Autophosphorylation-dependent Targeting of Calcium/ Calmodulin-dependent Protein Kinase II by the NR2B Subunit of theN-Methyl-d-aspartate Receptor

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 CaMKIIP-T286CaMKIIαCaMKIIα autophosphorylated at threonine 286P-T306CaMKIIαCaMKIIα 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-T286CaMKIIα) (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-T286CaMKIIα 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-32PATP (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 32PCaMKII 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-T286CaMKIIα 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-T306CaMKIIα. 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-T286CaMKIIα (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 32PCaMKIIα binding by overlay (Fig. 2 A). The NR2B cytoplasmic domain bound about six times more 32P-T286CaMKIIα 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-T306CaMKIIα) 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-T286CaMKIIα 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 in The CaMKII-binding domain in NR2B a high-affinity which is phosphorylated by CaMKII in and is also phosphorylated in 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). three lines of evidence indicate that the binding of CaMKII to NR2B-(1260–1309) is not on a substrate the model substrate only weakly CaMKII binding at of for NR2A residues are to and the are NR2A, 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 is overlay ± = 3, Fig. 2 suggesting that residues in NR2B-(1260–1309) are for high-affinity CaMKII binding. in which fusion protein was purified with glutathione-agarose, showed that calcium/calmodulin not CaMKII interaction with NR2B, but that interaction was strictly on CaMKIIα autophosphorylation at Thr286 (Fig. 2 D). the other calcium/calmodulin binding is sufficient for CaMKII activation, and Thr286 autophosphorylation the active 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). CaMKII residues the substrate binding are in the interaction with evidence for specific association of CaMKII with NR2B was by from brain cytosolic extracts. α and β isoforms of CaMKII were isolated following with but not GST CaMKIIα displayed of autophosphorylation (Fig.2 kinase a kinase with a similar M. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), as well as other and were not in the precipitated strongly indicating that NR2B-(1260–1309) binds selectively to CaMKII. The NR2B subunit of the NMDA receptor was to Thr286 autophosphorylated CaMKII in HEK293 cells. CaMKIIα was with NMDA receptor subunit and were by immunofluorescence NR1 not NMDA receptors in HEK293 of both and receptors to calcium influx M.J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). of CaMKIIα and NR1 in colocalization scores that were by with the receptor NMDA/glycine (Fig. 3 A). the but detectable CaMKII binding activity of NR2A (Fig. and expression of the NR2A subunit to a in CaMKIIα and colocalization, which was not increased by NMDA/glycine (Fig. 3 B). cells expressing NR2B with CaMKIIα and NR1, we observed a in colocalization in the absence of agonist with CaMKIIα and NR1 (Fig. 3, and D). to NMDA receptors, of receptors with NMDA/glycine a of CaMKIIα into (Fig. 3, and strongly suggesting that receptor the of a calcium with which is but binds only poorly to blocked the of NMDA (Fig. 3 D). of NMDA receptors is not sufficient for complex but calcium influx is to calcium/calmodulin-dependent autophosphorylation of CaMKII. with this of the Soderling T.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Schulman H. Neuron. Full Text PDF PubMed Scopus Google Scholar), expressed at similar levels of CaMKIIα to show colocalization with containing NMDA receptors D). NR2B targeting of CaMKII to NMDA receptors in a and in intact cells. a model in which dendritic calcium influx by synaptic activity triggers CaMKII autophosphorylation at Thr286 and binding to residues 1260–1309 in the NR2B subunit of the NMDA are the of this CaMKII in the PSD is by PSD-associated (18Strack S. Barban M.A. Wadzinski B.E. Colbran R.J. J. Neurochem. 1997; 68: 2119-2128Crossref PubMed Scopus (262) Google Scholar, J. Neurosci. 1985; 5: PubMed Google Scholar, A. J. Neurochem. 1993; PubMed Scopus Google Scholar). at CaMKII the of the NMDA receptor calcium is likely to during of NMDA receptor interaction of CaMKII with NMDA receptors is to autonomous kinase activity, to of of synaptic (15Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Science. 1997; 276: 2042-2045Crossref PubMed Scopus (884) Google Scholar). of CaMKII into 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), via association with NR2B, may a in the of synapses that LTP Y. L. Res. PubMed Scopus Google Scholar, Muller D. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). The developmental of NR2A and of NR2B in the with the of the of H. J. Neurosci. 1997; 17: PubMed Google Scholar, J. M. J. Neurosci. 1997; 17: PubMed Google Scholar). association of CaMKII with NR2B NR2A may provide a mechanism by which NMDA receptor subunit can developmental L. for M. Bass for V. and M. for NR1 antibodies and cortical D. for expression Soderling for CaMKII expression for of the expression L. S. and D. Lovinger for with and R. J. L. J. D. Lovinger, and B. Wadzinski for