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• W2149462562 abstract "Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689–20692). Here, we show that residues 1290–1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys1292, Leu1298, Arg1299, Arg1300, Gln1301, and Ser1303). Phosphorylation of NR2B at Ser1303 by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII·NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260–1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets. Calcium influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor and activation of calcium/calmodulin-dependent kinase II (CaMKII) are critical events in certain forms of synaptic plasticity. We have previously shown that autophosphorylation of CaMKII induces high-affinity binding to the NR2B subunit of the NMDA receptor (Strack, S., and Colbran, R. J. (1998) J. Biol. Chem. 273, 20689–20692). Here, we show that residues 1290–1309 in the cytosolic tail of NR2B are critical for CaMKII binding and identify by site-directed mutagenesis several key residues (Lys1292, Leu1298, Arg1299, Arg1300, Gln1301, and Ser1303). Phosphorylation of NR2B at Ser1303 by CaMKII inhibits binding and promotes slow dissociation of preformed CaMKII·NR2B complexes. Peptide competition studies imply a role for the CaMKII catalytic domain, but not the substrate-binding pocket, in the association with NR2B. However, analysis of monomeric CaMKII mutants indicates that the holoenzyme structure may also be important for stable association with NR2B. Residues 1260–1316 of NR2B are sufficient to direct the subcellular localization of CaMKII in intact cells and to confer dynamic regulation by calcium influx. Furthermore, mutation of residues in the CaMKII-binding domain in full-length NR2B bidirectionally modulates colocalization with CaMKII after NMDA receptor activation, suggesting a dynamic model for the translocation of CaMKII to postsynaptic targets. calcium/calmodulin-dependent protein kinase II calmodulin N-methyl-d-aspartate long-term potentiation postsynaptic density CaMKII autophosphorylated at Thr286 polymerase chain reaction glutathione S-transferase mitochondrion-targeting protein green fluorescent protein 2-amino-5-phosphonovaleric acid CaMKII1 is a family of ubiquitous, calcium/calmodulin-dependent kinases with broad substrate specificity (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (738) Google Scholar). The α and β isoforms are especially abundant in brain, constituting as much as 2% of total protein in the hippocampus (2Erondu N.E. Kennedy M.B. J. Neurosci. 1985; 5: 3270-3277Crossref PubMed Google Scholar). There is now overwhelming evidence that CaMKII is central to the mechanism of hippocampal, NMDA receptor-dependent long-term potentiation (LTP), a widely studied cellular model of learning and memory. Reduction of CaMKII activity by pharmacological or genetic means impairs LTP (3Silva A.J. Stevens C.F. Tonegawa S. Wang Y. Science. 1992; 257: 201-206Crossref PubMed Scopus (1183) Google Scholar), whereas injecting or overexpressing CaMKII increases synaptic strength, which occludes and is occluded by electrically induced LTP (4Pettit D.L. Perlman S. Malinow R. Science. 1994; 266: 1881-1885Crossref PubMed Scopus (268) Google Scholar, 5Lledo 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). Crucial to its function in LTP and spatial learning (6Cho Y.H. Giese K.P. Tanila H. Silva A.J. Eichenbaum H. Science. 1998; 279: 867-869Crossref PubMed Scopus (160) Google Scholar, 7Giese K.P. Fedorov N.B. Filipkowski R.K. Silva A.J. Science. 1998; 279: 870-873Crossref PubMed Scopus (884) Google Scholar), CaMKII undergoes rapid autophosphorylation following NMDA receptor-mediated calcium influx at a specific residue in its autoregulatory domain (Thr286 in the α isoform of CaMKII). This autophosphorylation renders the kinase calcium-independent and has been proposed as a form of molecular memory (8Lisman J.E. Goldring M.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5320-5324Crossref PubMed Scopus (207) Google Scholar). In support, recent in vitro studies show that CaMKII autophosphorylation permits integration of oscillating calcium signals (9De Koninck P. Schulman H. Science. 1998; 279: 227-230Crossref PubMed Scopus (1084) Google Scholar). We have recently demonstrated a second role for Thr286autophosphorylation, namely in promoting translocation of CaMKII to postsynaptic densities (PSDs) (10Strack 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), cytoskeletal scaffolds for the neurotransmitter receptor, ion channels, and their regulators. The search for proteins that target Thr286-autophosphorylated CaMKII ([P-T286]CaMKII) to the PSD initially identified a 190-kDa binding activity (11McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), corresponding in size to the highly PSD-enriched NR2A and NR2B subunits of the NMDA receptor. Indeed, we recently showed that NR2B is a binding protein for [P-T286]CaMKII and isolated a CaMKII·NMDA receptor complex from PSDs (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Subsequently, other laboratories implicated NR2A and NR2B (13Gardoni F. Caputi A. Cimino M. Pastorino L. Cattabeni F. Di Luca M. J. Neurochem. 1998; 71: 1733-1741Crossref PubMed Scopus (158) Google Scholar, 14Gardoni F. Schrama L.H. van Dalen J.J. Gispen W.H. Cattabeni F. Di Luca M. FEBS Lett. 1999; 456: 394-398Crossref PubMed Scopus (101) Google Scholar) and NR1 and NR2B (15Leonard A.S. Lim I.A. Hemsworth D.E. Horne M.C. Hell J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3239-3244Crossref PubMed Scopus (333) Google Scholar) as CaMKII-binding proteins. In this report, we identify amino acids critical for CaMKII binding in NR2B and investigate the regulation of CaMKII targeting to NR2B in vitro and in cells. The phospho-Thr286-specific CaMKII antibody was a generous gift from Said Goueli (Promega). The recombinant γ1 isoform of the protein phosphatase 1 catalytic subunit was generously provided by Dr. E. Lee (New York Medical College, Valhalla, NY). Sources of other materials are indicated below. Fragments of the rat NR2B cDNA were amplified by polymerase chain reaction (PCR) with primers incorporating 5′-BamHI and 3′-EcoRI sites and subcloned into pGEX-2T (Amersham Pharmacia Biotech) for expression of glutathioneS-transferase (GST) fusion proteins. All NR2B mutants were generated in the context of the NR2B-(1260–1339) sequence, which includes the core CaMKII-binding domain (see Fig. 2) flanked by AlwNI and NdeI sites, allowing for single-step ligation of the mutagenized fragment into the full-length NR2B cDNA (in a cytomegalovirus promoter-driven mammalian expression vector). Point mutants were generated by PCR using Pfu Turbo polymerase (Stratagene) and sense and antisense primers harboring the mutation as well as diagnostic silent restriction sites according to instructions supplied with the QuikChange kit (Stratagene). A cassette-based approach was used for the construction of internal deletion mutants and the B2A mutant. After disruption of the 3′-EcoRI site used for subcloning the NR2B-(1260–1339) fragment, unique silent restriction sites were introduced by PCR (see above) at the following NR2B amino acids: 1286–1288 (EcoRI), 1297–1298 (HindIII), and 1308–1310 (BglII). Mutagenic sense and antisense primers with compatible overhangs were ligated into cassettes generated by cutting the NR2B construct with two of the three restriction enzymes. Since NR2B-(1310–1339) includes at least one in vitrophosphorylation site for CaMKII in addition to Ser1303(data not shown), GST-NR2B mutants analyzed for Ser1303phosphorylation were truncated to NR2B-(1260–1316) by digestion with SfiI and NdeI and fill in/religation. GST-NR2B fusion proteins were bacterially expressed and purified on glutathione-agarose according to standard protocols. The CaMKII-(1–420) truncation mutant was PCR-amplified from the murine CaMKIIα cDNA with a sense primer incorporating a BamHI site and an antisense primer containing an EcoRI site and in-frame stop codon. The Δ380–420 internal deletion mutant was generated by three-step “loop-out” PCR utilizing primers spanning the deletion. Mutagenized cDNAs were ligated into the pVL1393 baculovirus transfer vector (Invitrogen). Sf9 cells were infected with recombinant baculovirus, and protein was expressed and purified by calmodulin-agarose affinity chromatography as described (11McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Point mutants of murine CaMKIIα were generated by PCR as described for NR2B mutants and subcloned into the pME18S mammalian expression vector (chimeric simian virus 40/retrovirus (SRα) promoter-driven; DNAX). The basis of this multidomain fusion protein is the mammalian green fluorescent protein (GFP) expression vector pEGFP-N1 (CLONTECH). The GST coding sequence including the C-terminal multiple cloning site, but excluding the stop codons from pGEX-2T, was PCR-amplified using a sense primer with an XhoI adaptor and an antisense primer with aHindIII adaptor. The GST fragment was ligated into pEGFP-N1, whose EcoRI and BamHI sites had previously been removed by fill in/religation, to create a GST-GFP fusion plasmid. Oligonucleotides encoding a mitochondrion-targeting sequence, the 15 amino-terminal amino acids of hexokinase I (16Gelb B.D. Adams V. Jones S.N. Griffin L.D. MacGregor G.R. McCabe E.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 202-206Crossref PubMed Scopus (82) Google Scholar), and a Myc epitope tag were ligated into N-terminal NheI and BglII sites, resulting in a hexokinase-Myc-GST-GFP fusion cDNA. Wild-type or mutant CaMKII-binding domains in the context of NR2B-(1260–1316) were ligated into BamHI and EcoRI sites between GST and GFP coding sequences. The resulting hexokinase-Myc-GST-NR2B-GFP fusion plasmid resulted in the expression of a 60-kDa protein and mitochondrion-localized GFP fluorescence (see Fig. 7 A), demonstrating that the protein was expressed intact in cells. Transfection of plasmids encoding CaMKII and NR2B mutants led to expression of proteins of the correct size at levels similar to those of the wild type. Sequences of all constructs were verified using an ABI 310 fluorescence sequencer at Center for Molecular Neuroscience, Vanderbilt University Medical Center. Recombinant murine CaMKIIα was autophosphorylated at Thr286 in the presence of calcium/calmodulin and [γ-32P]ATP (20–40,000 cpm/pmol) to a stoichiometry of 0.1–0.4 mol/mol and desalted as described (11McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Bacterial cultures (20 ml) were induced to express GST-NR2B-(1260–1339) wild-type and mutant proteins, and lysates (20 μg/lane) separated by SDS-polyacrylamide gel electrophoresis and blotted onto nitrocellulose were analyzed for binding of 200 nm [32P-T286]CaMKII by overlay (11McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), a concentration close to the KD of binding to wild-type NR2B (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Bound [32P-T286]CaMKII was quantified by a PhosphorImager (Molecular Dynamics, Inc.) and normalized to GST protein amount, detected by GST primary (Sigma) and iodinated secondary (Amersham Pharmacia Biotech) antibodies on duplicate blots. This solution binding assay is a modification of the Ni2+-coated microtiter plate assay previously described (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar) using GST fusion proteins adsorbed to glutathione-coated 96-well plates (Pierce) as the binding surface. Briefly, plates were adsorbed for 2–16 h with GST fusion proteins at room temperature or 4 °C (25 μg/ml, 200 μl/well, ∼50% of binding capacity) in wash buffer (5 mg/ml bovine serum albumin, 200 mm NaCl, 50 mmTris, pH 7.5, 0.1% Tween 20, 5 mm β-mercaptoethanol, and 0.1 mm EDTA). After extensive washes, 200 μl/well [32P-T286]CaMKIIα diluted in wash buffer was allowed to bind to the tethered fusion protein for 2 h at room temperature, followed by 10–12 more washes. Bound [32P-T286]CaMKII was solubilized in 1% SDS, 0.2 n NaOH, and 10 mm EDTA and quantified by liquid scintillation counting. Nonspecific binding to GST alone (5–20% of the total, same as wash buffer without GST) was subtracted from total binding to obtain specific binding. [32P-T286]CaMKIIα and GST-NR2B-(1260–1316) wild-type or S1303A fusion protein were incubated (30 min, 4 °C) at 1–2 μm each in binding buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.25 mg/ml bovine serum albumin, 0.1% Triton X-100, 1 mm dithiothreitol, and 1 mmEDTA). After addition of 0.1 volume of a 50% glutathione-agarose slurry and 15 min of continued incubation, CaMKII·NR2B complexes were recovered by brief centrifugation and washing in binding buffer. The agarose slurry was resuspended in dissociation buffer (0.5 ml of binding buffer with NaCl concentration reduced to 100 mm to permit efficient phosphorylation or dephosphorylation) supplemented with Mg·ATP or protein phosphatase as described in the figure legends and rotated at 25 or 30 °C. Aliquots were removed at the indicated time points and analyzed for soluble and glutathione-agarose-bound CaMKII by immunoblotting and/or autoradiography. [32P-T286]CaMKIIα dephosphorylation was quantified by adjusting aliquots to 20% (w/v) trichloroacetic acid and liquid scintillation counting the supernatant after high-speed microcentrifugation. HEK293 cells were seeded on coverslips (no. 1) in 35-mm dishes, transfected at 40–70% confluency with a total of 4–6 μg/dish DNA (2 μg of CaMKIIα expression plasmid plus either 2 μg of MTP plasmid or 2 μg of each NR1a and NR2B cytomegalovirus promoter plasmids) using TransIT-LT1 transfection reagent (Panvera) according to the manufacturer's instructions, and grown for 48 h in minimal essential medium with 10% fetal bovine serum and 1 mmglutamine. In experiments with MTP, dishes were either immediately fixed for immunofluorescence analysis or first incubated for variable amounts of time with 2 μm calcium ionophore A23187 (Sigma) in growth medium. When NMDA receptor subunits were transfected, the growth medium was supplemented with the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV; 1 mm), and cells were washed and incubated in Mg2+-free Hanks' balanced saline buffered with 20 mm Hepes, pH 7.5, containing 2 mm CaCl2 and either 50 μm APV or NMDA/glycine (100/10 μm) for 15 min prior to fixation. Cultures were fixed in 1:1 acetone/methanol for 10–15 min at 25 °C and processed for immunofluorescence using 1:500 antibody dilutions of goat anti-CaMKII antibody (11McNeill R.B. Colbran R.J. J. Biol. Chem. 1995; 270: 10043-10049Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and either mouse anti-Myc tag or rabbit anti-NR2A/B (Chemicon International, Inc.) antibody as described (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar,17Strack S. Kini S. Ebner F.F. Wadzinski B.E. Colbran R.J. J. Comp. Neurol. 1999; 413: 373-384Crossref PubMed Scopus (76) Google Scholar). Cultures were randomized and coded prior to sampling digital images on a confocal microscope, so the degree of colocalization between signals for CaMKII and MTP or NR2B could be estimated blindly. Cells (15–25/dish) were assigned a colocalization score from 0 to 4: 0, mutual exclusion; 1, coincidental overlap; 2 and 3, increasing degrees of colocalization; and 4, complete overlap of labels (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). CaMKII binding to NR2B is mediated by 50 amino acids (positions 1260–1309) in the NR2B C terminus (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). To investigate whether CaMKII binding to intact PSDs and to NR2B occur by similar mechanisms, co-sedimentation binding experiments (10Strack 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) were performed in which [32P-T286]CaMKIIα was allowed to interact with isolated native PSDs in the presence of increasing concentrations of NR2B-(1260–1309) fused to GST. GST-NR2B-(1260–1309) potently (IC50 = 50 nm) inhibited co-sedimentation of CaMKII with PSDs (Fig. 1). Inhibition was specific to NR2B because a GST fusion protein with the corresponding region of NR2A, which does not bind appreciably to CaMKII (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), had no effect. Although it is possible that NR2B allosterically interferes with the CaMKII/PSD association, we consider it more likely that a single domain in CaMKII interacts with both PSDs and NR2B since the two binding events share a dependence on CaMKII autophosphorylation and have similar affinities (10Strack 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, 12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The 30% residual binding observed at the highest NR2B concentrations could either be nonspecific (there is no meaningful blank for this assay) or reflect CaMKII associating with PSDs via a separate mechanism. To further elucidate the molecular determinants for CaMKII binding in NR2B, we initially narrowed down the CaMKII-binding domain by constructing a series of overlapping GST fusion proteins. Binding of [32P-T286]CaMKIIα by overlay depended on the presence of NR2B residues 1290–1309 (Fig.2 A), which thus constitute the “core” CaMKII-binding domain. However, we cannot formally rule out redundant stabilizing effects of NR2B residues 1260–1289 and 1310–1339, even though deleting either flanking region by itself had little effect on CaMKII binding. To further define the core domain, small internal deletion mutants were analyzed in the context of NR2B-(1260–1339). Whereas deletion of residues C-terminal of Ser1303 (Δ1304–1307 and Δ1306–1309) had a modest to no effect, incremental deletions of N-terminal amino acids from positions 1291 to 1296 reduced CaMKII binding by up to 75% (Fig.2 B). Since CaMKII binding to NR2A-(1255–1298) is 10 times weaker than to NR2B-(1260–1309) and NR2B-(1260–1339) by overlay (Fig. 2 B) (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), nonconserved amino acids in NR2B must play a critical role in the high-affinity interaction with CaMKII. Interestingly, nine amino acids within the core CaMKII-binding domain of NR2B (positions 1290–1309) are identical in NR2A-(1279–1298) (Fig. 2 B), including residues surrounding a high-affinity CaMKII phosphorylation site at Ser1303 in NR2B (18Omkumar 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). Homology-scanning or “reversal” mutations were generated to probe the role of residues unique to NR2B. Replacing Lys1292 or Arg1299 with the corresponding NR2A residues (K1292Q or R1299N) compromised binding by 30–40% and by 50% in the double mutant. Changing four additional residues to their NR2A counterparts (A1290Q, Q1291F, R1295K, and N1296L) in the B2A mutant led to a 65% reduction in CaMKII binding. NR2B residues conserved in NR2A were also subjected to mutational analysis. Mutation of the phosphorylation site Ser1303 to Ala had little effect, whereas introduction of a negative charge (S1303D) or a hydrophobic side chain (S1303L) severely interfered with the CaMKII interaction (75–80% reduction). NR2B Leu1298and Arg1300 align with the predicted CaMKII substrate recognition motif (I/L)XRXX(S/T) (19White 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). Replacing Arg1300 with Glu or Gln diminished binding by >85%, and the L1298A mutation almost completely obliterated the interaction with CaMKII, similar to the R1300Q/S1303D double mutant. As expected, the conservative substitution mutant L1298I displayed CaMKII binding indistinguishable from the wild-type. In agreement with White et al. (19White 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), who noted a preference of CaMKII for substrates containing Gln at position −2, replacing Gln1301 with Ala in NR2B reduced CaMKII binding by 50%. To verify that critical mutations affect CaMKII binding, as opposed to the ability of fusion proteins to renature on the blot prior to CaMKII overlay, solution-phase binding assays were performed with native GST-NR2B fusion proteins. Whereas the S1303A mutant bound [32P-T286]CaMKIIα similarly to the wild type, the R1300Q, R1300E, and B2A mutants were severely binding-impaired (Fig.2 C and data not shown), confirming results from overlay experiments. None of the NR2B mutants displayed specific binding to CaMKII autophosphorylated at Thr305/306 in the absence of Ca2+/calmodulin (data not shown), in agreement with previous results demonstrating that interaction with NR2B requires autophosphorylation at the autonomy site, Thr286 (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). The finding that NR2B amino acids important for the interaction with CaMKII include the substrate recognition motif (Ser1303, Arg1300, and Leu1298) prompted us to examine whether peptide substrates are effective competitors for the CaMKII/NR2B interaction. Syntide-2, derived from a CaMKII phosphorylation site in glycogen synthase, at concentrations of up to 400 μm (20 times the Km for phosphorylation (20Hashimoto Y. Soderling T.R. Arch. Biochem. Biophys. 1987; 252: 418-425Crossref PubMed Scopus (151) Google Scholar)) did not compete for CaMKII binding to NR2B (Fig.3 B), in agreement with previous, more qualitative data (12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). In contrast, autocamtide-2, a peptide substrate modeled after the autoregulatory domain surrounding Thr286 in CaMKIIα, was an effective competitor, with an IC50 of 10 μm similar to the Km for its phosphorylation (21Hanson 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). A possible explanation for this dramatic difference in inhibitory potency is provided by an alignment of the two peptides with the corresponding sequence in NR2B, revealing more extensive homology of NR2B to autocamtide-2 compared with syntide-2 N-terminal of the phosphorylated residue (Fig. 3 A). In particular, autocamtide-2 contains residues corresponding to the mutation-sensitive NR2B residues Gln1301 and Arg1299 but syntide-2 does not. Since our mutational analysis implicated an important role for NR2B Ser1303, the effect of Ser1303 phosphorylation on CaMKII binding was investigated. In agreement with Omkumar et al. (18Omkumar 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), GST-NR2B-(1260–1309), but not GST-NR2B-(1260–1299), could be phosphorylated by CaMKII to ∼1 mol/mol stoichiometry (Fig.4). Furthermore, mutating Ser1303 to Ala in the context of GST-NR2B-(1260–1316) completely abrogated phosphorylation (data not shown). Stoichiometric phosphorylation of GST-NR2B-(1260–1309) reduced CaMKII interaction by overlay by 59 ± 6% (n = 3) (Fig. 4). To investigate whether NR2B Ser1303 phosphorylation not only inhibited initial association of CaMKII, but also could dissociate CaMKII previously bound to NR2B, release of CaMKII from CaMKII·GST-NR2B complexes was monitored with or without ATP. In the absence of ATP, CaMKII remained stably associated with GST-NR2B for >1 h under these conditions (Fig. 5). Addition of ATP led to stoichiometric phosphorylation of NR2B Ser1303 by 5 min, revealed by the appearance of a lower mobility band (Figs. 4 and 5 A, compareasterisks). Phosphorylation of NR2B was accompanied by dissociation of CaMKII, albeit incomplete and with a protracted time course (10% released after 75 min). No ATP-dependent release of CaMKII from complexes with the NR2B S1303A mutant was detected (Fig. 5 B), demonstrating that this dissociation is a consequence of NR2B Ser1303 phosphorylation, as opposed to continued, calcium/calmodulin-independent autophosphorylation of CaMKII. Autophosphorylation of CaMKII at Thr286 is required for high-affinity binding to PSDs and to NR2B (10Strack 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, 12Strack S. Colbran R.J. J. Biol. Chem. 1998; 273: 20689-20692Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). We therefore investigated the reversibility of the CaMKII/NR2B interaction by dephosphorylation of Thr286. Incubation of kinase·NR2B complexes with a 2 μg/ml concentration of the catalytic subunit of protein phosphatase 1 resulted in >70% dephosphorylation of [32P-T286]CaMKIIα in 2 h, measured as release of trichloroacetic acid-soluble radioactivity (Fig. 5 C), as well as a decrease in immunoreactivity with a phospho-Thr286-specific CaMKII antibody (data not shown). Paralleling the time course of dephosphorylation, ∼20% of the CaMKII·NR2B complexes dissociated during this time period. Higher protein phosphatase 1 concentrations (10 μg/ml) led to release of up 40% CaMKII under otherwise identical conditions (data not shown). Both dephosphorylation and dissociation were blocked by inhibiting protein phosphatase 1 with microcystin, demonstrating that Thr286dephosphorylation promotes release of CaMKII from NR2B. In an effort to delineate domains in CaMKII important for the interaction with NR2B, we constructed deletion mutants of CaMKIIα. Two mutations in the C-terminal domain required for formation of a holoenzyme consisting of 10–12 subunits (22Kolb S.J. Hudmon A. Ginsberg T.R. Waxham M.N. J. Biol. Chem. 1998; 273: 31555-31564Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 23Shen K. Meyer T. J. Neurochem. 1998; 70: 96-104Crossref PubMed Scopus (39) Google Scholar) were expressed in insect cells. The 1–420 mutant lacks the C-terminal 58 amino acids, whereas the Δ380–420 mutant lacks 41 residues in the middle of the oligomerization domain (Fig. 6 A). As expected, both deletions disrupt holoenzyme formation since the mutants migrated as monomers by gel filtration chromatography and sucrose gradient centrifugation (data not shown). Both mutants underwent calcium/calmodulin-dependent autophosphorylation at Thr286 and attained levels of autonomous activity similar to those of the wild type (30–50% of calcium/calmodulin-dependent activity). However, whereas autophosphorylation of wild-type CaMKII is rapid (seconds), occurring between adjacent subunits of the same holoenzyme (24Mukherji S. Soderling T.R. J. Biol. Chem. 1994; 269: 13744-13747Abstract Full Text PDF PubMed Google Scholar), maximal autophosphorylation of the monomeric mutants required high enzyme concentration and prolonged incubation (1–3 min) at 30 °C, consistent with an intermolecular reaction. Both mutants displayed normal catalytic activity toward syntide-2 peptide substrate (wild-type: Km = 12.3 μm,K cat = 324 min−1; 1–420: Km = 13.2 μm,K cat = 236 min−1; and Δ380–420, Km = 14.8 μm,K cat = 348 min−1;n = 2–3) and phosphorylated GST-NR2B-(1260–1316) with comparable efficiency (Fig. 6 B). Moreover, a detailed comparison of GST-NR2B-(1260–1316) phosphorylation kinetics failed to reveal significant differences between the wild type and the Δ" @default.
• W2149462562 created "2016-06-24" @default.
• W2149462562 creator A5045487847 @default.
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