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• W2080213202 abstract "Activation and Thr286autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx viaN-methyl-d-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation. Activation and Thr286autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx viaN-methyl-d-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation. CaMKII is a multifunctional, calcium-activated kinase (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar, 2Soderling T.R. Adv. Second Messenger Phosphoprotein Res. 1995; 30: 175-189Crossref PubMed Scopus (39) Google Scholar), whose α and β isoforms are particularly abundant in brain cytosol and in postsynaptic densities (PSDs), 1The abbreviations used are: PSDpostsynaptic densityCaMKIIcalcium/calmodulin-dependent protein kinase IICaMKIIα/βα/β isoform of CaMKII[P-T286]CaMKIIαCaMKIIα autophosphorylated at threonine 286[P-T306]CaMKIIαCaMKIIα autophosphorylated at threonine 305 and/or threonine 306NMDAN-methyl-d-aspartateAPV2-amino-5-phosphonovaleric acidGSTglutathioneS-transferaseLTPlong term potentiation. submembranous scaffolds for receptors, ion channels, and signal transducers (3Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 4Ziff E.B. Neuron. 1997; 19: 1163-1174Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Postsynaptic calcium influx triggers autophosphorylation of CaMKII at a threonine residue in the autoinhibitory domain (Thr286 in CaMKIIα) (5Fukunaga K. Stoppini L. Miyamoto E. Muller D. J. Biol. Chem. 1993; 268: 7863-7867Abstract Full Text PDF PubMed Google Scholar), which renders the kinase persistently active and causes a translocation of soluble CaMKII to the PSD (6Strack S. Choi S. Lovinger D.M. Colbran R.J. J. Biol. Chem. 1997; 272: 13467-13470Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). Multiple lines of evidence indicate Thr286 autophosphorylation of postsynaptic CaMKII is necessary for NMDA receptor-dependent LTP (7Silva A.J. Stevens C.F. Tonegawa S. Wang Y. Science. 1992; 257: 201-206Crossref PubMed Scopus (1183) Google Scholar, 8Pettit D.L. Perlman S. Malinow R. Science. 1994; 266: 1881-1885Crossref PubMed Scopus (268) Google Scholar, 9Lledo P.-M. Hjelmstad G.O. Mukherji S. Soderling T.R. Malenka R.C. Nicoll R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11175-11179Crossref PubMed Scopus (360) Google Scholar, 10Otmakhov N. Griffith L.C. Lisman J.E. J. Neurosci. 1996; 17: 5357-5365Crossref Google Scholar, 11Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (884) Google Scholar), a cellular model of learning and memory. PSD-associated CaMKII phosphorylates ionotropic glutamate receptors (6Strack S. Choi S. Lovinger D.M. Colbran R.J. J. Biol. Chem. 1997; 272: 13467-13470Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 12McGlade-McCulloh E. Yamamoto H. Tan S.E. Brickey D.A. Soderling T.R. Nature. 1993; 362: 640-642Crossref PubMed Scopus (337) Google Scholar, 13Omkumar R.V. Kiely M.J. Rosenstein A.J. Min K.T. Kennedy M.B. J. Biol. Chem. 1996; 271: 31670-31678Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 14Mammen A.L. Kameyama K. Roche K.W. Huganir R.L. J. Biol. Chem. 1997; 272: 32528-32533Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), providing a mechanism for increased synaptic strength during LTP (15Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Science. 1997; 276: 2042-2045Crossref PubMed Scopus (884) Google Scholar). postsynaptic density calcium/calmodulin-dependent protein kinase II α/β isoform of CaMKII CaMKIIα autophosphorylated at threonine 286 CaMKIIα autophosphorylated at threonine 305 and/or threonine 306 N-methyl-d-aspartate 2-amino-5-phosphonovaleric acid glutathioneS-transferase long term potentiation. Mechanisms by which CaMKII is targeted to its postsynaptic substrates are poorly understood. Previous gel overlay analyses revealed a candidate PSD-associated CaMKII-anchoring protein, p190, that binds selectively to the Thr286-autophosphorylated kinase ([P-T286]CaMKIIα) (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The NR2A and NR2B subunits of the NMDA receptor share several properties with this CaMKII-binding activity, including apparent size, enrichment in PSDs, and regional and developmental expression profiles 2S. Strack, R. B. McNeill, and R. J. Colbran, unpublished data. (17Sheng M. Cummings J. Roldan L.A. Jan Y.N. Jan L.Y. Nature. 1994; 368: 144-147Crossref PubMed Scopus (1110) Google Scholar). Here, we demonstrate a direct and specific interaction between [P-T286]CaMKIIα and NR2B and show that NR2B targets CaMKII in intact cells. PSD isolation and immunoprecipitation of sodium dodecyl sulfate (SDS)-solubilized PSD proteins were carried out as described (6Strack S. Choi S. Lovinger D.M. Colbran R.J. J. Biol. Chem. 1997; 272: 13467-13470Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar) using 2 μg/ml NR2A/B antibodies (Chemicon) and protein phosphatase 1 antibodies (18Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (262) Google Scholar). For CaMKII·NMDA receptor coimmunoprecipitation, PSDs (1 mg/ml) were cross-linked (45 min, 4 °C) with 0.25 mm dithiobis(succinimidyl suberate), dissolved by sonication in 2% SDS, and diluted 15-fold in 1% Nonidet P-40, 200 mm NaCl, 50 mm Tris, pH 7.5, 2 mm EDTA, 2 mm EGTA, 1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, 1 μm microcystin-LR. The supernatant after ultracentrifugation (30 min, 100,000 × g) was immunoprecipitated with 3 μg/ml goat anti-CaMKII (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) or preimmune IgG (19Colbran R.J. Bass M.A. McNeill R.B. Bollen M. Zhao S. Wadzinski B.E. Strack S. J. Neurochem. 1997; 69: 920-929Crossref PubMed Scopus (46) Google Scholar). The cross-linker was cleaved and proteins eluted from the beads by boiling in reducing SDS sample buffer. Purified recombinant CaMKIIα was autophosphorylated with [γ-32P]ATP (8,000–40,000 cpm/pmol) in the presence of calcium/calmodulin or EGTA at Thr286 or Thr305/306, respectively, and desalted (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Stoichiometries ranged between 0.17 and 0.39 (Thr286) and 0.24 and 0.47 (Thr305/306). Protein blots to be analyzed for CaMKII binding were blocked and incubated with 100–200 nm [32P]CaMKII in 5% milk for 3 h, washed extensively, and autoradiographed. 18-Day-old cultures of dissociated neonatal rat cortex were fixed in acetone:methanol (1:1), blocked, and incubated 10–14 h in 1:500 dilutions of goat anti-CaMKII (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), rabbit anti-NR1 (20Rema V. Ebner F.F. J. Comp. Neurol. 1996; 368: 165-184Crossref PubMed Scopus (36) Google Scholar), and mouse anti-synaptophysin (Boehringer Mannheim) in 1% normal donkey serum, 10 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Triton X-100. Cultures were treated with species-specific donkey antibodies conjugated to Cy3, Cy2, and Cy5 (Jackson Laboratories) and imaged on a Zeiss laser scanning confocal microscope. The entire cytoplasmic domains (C terminus starting immediately after transmembrane region IV) of NR1 (splice variant A containing both C1 and C2 exon cassettes), NR2A, and NR2B subunits, as well as shorter NR2B constructs, were subcloned from full-length cDNAs by polymerase chain reaction using Pfu polymerase and primers containing restrictions sites or by restriction digests. Fragments were sequenced and ligated into pRSET-A His6 tag (Qiagen) or pGEX-2T glutathione S-transferase (GST) (Amersham Pharmacia Biotech) fusion vectors. His6 tag fusions were expressed, and GST fusions were expressed and purified according to the manufacturers' instructions. His6 tag fusion protein lysates were subjected to CaMKII overlay (see above) or immunoblotted with anti His6 tag antibodies (CLONTECH) and 125I-labeled secondary antibodies for expression levels, followed by PhosphorImager quantification. Ni2+-coated 96-well plates (HisSorb strips, Qiagen) were adsorbed for 2 h with soluble His6 tag NR2B fusion protein expressing or nonexpressing bacterial extracts (0.25 mg/ml) in blocking buffer (5 mg/ml bovine serum albumin, 200 mm NaCl, 50 mm Tris, pH 7.5, 0.1% Tween 20, 5 mm β-mercaptoethanol). After extensive washes, [32P-T286]CaMKIIα diluted in blocking buffer (200 μl) was allowed to bind to the tethered fusion protein for 2 h, followed by 10–12 more washes. Bound CaMKII was solubilized in 1% SDS, 0.2 n NaOH, 50 mm EDTA, and quantified by liquid scintillation counting. Nonspecific binding to control bacterial extracts was subtracted from total binding to obtain specific binding. No specific binding was observed using [32P-T306]CaMKIIα. GST fusion proteins were incubated (1 h, 4 °C) with either purified CaMKIIα (Fig. 2 D, see caption) or with a freshly prepared rat brain cytosolic extract (∼3 mg/ml extract protein, 10 μg/ml GST fusion protein) containing 2 μm microcystin-LR and 0.5% Triton X-100, precipitated with glutathione-agarose, washed extensively, and eluted with SDS sample buffer. CaMKIV antibodies were from Transduction Laboratories. HEK293 cells were seeded on coverslips in 35-mm dishes, transfected with a total of 3 μg/dish DNA (1 μg of SRα promotor-CaMKIIα expression plasmid, 2 μg of cytomegalovirus promotor plasmids with NMDA receptor subunits at a mass ratio of 1:3 NR1a and NR2A/B subunits), and grown for 48 h as described (21Lovinger D.M. J. Pharmacol. Exp. Ther. 1995; 274: 164-172PubMed Google Scholar). Robust expression of NMDA currents was verified by patch-clamp recording of parallel cultures. 3R. L. Popp and D. M. Lovinger, personal communication. Cells were washed and incubated in Mg2+-free Hanks' balanced saline containing 2 mm CaCl2 and either the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV, 50 μm) or NMDA/glycine (100/10 μm) for 15 min. Cultures were fixed and processed for immunofluorescence (see above) using 1:500 antibody dilutions of goat anti-CaMKII (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), mouse anti-NR1 (PharMingen), and rabbit anti-NR2A/B (Chemicon). Between 2 and 5% of cells were strongly positive for at least one label; only those cells expressing high levels of each antigen (>50% of transfected cells) were included in the analyses. Under basal conditions, CaMKIIα expression was diffusely cytoplasmic. Irrespective of agonist treatment, NR1 and NR2A/B strictly colocalized (mean scores >3.4, see below) in a patchy or reticular, often perinuclear pattern as seen previously in heterologous cells (22Kim E. Cho K.O. Rothschild A. Sheng M. Neuron. 1996; 17: 103-113Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). Cultures were randomized prior to sampling digital images on a confocal microscope to prevent operator bias. Coded images (as in Fig. 3) were assigned a colocalization score by a second, naive observer: 0, mutual exclusion; 1, coincidental overlap; 2 or 3, increasing degrees of colocalization, 4, complete overlap of labels. For reference, the cells in Fig. 3 scored a 0, 1, 2, 2, and a 3 (from left to right, top tobottom). To determine whether NR2 subunits contribute to the previously characterized “p190” overlay binding activity (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), we analyzed immunoprecipitated NR2A/B by gel overlay with [32P-T286]CaMKIIα (Fig.1 A). A CaMKII-binding activity comigrating with NR2A and NR2B was immunoprecipitated with NR2A/B antibodies, but not control antibodies, indicating that NR2A and/or NR2B are CaMKII-binding proteins. This interaction may be physiologically relevant, because triple immunofluorescent labeling of cultured cortical neurons demonstrated that CaMKII colocalizes with NMDA receptors in many punctae along dendritic shafts, identified as synapses by the adjacent or overlapping presence of synaptophysin (Fig. 1 B). Higher magnification revealed a mostly postsynaptic localization of CaMKII in dendritic spines (Fig. 1 C). Moreover, a complex of CaMKII with NMDA receptor subunits can be immunoprecipitated from PSDs using CaMKII antibodies, but not preimmune IgG (Fig. 1 D). NR2B was more efficiently coprecipitated than NR1, likely because association of CaMKII with NR1 is indirect (i.e. via NR2B, see below). Recovery of the receptor-kinase complex required pretreatment of PSDs with a reversible cross-linker prior to essentially complete PSD solubilization in 2% SDS, indicating that the interaction of CaMKII with NMDA receptors is not stable in harsh detergents. The specificity of the cross-linking procedure was demonstrated by the absence of other abundant PSD proteins in the immunoprecipitate, including the catalytic subunit of protein phosphatase 1 (Fig. 1 D). NMDA receptor subunits have a common transmembrane topology with three membrane-spanning regions and a C-terminal tail of variable length, which forms the intracellular portion of the receptor (Fig.2 A, diagram). Bacterial lysates expressing the cytoplasmic domains of the predominant forebrain NMDA receptor subunits, NR1, NR2A, and NR2B, as His6 tag fusion proteins were screened for [32P]CaMKIIα binding by overlay (Fig. 2 A). The NR2B cytoplasmic domain bound about six times more [32P-T286]CaMKIIα than the corresponding region of NR2A; neither NR1 nor any endogenous bacterial proteins showed detectable binding. Interactions with NR2A and NR2B were specific for autonomously active CaMKII, as CaMKIIα phosphorylated in the absence of calcium/calmodulin at Thr305/306 ([P-T306]CaMKIIα) bound only weakly (<5%). Because NR2B displayed the most robust interaction with CaMKII, we mapped its CaMKII-binding domain by creating a series of truncation and internal deletion constructs. Only constructs containing NR2B residues 1260–1309 showed CaMKII binding similar to the full-length cytoplasmic tail. Fusion of NR2B-(1260–1309) to GST demonstrated that this domain is also sufficient for interaction with autonomous CaMKII (Fig.2 B). A solution interaction assay was employed to examine binding of CaMKII to NR2B that had not undergone denaturation/renaturation for gel overlay analysis. [32P-T286]CaMKIIα bound saturably to a His6 tag NR2B fusion protein containing residues 1260–1309, but not to a construct that starts at residue 1310, C-terminal of this domain (Fig. 2 C). Scatchard analysis indicated that binding involves a simple bimolecular interaction with aKd of 138 ± 60 nm(n = 3) (Fig. 2 C, inset). ThisKd is ∼100 times lower than the average concentration of CaMKIIα in forebrain (16McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 23Erondu N.E. Kennedy M.B. J. Neurosci. 1985; 5: 3270-3277Crossref PubMed Google Scholar), suggesting that the interaction can readily occur in neurons. The CaMKII-binding domain in NR2B contains a high-affinity phosphorylation site, Ser1303, which is phosphorylated by CaMKII in vitro and is also phosphorylated in vivo (13Omkumar R.V. Kiely M.J. Rosenstein A.J. Min K.T. Kennedy M.B. J. Biol. Chem. 1996; 271: 31670-31678Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). However, three lines of evidence indicate that the binding of CaMKII to NR2B-(1260–1309) is not dependent on a substrate interaction. First, the model peptide substrate syntide-2 only weakly inhibits CaMKII binding (∼30%) at concentrations of ∼100-fold theKm for phosphorylation (not shown). Second, even though NR2A residues 1255–1298 are 36% identical to NR2B-(1260–1309), and sequences surrounding the phosphorylation site are almost perfectly conserved (NR2B, LRRQHSYD; NR2A, INRQHSYD) (13Omkumar R.V. Kiely M.J. Rosenstein A.J. Min K.T. Kennedy M.B. J. Biol. Chem. 1996; 271: 31670-31678Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), CaMKII binding to NR2A-(1255–1298) is ∼10-fold weaker under our overlay conditions (10.7 ± 1.8%,n = 3, Fig. 2 B), suggesting that nonconserved residues in NR2B-(1260–1309) are important for high-affinity CaMKII binding. Third, “pull-down” experiments, in which GST-NR2B fusion protein was purified with glutathione-agarose, showed that calcium/calmodulin alone did not promote CaMKII interaction with NR2B, but that stoichiometric interaction was instead strictly dependent on CaMKIIα autophosphorylation at Thr286 (Fig. 2 D). On the other hand, calcium/calmodulin binding is sufficient for full CaMKII activation, and Thr286 autophosphorylation stabilizes the active conformation of the kinase in the absence of calcium/calmodulin (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar, 2Soderling T.R. Adv. Second Messenger Phosphoprotein Res. 1995; 30: 175-189Crossref PubMed Scopus (39) Google Scholar). Thus, CaMKII residues outside the substrate binding site are involved in the interaction with NR2B. Further evidence for specific association of CaMKII with NR2B was obtained by performing GST-NR2B pull-downs from brain cytosolic extracts. α and β isoforms of CaMKII were isolated following incubation with GST-NR2B-(1260–1309), but not GST alone. Affinity-purified CaMKIIα displayed an upward electrophoretic mobility shift characteristic of autophosphorylation (Fig.2 E). CaM kinase IV, a related kinase with a similar phosphorylation site preference (24White R.R. Kwon Y.-G. Taing M. Lawrence D.S. Edelman A.M. J. Biol. Chem. 1998; 273: 3166-3179Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), as well as other kinases and phosphatases tested, were not detected in the precipitated material, strongly indicating that NR2B-(1260–1309) binds selectively to CaMKII. The NR2B subunit of the NMDA receptor was shown to target Thr286 autophosphorylated CaMKII in HEK293 cells. CaMKIIα was coexpressed with various NMDA receptor subunit combinations, and their distributions were compared by immunofluorescence (Fig.3). Whereas NR1 alone does not form functional NMDA receptors in HEK293 cells, activation of both NR1/NR2A and NR1/NR2B receptors leads to massive calcium influx (25Grant E.R. Bacskai B.J. Pleasure D.E. Pritchett D.B. Gallagher M.J. Kendrick S.J. Kricka L.J. Lynch D.R. J. Biol. Chem. 1997; 272: 647-656Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Coexpression of CaMKIIα and NR1 alone resulted in low colocalization scores that were unaffected by acute treatment with the receptor coagonists NMDA/glycine (Fig. 3 A). Perhaps reflecting the low but detectable CaMKII binding activity of NR2A (Fig. 2,A and B), additional expression of the NR2A subunit led to a small increase in CaMKIIα and NR1/NR2A colocalization, which was not significantly increased by NMDA/glycine treatment (Fig. 3 B). In cells expressing NR2B with CaMKIIα and NR1, we observed a similarly modest increase in colocalization in the absence of agonist treatment compared with CaMKIIα and NR1 alone (Fig. 3, C and D). In contrast to NR2A-containing NMDA receptors, activation of NR1/NR2B receptors with NMDA/glycine caused a highly significant redistribution of CaMKIIα into receptor-positive patches (Fig. 3, C and D), strongly suggesting that receptor activation induced the formation of a CaMKII·NR2B complex. Replacing extracellular calcium with barium, which is receptor-permeable but binds only poorly to calmodulin, completely blocked the effect of NMDA (Fig. 3 D). Thus, opening of NMDA receptors is not sufficient for complex formation, but calcium influx is essential, presumably to stimulate calcium/calmodulin-dependent autophosphorylation of CaMKII. Consistent with this interpretation, an autophosphorylation-incompetent form of the kinase, T286A-CaMKIIα (26Fong Y.L. Taylor W.L. Means A.R. Soderling T.R. J. Biol. Chem. 1989; 264: 16759-16763Abstract Full Text PDF PubMed Google Scholar, 27Hanson P.I. Kapiloff M.S. Lou L.L. Rosenfeld M.G. Schulman H. Neuron. 1989; 3: 59-70Abstract Full Text PDF PubMed Scopus (237) Google Scholar), expressed at similar levels of wild-type CaMKIIα failed to show activity-induced colocalization with NR1/NR2B containing NMDA receptors (Fig.3 D). Thus, NR2B mediates targeting of CaMKII to NMDA receptors in a calcium- and Thr286autophosphorylation-dependent manner in intact cells. Our data support a model in which dendritic calcium influx induced by synaptic activity triggers CaMKII autophosphorylation at Thr286 and subsequent binding to residues 1260–1309 in the NR2B subunit of the NMDA receptor. What are the functional consequences of this interaction? Autonomous CaMKII in the PSD is inactivated by PSD-associated serine/threonine phosphatases (18Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (262) Google Scholar, 28Shields S.M. Ingebritsen T.S. Kelly P.T. J. Neurosci. 1985; 5: 3414-3422Crossref PubMed Google Scholar, 29Dosemeci A. Reese T.S. J. Neurochem. 1993; 61: 550-555Crossref PubMed Scopus (60) Google Scholar). Once dephosphorylated at Thr286, CaMKII positioned near the mouth of the NMDA receptor calcium channel is likely to undergo rapid re-autophosphorylation even during periods of low level NMDA receptor activation. Thus, an interaction of CaMKII with NMDA receptors is predicted to boosts autonomous kinase activity, leading to enhanced phosphorylation of nearby downstream effectors of synaptic plasticity (15Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Science. 1997; 276: 2042-2045Crossref PubMed Scopus (884) Google Scholar). Furthermore, recruitment of CaMKII into the PSD structure (6Strack S. Choi S. Lovinger D.M. Colbran R.J. J. Biol. Chem. 1997; 272: 13467-13470Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), possibly via association with NR2B, may play a role in the rapid ultrastructural changes of synapses that undergo LTP (30Geinisman Y. deToledo-Morrell L. Morrell F. Brain Res. 1991; 566: 77-88Crossref PubMed Scopus (233) Google Scholar, 31Buchs P.-A. Muller D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8040-8045Crossref PubMed Scopus (228) Google Scholar). The developmental appearance of NR2A and down-regulation of NR2B in the mammalian visual system correlate with the end of the “critical period” of synapse maturation (32Flint A.C. Maisch U.S. Weishaupt J.H. Kriegstein A.R. Monyer H. J. Neurosci. 1997; 17: 2469-2476Crossref PubMed Google Scholar, 33Shi J. Aamodt S.M. Constantine-Paton M. J. Neurosci. 1997; 17: 6264-6276Crossref PubMed Google Scholar). Preferential association of CaMKII with NR2B over NR2A may therefore provide a mechanism by which NMDA receptor subunit composition can impact developmental plasticity. We thank L. MacMillan for scoring cells; M. Bass for invaluable technical assistance; V. Rema, F. Ebner, and M. Maguire (Vanderbilt) for NR1 antibodies and cortical cultures; D. Lynch (Penn State) for NR expression plasmids; T. Soderling (Vollum Institute) for CaMKII expression plasmids; DNAX, Inc. (Palo Alto, CA) for use of the pME18S expression vector; L. Popp, S. Sessoms, and D. Lovinger (Vanderbilt) for help with HEK cell transfections; and R. Blakely, F. Ebner, J. Exton, L. Limbird, J. Lisman, D. Lovinger, and B. Wadzinski for helpful suggestions." @default.
• W2080213202 created "2016-06-24" @default.
• W2080213202 creator A5045487847 @default.
• W2080213202 creator A5070992833 @default.
• W2080213202 date "1998-08-01" @default.
• W2080213202 modified "2023-10-10" @default.
• W2080213202 title "Autophosphorylation-dependent Targeting of Calcium/ Calmodulin-dependent Protein Kinase II by the NR2B Subunit of theN-Methyl-d-aspartate Receptor" @default.
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• W2080213202 doi "https://doi.org/10.1074/jbc.273.33.20689" @default.
• W2080213202 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9694809" @default.
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