck, and Abl SH2-kinase linker domains result in kinase activation (24.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar, 42.Gonfloni S. Williams J.C. Hattula K. Weijland A. Wierenga R.K. Superti-Furga G. EMBO J. 1997; 16: 7261-7271Crossref PubMed Scopus (127) Google Scholar, 43.Briggs S.D. Smithgall T.E. J. Biol. Chem. 1999; 274: 26579-26583Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), presumably by disrupting the intramolecular SH3-linker interaction required for maintenance of the inactive state. Other work has shown that interaction with a ligand that binds exclusively to the SH3 domain of Hck induces kinase activation due to displacement of the linker region from the SH3 domain (44.Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar, 45.Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Because 2D866–1064inhibits rather than stimulates kinase activity, and inhibition requires the SH3 domain of Abl, we conclude that 2D866–1064 must make additional contacts with Abl outside of the SH3 domain, possibly within the kinase domain itself. Furthermore, the inability of the NR2D proline-rich fragment to inhibit the activated linker mutant of Abl suggests that inhibition requires the wild-type structural relationship between the Abl SH3, linker, and kinase domains. In addition to the NR2D subunit of the NMDA receptor, a number of other proteins have been reported to interact with the Abl SH3 domain. Among these proteins, both AAP-1 (46.Zhu J. Shore S.K. Mol. Cell Biol. 1996; 16: 7054-7062Crossref PubMed Scopus (37) Google Scholar) and PAG (47.Wen S. Van Etten R.A. Genes Dev. 1997; 11: 2456-2467Crossref PubMed Scopus (239) Google Scholar) have been shown to suppress kinase activity. Data presented in this report provide the first demonstration of a direct effect of a ligand gated ion channel subunit on tyrosine kinase function and may have important implications for the significance of Abl in the brain. Although specific functions for the Abl tyrosine kinase have not been determined, Abl plays a role in the negative regulation of cell growth (35.Sawyers C.L. McLaughlin J. Goga A. Havlik M. Witte O. Cell. 1994; 77: 121-131Abstract Full Text PDF PubMed Scopus (250) Google Scholar) and interacts with cell cycle regulatory proteins (48.Wang J.Y.J. Curr. Opin. Genet. Dev. 1993; 3: 35-43Crossref PubMed Scopus (154) Google Scholar, 49.Baskaran R. Chiang G.G. Mysliwiec T. Kruh G.D. Wang J.Y. J. Biol. Chem. 1997; 272: 18905-18909Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 50.Welch P.J. Wang J.Y.J. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In addition, recent evidence suggests a role for Abl in mediating the cellular stress response by activating the c-Jun NH2-terminal kinase pathway (51.Kharbanda S. Pandey P. Ren R. Mayer B. Zon L. Kufe D. J. Biol. Chem. 1995; 270: 30278-30281Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Interestingly, the c-Jun NH2-terminal kinase pathway is also activated by NMDA receptor stimulation (52.Schwarzschild M.A. Cole R.L. Hyman S.E. J. Neurosci. 1997; 17: 3455-3466Crossref PubMed Google Scholar, 53.Ko H.W. Park K.Y. Kim H. Han P.L. Kim Y.U. Gwag B.J. Choi E.J. J. Neurochem. 1998; 71: 1390-1395Crossref PubMed Scopus (104) Google Scholar), suggesting the possibility that NMDA receptor-mediated activation of cellular stress pathways may be Abl-mediated. The localization of Abl and NMDA receptor subunits show several parallels. Subcellularly, Abl localizes to both the cytoskeleton and the nucleus (54.Van Etten R.A. Jackson P.K. Baltimore D. Sanders M.C. Matsudaira P.T. Janmey P.A. J. Cell Biol. 1994; 124: 325-340Crossref PubMed Scopus (237) Google Scholar, 55.Wen S. Jackson P.K. Van Etten R.A. EMBO J. 1996; 15: 1583-1595Crossref PubMed Scopus (186) Google Scholar). The NMDA receptor associates with structural proteins that link it to the cytoskeleton (17.Kornau H. Schenker L.T. Kennedy M.B. Seeburg P.H. Science. 1995; 269: 1737-1740Crossref PubMed Scopus (1631) Google Scholar, 25.Gomperts S.N. Cell. 1996; 84: 659-662Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The co-localization of Abl and NMDA receptors to the same subcellular compartment may provide an opportunity for these proteins to interact in a biologically relevant manner. Abl has been identified in the brain, in regions known to express NMDA receptor subunits (56.Grant S.G.N. O'Dell T.J. Karl K.A. Stein P.L. Soriano P. Kandel E.R. Science. 1992; 258: 1903-1910Crossref PubMed Scopus (966) Google Scholar, 57.Renshaw M.W. Capozza M.A. Wang J.Y.J. Mol. Cell. Biol. 1988; 8: 4547-4551Crossref PubMed Scopus (41) Google Scholar, 58.Koleske A.J. Gifford A.M. Scott M.L. Nee M. Bronson R.T. Miczek K.A. Baltimore D. Neuron. 1998; 21: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar), and theDrosophila homolog of Abl plays a role in the development of the nervous system (59.Henkemeyer M. West S.R. Gertier F.B. Hoffman F.M. Cell. 1990; 63: 949-960Abstract Full Text PDF PubMed Scopus (86) Google Scholar, 60.Hill K.K. Bedian V. Juang J. Hoffmann F.M. Genetics. 1995; 141: 595-606Crossref PubMed Google Scholar). Recently, a critical role for Abl and the Abl-related gene product Arg, in the development of the mammalian CNS has been demonstrated (58.Koleske A.J. Gifford A.M. Scott M.L. Nee M. Bronson R.T. Miczek K.A. Baltimore D. Neuron. 1998; 21: 1259-1272Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Given the unique developmental distribution of the NR2D subunit (13.Watanabe M. Inoue Y. Sakimura K. Mishina M. Neuroreport. 1992; 3: 1138-1140Crossref PubMed Scopus (617) Google Scholar, 61.Wenzel A. Schuerer L. Kunzi R. Fritschy J.M. Mohler H. Benke D. Neuroreport. 1995; 7: 45-48Crossref PubMed Scopus (132) Google Scholar), it is interesting to speculate that an interaction between NR2D and Abl may occur during development. We thank Dr. Peter Seeburg for the gift of the NR2D cDNA. We also thank Scott Briggs for generating the SH3 domain-GST fusion proteins and Michelle Bobo and Mavrothi Kontanis for excellent technical assistance." @default.
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