Regulation of CaMKII Docking to NMDA Receptors by Calcium ... · the T site and the inhibitory...

Regulation of CaMKII Docking to NMDA Receptors by Calcium/Calmodulin and α-Actinin

A. Soren Leonard1,2, K.-Ulrich Bayer3, Michelle A. Merrill1,2, Indra A. Lim1,2, Madeline

A. Shea4, Howard Schulman3, and Johannes W. Hell1,2*

From

1The Department of Pharmacology, University of Wisconsin,

Madison, Wisconsin 53706-1532,

2The Department of Pharmacology, University of Iowa,

Iowa City, Iowa 52242-1109

3The Department of Neurobiology, Stanford University,

Stanford, CA 94305-5125

And

4The Department of Biochemistry, University of Iowa,

Iowa City, Iowa 52242-1109

Running Title: CaMKII Docking on NMDA Receptors

*Correspondence to: Johannes W. Hell Phone: (319) 384 47322-512 BSB FAX: (319) 335 8930Department of Pharmacology E-mail: [email protected] of Iowa 51 Newton Road Iowa City, IA 52242-1109

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Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

Ca2+ influx through the NMDA-type glutamate receptor leads to activation and

postsynaptic accumulation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and

ultimately to long-term potentiation, which is thought to be the physiological correlate of

learning and memory. The NMDA receptor also serves as a CaMKII docking site in

dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We

demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of

T286. This autophosphorylation reduces the off rate to a level (t1/2 ~23 min) that is similar

to that observed for dissociation of the T286D mutant CaMKII (t1/2 ~30 min) from spines

after its glutamate-induced accumulation (Shen, K., Teruel, M.N., Connor, J.H., Shenolikar,

S., and Meyer, T. (2000) Nature Neurosci. 3, 881-886). CaMKII as well as the previously

identified NR1 binding partners calmodulin and α-actinin bind to the short C-terminal

portion of the C0 region of NR1. Like Ca2+/calmodulin, autophosphorylated CaMKII

competes with α-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a

key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with

calmodulin to modulate the stimulatory role of α-actinins interaction with the NMDA

receptor.

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Long-term potentiation (LTP) in the hippocampus is a long-lasting increase in

synaptic transmission, which likely constitutes a physiological correlate of learning and

memory (1,2). LTP usually requires Ca2+ influx through the NMDA receptor and

subsequent activation of CaMKII by Ca2+/calmodulin (2-4). CaMKII is concentrated at

postsynaptic densities (5,6). This localization is strategically ideal for activation by Ca2+

influx through NMDA receptors and for phosphorylation of neighboring AMPA receptors,

which contributes to LTP (3,7,8). Simultaneous binding of two or more Ca2+/calmodulin

molecules to neighboring subunits within the same dodecameric CaMKII holoenzyme leads

to inter-subunit phosphorylation at T286 (4,9). This mechanism enables CaMKII to decode

Ca2+-spike frequency (10). T286 phosphorylation keeps the enzyme in a catalytically active

conformation independent of the presence of Ca2+/calmodulin in the holoenzyme and

thereby of cytosolic Ca2+ levels (4,9). Coincident with its activation, CaMKII associates

with the NMDA receptor subunits NR1 (11), NR2A (12), and NR2B (11,13) and

accumulates at postsynaptic spines (14,15).

NMDA receptors in cortex and hippocampus mainly consist of one or two NR1 and

two or three NR2A and 2B subunits (16-19). Specific interaction of CaMKII with NR2A

(12,20) is about an order of magnitude weaker than with NR1 or NR2B (Leonard and Hell,

unpublished observations; see also (13)) and not easily detectable under several conditions

(11,21). CaMKII robustly binds to two independent sites on the 644 amino acid-long

intracellular C-terminus of NR2B. One site (NR2B/C) is formed by residues 1290-1309;

the other site (NR2B/P) is localized within residues 839-1120 but has evaded further

identification up to now (11,13,21,22). Ca2+/calmodulin is sufficient to induce high affinity

binding of CaMKII to NR2B/C (22). Ca2+/calmodulin binding and T286 phosphorylation

remove the autoinhibitory segment of CaMKII to various degrees from its catalytic substrate

binding site (S site) and from the endogenous attachment site for T286, the T site (23). The

NR2B1289-1310 sequence binds to the T site in the presence of Ca2+/calmodulin. The

resulting interaction outlasts dissociation of Ca2+/calmodulin, thereby displacing T286 from

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the T site and the inhibitory segment from the catalytic S site for an extended period of time

(22). The result is a lasting increase in the activity of NR2B/C-associated CaMKII

independent of cytosolic Ca2+ or T286 phosphorylation.

The initial Ca2+-induced translocation of CaMKII to postsynaptic sites requires

Ca2+/calmodulin binding to CaMKII but not T286 phosphorylation (14,15). These studies did

not identify the postsynaptic attachment sites for CaMKII but our earlier evidence indicates

that the initial Ca2+-induced postsynaptic accumulation is at least in part due to

Ca2+/calmodulin-dependent association of CaMKII with the NR2B/C site (22). However, the time

course of persistent CaMKII translocation to the postsynaptic site does depend on T286

phosphorylation; prolonging autophosphorylation by inhibiting phosphatase 1 and mimicking

T286 phosphorylation by introducing a T286D mutation resulted in a much reduced

dissociation rate of CaMKII from postsynaptic sites (14,15). Autophosphorylation of

CaMKII increases its affinity for NR2B/C (13,21,22). This autophosphorylation-induced

increase in NR2B/C binding is comparable to that caused by Ca2+/calmodulin (22) (see Fig.

1C). This autophosphorylation-dependent interaction could, therefore, contribute to a more

enduring association of CaMKII with the postsynaptic site. However, serine 1303 in

NR2B/C is the major CaMKII phosphorylation site on NR2B (24) and phosphorylation of

this serine inhibits CaMKII binding to this site (21), thereby antagonizing an otherwise

potentially lasting interaction between NR2B/C and CaMKII. We now show that

autophosphorylation of T286 decreases the off-rate for CaMKII release from NR1 by a

factor of 10 and brings it into a time range similar to that observed for dissociation of T286-

phosphorylated CaMKII from synaptic sites in neuronal cultures. Together with previous

work our results argue for a complex regulatory mechanism of the CaMKII-NMDA receptor

interaction.

Previous work indicates that calmodulin itself and also α-actinin-2 can directly bind

to the C-terminus of NR1 (25-27). Furthermore, Ca2+/calmodulin appears to reduce

NMDA receptor activity by binding to the C0 region (25,27), thereby displacing α-actinin

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(26,28). The physiological significance of these findings is highlighted by earlier results that

demonstrated that an intact actin cytoskeleton and interaction with α-actinin support NMDA

receptor activity (28,29). Using peptide libraries to precisely map the NR1 interaction sites

for α-actinin, calmodulin and CaMKII we demonstrate that all three proteins bind to the

membrane-distal portion (residues 845-863) of the 30 residue-long C0 region in the NR1

C-terminus. Not only calmodulin but also CaMKII competes with α-actinin for NR1

binding. These results indicate that NR1 constitutes a critical interaction site for lasting

relocation of CaMKII to the postsynaptic site, which is regulated in a complex manner by

postsynaptic calcium concentrations and the actin cytoskeleton. Our results suggest that

CaMKII, in conjunction with calmodulin, displaces α-actinin from NR1, providing a novel

mechanism for modulating the channel activity of NR1.

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METHODS

Materials. [γ-32P]ATP (111Tbq/mmol) was obtained from New England

Nuclear/DuPont (Wilmington, DE). Glutathione sepharose and enhanced

chemiluminescence detection kit were purchased from Amersham-Pharmacia (Piscataway,

NJ). Bovine brain calmodulin, protein A-Sepharose and all organic solvents were purchased

from Sigma (St. Louis, MO). Antibodies against CaMKIIα (11) and GST (30) were as

described. Mouse anti-calmodulin antibodies were purchased from Zymed, anti-His6

antibodies from Qiagen and secondary HRP-conjugated antibodies and Protein A from

BioRad (Richmond, CA). F-moc L-amino acids were obtained from AnaSpec (San Jose,

CA), blocks of solid support polypropylene pins were purchased from Chiron Mimotopes

(Clayton Victoria, Australia). Streptavidin coated plates and developing reagents were

purchased from Pierce (Rockford, IL).

Production of fusion proteins. Recombinant Glutathione S-transferase (GST) fusion

proteins of the various NR1 C termini were produced by digesting pBS SK(-) vectors

containing NR1-1B (Accession Number: U0-8263), NR1-2B (U0-8264), NR1-3B (U0-

8266), and NR1-4B (U0-8268) (kindly provided by S. F. Heinemann; Salk Institute, San

Diego, CA) with SphI/HinDIII. The resulting C-terminal fragments were treated with T4

DNA polymerase to obtain blunt ends, gel-purified, ligated into pGEX-5X2 (Amersham-

Pharmacia), and transformed into E. coli (Novablue; Novagen). All constructs were

confirmed by DNA sequencing and expressed along with nonchimeric GST. The C-terminal

fusion proteins GST-NR2A (residues 1222-1464 of full length NR2A were amplified by

PCR and inserted into pGEX-6P-3; Accession Number: D13211 (31); kindly provided by

Drs. F. Zheng and P. J. Conn, Emory University, Atlanta, GA), GST-NR2B (839-1482),

GST-NR2B/P (839-1120), GST-NR2B/C (1120-1482) (11,22), and poly-His6-tagged α-

actinin-2 (clone A2.10 subcloned into the EcoR1site of pRSETB and encoding residues

344-894 of human α-actinin-2 (26)) in E. coli (BL21-CodonPlus; Stratagene) as described

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(11).

Expression and purification of recombinant CaMKIIα. Spodoptera frugiperda (Sf9)

cells adapted for serum-free growth (Gibco-Invitrogen) were cultured in Ex-cell 420

Serum-free media (JRH Biosciences). Cultured Sf9 cells (200 ml of 7 × 106 cells/ml) were

infected with recombinant baculovirus expressing mouse CaMKIIα (1 × 108 plaque-forming

units/ml; a gift from Dr. T. R. Soderling, Vollum Institute, Portland, OR (32)). After 1 hour

at room temperature, the cells were diluted to 1l with media and grown for 72 h at 27°C in an

orbital shaker. Cells were harvested by centrifugation (750 × g for 15 min) and decanted

pellets were stored at -80°C until purification (32). Cells were resuspended in lysis buffer

(10 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 5 mm DTT, 0.2 mM PMSF) and

sonicated on ice for 3 × 10 seconds at 10 second intervals with a large tip sonicator (power

setting: 7). The lysate was centrifuged at 118,000 × g for 30 minutes and (NH4)2SO4 was

added to 60% to the supernatant while stirring on ice. The precipitated proteins were

collected by centrifugation at 118,000 × g for 20 minutes and the pellet was resuspended in

buffer A (50 mM HEPES, pH 7.4, 50 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 1 mM DTT).

The suspension was added to a 5 ml calmodulin-Sepharose (Sigma) column, equilibrated

with buffer A, and incubated for 1 h under tilting. The column was washed with 10 column-

volumes of buffer A followed by a high salt wash (buffer A plus 950 mM NaCl). The

CaMKII was eluted by chelating Ca2+ via incubating the column for 1 h in one column

volume 50 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1 mM DTT.

Additional fractions were eluted with several more column volumes of the same buffer. The

protein concentration was determined by OD280 reading and with the bicinchoninic acid

assay (Pierce). The various fractions were analyzed by SDS-PAGE, followed by Coomassie

staining to control for purity and potential degradation and by immunoblotting to identify

CaMKII. CaMKII was dialyzed against 100 mM HEPES, pH 7.5, 5 mM DTT, 1 mM EDTA

and stored at -20°C in the dialysis buffer with 50% glycerol added to it.

CaMKII and α-actinin binding and competition assays with NMDA receptor fusion

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proteins. Recombinant CaMKIIα (1-2 µg) was preincubated for 5 min at 0ÚC (to promote

T286 autophosphorylation in selected samples) in basic phosphorylation buffer (50 mM

Hepes-NaOH, pH 7.4, 10 mM MgCl2) containing, if indicated, 500 µM CaCl2, 1 µM

calmodulin, and 500 µM ATP. In some samples EGTA was added, which completely

remove calcium/calmodulin from the kinase (22). To induce T305/306 phosphorylation in

selected samples (Fig. 1 only), the incubation was continued for another 15 min at 30ÚC.

The kinase mixture was then added to 20 µl glutathione Sepharose resin samples loaded with

about 1.0 µg GST fusion proteins or GST alone (as estimated by SDS-PAGE and subsequent

staining with Coomassie brilliant blue in addition to a quantitative bicinchoninic acid -

protein assay (Pierce) (11)). After incubation for 2-3 h at 0-4ÚC in and washing of the

samples with TBS (10 mM Tris-Cl, pH 7.4, 150 mM NaCl) containing 0.1% Triton X-100,

proteins were separated by SDS-PAGE, transferred onto nitrocellulose for immunoblotting

and quantification by ImageQuant (Molecular Dynamics) analysis. Competition by α-

actinin was tested by the addition of up to 10 µM α-actinin to the resin samples during the

CaMKII binding step. Binding of α-actinin to GST-NR1 was measured in the same way in

the absence and presence of Ca2+ and increasing calmodulin concentrations.

To determine dissociation rates, unphopshorylated (no ATP present during

preincubation and binding) and autophosphorylated recombinant CaMKIIα was incubated

with immobilized GST-NR1 as described in the previous paragraph for 1.5 h at 4ÚC (initial

experiments revealed that binding equilibrium was reached within less than 1 h; 24 h

equilibriation time did not result in more CaMKII binding). Matrix bound NR1-CaMKII

complexes were obtained by centrifugation, followed by three quick washing steps, which

took less than 3 min. Resin samples were then resuspended in either 1 or 10 ml TBS

containing 0.1% Triton X-100 corresponding to at least 50X and 500X dilution factors,

respectively. Results with the two different dilution factors were comparable indicating that

dissociation rates were independent of final buffer volume following at least a 50X dilution.

No molecular weight shifts were visible after 138 min post dilution, indicating that no

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hyperphosphorylation had occurred. Matrix bound CaMKII was obtained by centrifugation

at the indicated time points, followed by two quick washing steps taking less than 60 sec,

extraction with SDS sample buffer, immunoblotting, and quantification as described. The

koff value was obtained from single-exponential best-fit curves of experimental data. All data

fits were performed with PRISM software (Ver. 3.02, GraphPad Software, Inc. San Diego,

CA).

Peptide synthesis. F-moc (9-fluorenylmethyloxycarbonyl) peptide synthesis was

performed manually in a solid-phase approach as described by the manufacturer (Chiron

Mimotopes). In brief, peptides were synthesized on derivatized polypropylene pins arranged

in a 96-well plate format. Each condensation reaction began with the removal of the F-moc

protecting group with base (20% piperidine/DMF) and adding a new F-moc amino acid

activated ester along with the appropriate activator (diisopropylcarbodiimide (DIC)/1-

hydroxybenzotriazole (HOBt)). After synthesis, the final F-moc protecting group was

removed and the terminal amino group was capped by acetylation (10% (CH3CO)2O in

DMF for 10-15 min and 1 drop of pyridine). Removal of side chain protecting groups was

accomplished with trifluoroacetic acid (50% in DMF, 1-2% ethanedithiol or anisole) for 20

min. A wash with isopropyl alcohol (containing 1% ethanedithiol or anisole) followed TFA

treatment, and successive washes with 10% triethylamine in DMF (TEA), methanol, TEA,

methanol, and DMF completed the neutralization sequence.

Peptide binding assays. Pins with the peptides were utilized for binding assays in 96-

well plates by an ELISA procedure with recombinant CaMKIIα, poly-His6-α-actinin-2, or

brain calmodulin, followed by antibodies against CaMKIIα, the poly-His6 tag, or

calmodulin, respectively, and ultimately by HRP-coupled goat anti-mouse IgG antibodies

for all three proteins. All binding assays and antibody incubations were done in 20 mM

phosphate buffer, pH 7.4, containing 150 mM NaCl, 2 % bovine serum albumin, and 0.1 %

Tween 20. To detect the HRP-labeled antibodies, 150 µl of the 1-step slow TMB-ELISA

solution (Pierce) was added to each well. OD650 readings were taken every 5 min for up to

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30 min to follow the reaction. Finally 100 µl 1M H2SO4 was added to stop the reaction for

OD450 readings, which often showed reduced background compared to the readings at

OD650 taken without H2SO4. Because the peptides were synthesized on a non-cleavable peptide

(NCP) block, repeated binding assays could be performed after regenerating the block by

ultrasound treatment in a bath sonicator in 62.5 mM Tris-Cl, pH 6.8, 1% SDS, 20 mM

dithiothreitol, for 30 min at 55oC (power setting 6). This procedure removed bound target

and antibody from the peptide without breaking the bond between the peptide and the pin.

Subsequent control ELISA tests with primary and secondary antibodies only confirmed that

no antigen remained on the pins. After sonication, pins were washed five times with water

and five times with TBS and stored in TBS containing 0.02% azide. All NCP blocks

contained positive control peptides, including AC3-I (KKALHRQETVID) (11) and

NR2B1295-1306 (RNKLRRQHSYDT) (21,22).

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RESULTS

Regulation of CaMKII binding to NR1 and NR2B by calmodulin and

autophosphorylation. The NMDA receptor appears to be a major docking site for CaMKII at

postsynaptic sites (11-13,20-22). The initial accumulation of CaMKII at postsynaptic sites

requires Ca2+/calmodulin binding to CaMKII but persistent CaMKII clustering depends on

T286 phosphorylation (14,15). To evaluate the binding partners and molecular mechanisms

of CaMKII recruitment and retention at postsynaptic sites we determined the effect of

Ca2+/calmodulin and of autophosphorylation of CaMKII on its binding to the three high affinity

interaction sites NR1, NR2B/P, and NR2B/C (Fig.1).

GST fusion proteins of NR1, NR2B/P, and NR2B/C were immobilized on glutathione

Sepharose and incubated with recombinant CaMKII in the absence and presence of Ca2+ and

calmodulin and ATP before washing and detection of CaMKII by immunoblotting. CaMKII

bound to all three fusion proteins even in the absence of Ca2+/calmodulin and ATP. The

resulting interactions were rather weak explaining why they have not been observed in other

studies (13,21). These Ca2+/calmodulin- and autophosphorylation-independent interactions

were nevertheless specific because negative controls with plain GST performed in parallel

did not yield signals for CaMKII (data not shown; but see (11)). Ca2+ by itself had no effect

but together with calmodulin it increased association of CaMKII with NR2B/C (Fig. 1C).

Ca2+/calmodulin was not sufficient to stimulate CaMKII binding to NR2B/P or NR1 (Fig. 1A,

B). Preincubation of CaMKII with Ca2+/calmodulin and ATP under conditions that result in

T286 phosphorylation was necessary to induce high affinity binding of CaMKII to NR1 and

NR2B/P (Fig. 1A, B). These results indicate high affinity interactions of NR2B/C with the

Ca2+/calmodulin-bound form of CaMKII and of NR1 and NR2B/P with T286 phosphorylated

CaMKII.

Focusing on NR1 we tested whether T286 phosphorylation is not only necessary but

also sufficient for high affinity interaction of CaMKII with NR1. GST-NR1 and, as negative

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control, GST alone were immobilized on anti-GST antibody-coated 96-well plates. The

samples were incubated with wt, T286A-, and T286D-mutated CaMKII before washing and

immunoblotting. This procedure is less sensitive in our hands than the pull-down

experiments with fusion proteins on glutathione Sepharose and only permits detection of the

high affinity interaction between CaMKII and NR1. When indicated, CaMKII had been

pretreated with Ca2+/calmodulin and ATP under conditions that result in T286

phosphorylation in the wild type form. As before, wt CaMKII bound with high affinity to

GST-NR1 only after T286 phosphorylation (Fig. 2, lanes 1,2). CaMKII did not show an

interaction with NR1 when ATP was omitted during the procedure to prevent T286

phosphorylation, whether Ca2+/calmodulin was present or not (lanes 3,4). Addition of

EGTA after pretreatment completely removes Ca2+/calmodulin from CaMKII (22) but did

not result in a reduction of T286-phosphorylated CaMKII binding to NR1 (compare lanes 1

and 2). These results show that high affinity binding of CaMKII to NR1 requires

autophosphorylation of CaMKII but not the continued presence of Ca2+/calmodulin in the

CaMKII complex. The T286A mutant did not bind to GST-NR1 under any conditions in

this assay (lanes 5-8) indicating that autophosphorylation on T286 is critical for this

interaction. In contrast, CaMKII with a T286D mutation, which partially mimics T286

autophosphorylation, interacted with GST-NR1 without any preceding autophosphorylation

step. This finding demonstrates that T286 phosphorylation is sufficient for the high affinity

interaction of CaMKII with NR1. Analogous experiments had been performed earlier for

CaMKII binding to NR2B/P with comparable results (22) indicating that T286

phosphorylation is necessary and sufficient for high affinity interaction of CaMKII with this

site as well. No binding was observed under any of these conditions to GST alone, which

served as negative control (data not shown, but see ref. (22)).

Regulation of CaMKII dissociation from NR1 by autophosphorylation. We

hypothesize that the increase in affinity of the CaMKII interaction upon T286

phosphorylation could at least in part be responsible for the very slow redistribution rate of

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the T286D mutant of CaMKII after postsynaptic accumulation. CaMKII was incubated with

GST-NR1 adsorbed onto glutathione Sepharose in the presence and absence of

Ca2+/calmodulin and ATP. Dissociation of the resulting CaMKII-NR1 complexes were measured

after 50 fold dilution by determining how much CaMKII was left on the beads at various

time points. Phosphorylation increased the half life of the NR1-CaMKII complex by a factor

of 10 from 2.4 to 23 min (Fig. 3). There was no significant difference in these off rates when

dissociation was initiated by a 500 instead of a 50 fold dilution indicating that the observed

off rates were true off rates and not contaminated by substantial rebinding or other factors

(Leonard and Hell, data not shown). These results link T286 phosphorylation and the

resulting decrease in the dissociation rate of an NR1-CaMKII complex to a strongly reduced

relocation rate of synaptically clustered CaMKII. The relocation rate of wt CaMKII is much

faster than that of the T286D mutant and depends at least in part on phosphatase activity

(14,15). Our results suggest that phosphorylation of T286 reduces dissociation rates for

CaMKII from NR1 and thereby from the postsynaptic site. Accordingly, dephosphorylation

of T286 should increase NR1 dissociation and, thereby, increase relocation rate.

Autophosphorylation of T305 or T306 is induced when the Ca2+ concentration

returns to a basal level and calmodulin dissociates from T286-phosphorylated CaMKII (9).

T305 and T306 phosphorylation results in inhibition of calmodulin binding (9). A

T305/306A double mutant of CaMKII has a very slow redistribution rate after its

postsynaptic clustering induced by glutamate receptor-mediated Ca2+ influx (15). The latter

finding suggests that T305/306 phosphorylation contribute to acceleration in relocation of

postsynaptically clustered CaMKII. T305/306 phosphorylation should decrease affinity for

the NMDA receptor if the NMDA receptor is to a substantial degree responsible for CaMKII

anchoring at postsynaptic sites. We induced T305/306 phosphorylation by adding EGTA

after the initial 5 min preincubation of CaMKII with Ca2+/calmodulin and ATP to the

phosphorylation reaction mixture and by continuing the preincubation for 15 min at increased

temperature (30ÚC). The effectiveness of this established protocol in causing

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hyperphosphorylation is demonstrated by the expected decrease in electrophoretic mobility

of CaMKII (Fig. 1, right lanes). The hyperphosphorylation clearly reduced the affinity of

CaMKII for NR1, NR2B/P, and NR2B/C, although the affinity was in each case still more

than two fold higher than the weak basal binding (Fig. 1A-C). Accordingly, T305/306

phosphorylation may contribute to a reduction in the affinity of CaMKII for the NMDA

receptor and may foster release of CaMKII from the postsynaptic site (15). We note that

addition of EGTA alone to remove calcium/calmodulin from CaMKII without raising the

temperature did, as expected, not result in hyperphosphorylation as the electrophoretic

mobility did not shift without the treatment at elevated temperature (see Fig. 2, lane 2).

Identification of the binding site for CaMKII, calmodulin and α-actinin on NR1. The

precise CaMKII attachment site has been defined for NR2B/C (21), which may play a crucial

role in the initial Ca2+/calmodulin-induced CaMKII binding (22), but not for NR1, which

may be critical for sustained CaMKII anchoring upon T286 phosphorylation (see above).

The intracellular C-terminus of NR1 exists in four splice variants. All possess the C0 region

(residues 834-863) (33). The 37 residue-long C1 region is encoded by an exon that is

spliced out in some isoforms. Two alternative C terminal regions, C2 (38 residues) and C2’

(22 residues), are created by the use of two distinct splice acceptor sites, the one for C2’

being downstream of the stop codon at the end of C2. We expressed all four carboxyl-

termini (NR11-4) as GST fusion proteins. After autophosphorylation, recombinant CaMKII

interacted with the four different NR1-GST fusion proteins to comparable degrees (Fig. 4

upper panel, lanes 1-4). We detected only high affinity CaMKII interaction in these sets of

experiments and no CaMKII binding was observed with GST alone and a GST fusion protein

carrying the partial C-terminus of NR2A (residues 1222-1464); GST-NR2B/C (residues

1120-1482) served as positive control. Accordingly, the common C0 cassette of NR1

contains a CaMKII-association site. The observation that CaMKII binds all four splice

variants equally well suggests that the C1 cassette is not necessary and may not directly

influence this interaction. This observation also suggests that the C2 and C2’ cassettes may

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either not contribute to the CaMKII – NR1 interaction or may do so to the same extent so that

they can substitute for each other.

The C0 region interacts with calmodulin in the presence of Ca2+ (25) and α-actinin-

2 (26). Ca2+/calmodulin competes with α-actinin-2 for binding to C0 (26), thereby

reducing the activity of the NMDA receptor (27,28). Ion flux through this receptor channel

depends on an intact actin cytoskeleton for optimal activity (29). To define the precise

interaction sites for CaMKII, calmodulin, and α-actinin within NR1-C0, we synthesized a

peptide library encompassing the C-terminus of NR1. The sequence of the first peptide

corresponded to a short linker sequence followed by the membrane-proximal 12 residues of

C0; subsequent peptides were progressively shifted by one position towards the C-terminus

and covered all sequences from all four NR1 splice variants (including all possible C0-C1-

C2 junctions). Our coupling method and protocol left peptides covalently attached to the

solid phase synthesis matrix during the peptide deprotection step. This strategy permitted

ELISA-styled interaction assays with autophosphorylated CaMKII, Ca2+/calmodulin, and

α-actinin-2 in independent experiments, which identified the hitherto unknown exact binding

sites for these three proteins (Fig. 5). Only peptides in the C0 region displayed significant

binding to CaMKII and are shown in the histogram. The Gaussian-like distributions of these

histograms define the core sequences that are necessary and neighboring residues that help to

support the observed interactions. For example, specific CaMKII binding starts with peptide

12 and ends with peptide 17 as designated in Fig. 5. The stretch of residues that is common

to these two peptides (and to peptides 13-16; LAFAAVNV) is, therefore, necessary for

CaMKII binding and constitutes the minimal or core sequence. However, peptides 12 and 17

show only a relatively low level of CaMKII binding compared to peptides 13-16 indicating

that residues next to the core sequence also contribute to binding (i.e., QMQ N-terminal and

WRK C-terminal to the core sequence). The optimal binding peptide covered NR1(847-

858) for CaMKII and for Ca2+/calmodulin and α-actinin-2 NR1(850-862). The overlap in

the binding segment for Ca2+/calmodulin and α-actinin-2 explains their competition for C0

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association.

Competition between CaMKII and α-actinin for association with NR1. The overlap

of the association segments for CaMKII, Ca2+/calmodulin, and α-actinin-2 suggest that

these three proteins might compete for C0 binding. As demonstrated earlier (26), we found

that Ca2+/calmodulin can displace α-actinin-2 (Fig. 6). The important point of this

experiment is that at least 0.5-1 µM calmodulin is required for substantial displacement of

α-actinin-2; 100 nM calmodulin had little, if any, effect. This observation allowed us to

preincubate CaMKII with Ca2+ and ATP in the presence of 100 nM calmodulin to stimulate

T286 phosphorylation without subsequent removal of calmodulin before the binding

competition assays with α-actinin-2. Increasing amounts of α-actinin-2 prevented more

and more CaMKII binding (Fig. 7). These results demonstrate a competitive relationship

between these two proteins for C0 association.

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DISCUSSION

Previous studies indicate that the NR1, NR2A and NR2B subunits of the NMDA

receptor complex serve as postsynaptic anchor sites for CaMKII and that these interactions

are directly regulated by the autophosphorylation state of the kinase (11-13,21,34). Binding

to the NR2B/C site has received most attention up to now and was further evaluated in this

study with respect to dependence on calmodulin and autophosphorylation on T286 as well as

on T305/306. Similar to earlier publications (13,21,22) we find that CaMKII binds with high

affinity to NR2B/C when autophosphorylated on T286 independent of the presence of Ca2+

and calmodulin (Fig. 1). A comparable level of binding was observed without

autophosphorylation as long as Ca2+ and calmodulin were present; binding in the absence of

Ca2+/calmodulin and T286 phosphorylation was weak. These results indicate that

displacement of the autoinhibitory loop of CaMKII by either Ca2+/calmodulin binding or

T286 phosphorylation induces high affinity binding to NR2B/C (see also (22)). Once

CaMKII is bound to NR2B/C, Ca2+/calmodulin can be removed from the non-

phosphorylated kinase without displacing NR2B/C. NR2B/C association keeps CaMKII at a

high activity level even after calmodulin is removed, whether or not the kinase underwent

T286 phosphorylation. This mechanism constitutes a second way to generate a lasting,

autonomous activity of CaMKII beyond the initial binding of calmodulin as occurring during

a rise in intracellular Ca2+, which is usually transient (22).

Redistribution of CaMKII after its Ca2+-induced accumulation at postsynaptic sites

is quite fast (t1/2 is ~1min; (14,15)) raising the question, which mechanisms contribute to a

reversal of the CaMKII-NMDA receptor interaction. S1303 is part of the NR2B/C site,

which comprises residues 1290-1309 (13,21). S1303 is the major CaMKII phosphorylation

site in NR2B (24). In the presence of ATP, NR2B in complex with CaMKII becomes

phosphorylated, resulting in the release of a portion of CaMKII, although this release is by

far not complete (21). An additional phosphorylation event that occurs upon removal of

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Ca2+ and calmodulin from CaMKII is auto-hyperphosphorylation of CaMKII on several other

residues, including T305 and T306. Phosphorylation of T305 and T306 blocks subsequent

Ca2+/calmodulin binding (9). We observed a substantial reduction in the association of

T286-phosphorylated CaMKII with NR2B/C when the kinase was hyperphosphorylated

(Fig. 1C). If CaMKII was preincubated under conditions that promoted mainly T305/306 but

not T286 phosphorylation, high affinity binding was reduced by more than 95% (13). The

difference between the more modest effect of hyperphosphorylation on NR2B/C binding in

our hands as compared to Strack et al. (13) may be due to the higher level of T286

phosphorylation in our experiments (we allowed near stoichiometric T286 phosphorylation at

low temperatures before inducing T305/306 phosphorylation by adding EGTA and raising

the temperature for 15 min to 30ÚC). T305/306 phosphorylation prevents Ca2+/calmodulin

from associating with CaMKII and Ca2+/calmodulin by itself can stimulate CaMKII

interaction with NR2B/C. Therefore, it is possible that the reduction of CaMKII binding to

NR2B/C upon T305/306 phosphorylation is in part due to the irreversible displacement of

calmodulin from the kinase. In any case, collectively the study by Strack et al. (13) and our

results argue that T305/306 phosphorylation results in reduced CaMKII affinity for NR2B/C.

This effect, together with S1303 phosphorylation of NR2B, promotes dissociation of CaMKII

from this site after increased Ca2+ concentrations will return to basal levels inside the

neuron. Phosphorylation of S1303 in the NR2B subunit and of T305 and T306 in the

CaMKII autoinhibitory loop may, therefore, promote redistribution of CaMKII after its

Ca2+-induced accumulation at postsynaptic sites.

We previously identified two additional high affinity binding sites for CaMKII,

NR2B/P, which is upstream of NR2B/C, and the C-terminus of NR1 (11). In contrast to

NR2B/C-binding, Ca2+/calmodulin is neither sufficient nor necessary to stimulate

association of CaMKII with NR1 or NR2B/P (Fig. 1,2). Rather, autophosphorylation of

T286 or its mutation to aspartate induces high affinity interaction with NR1 and NR2B/P,

whether Ca2+/calmodulin is present or not (Fig. 1,2; see also (22)). We demonstrated that

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T286 autophosphorylation reduced the dissociation rate of CaMKII from the NR1 site from

t1/2 of 2.4 min to 23 min (Fig. 3). Compared to wt, the T286D mutant CaMKII exhibits a

prolonged accumulation at the postsynaptic site following Ca2+ influx in intact neurons (t1/2

is ~1 min for wt and ~30 min for T286D; (14,15)). Furthermore, the T286A mutant of

CaMKII, which can only bind to NR2B/C but not to NR2B/P or NR1, has a very fast

redistribution rate after postsynaptic accumulation (t1/2 is <10 sec; (15)). Our results indicate

that T286 phosphorylation-induced high affinity binding to NR1 and NR2B/P may act by

prolonging the postsynaptic residence time of CaMKII.

The data described in the preceding paragraph collectively also suggest that

dephosphorylation of T286 in wt CaMKII is important to reverse CaMKII clustering on a

time scale in the range of 1 min. In fact, the phosphatase inhibitor calyculin A strongly

decreases the redistribution rate (t1/2 for wt CaMKII is ~17 min in the presence of calyculin

A (15)). A second mechanism that contributes to this redistribution is phosphorylation of

T305 and T306 of CaMKII; when phosphorylation of these two residues is prevented by

mutating both residues to alanine, t1/2 is increased to ~9 min (15). We found that high

affinity binding to all three sites (i.e., NR1, NR2B/P, and NR2B/C) was decreased, though

clearly not abolished, when CaMKII was pretreated under conditions that promoted

T305/306 autophosphorylation (preincubation after EGTA addition at 30ÚC) subsequent to

T286 autophosphorylation (Fig. 1). Distinct phosphatases may be responsible for

dephosphorylation of these sites or the sites may be differentially dephosphorylated when

CaMKII is associated with the NMDA receptor complex. In fact, soluble CaMKII is more

effectively dephosphorylated by the phosphatase PP2A than by PP1 but the reverse is true

when CaMKII is bound to a postsynaptic density fraction (34,35). A preferential

dephosphorylation of T286 over T305/306 on CaMKII bound to the NMDA receptor would

reduce binding affinity and promote the release of CaMKII from postsynaptic sites.

Using GST-fusion proteins and different peptide libraries, which spanned the C-

terminus of NR1 and also the region of NR2B that contained the NR2B/P site, we were able

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to precisely define the CaMKII binding site in NR1 (Figs. 4, 5), but not NR2B/P (Merrill and

Hell, data not shown). CaMKII binds to the C-terminal portion of the C0 region of NR1

(residues 845-862). The same region also interacts with α-actinin-2 and Ca2+/calmodulin

(Fig. 5). These data are in agreement with earlier findings that calmodulin and α-actinin-2

bind to the C0 region (25-27) and also identify the exact binding site for those two proteins

within this region. The overlap of the α-actinin and Ca2+/calmodulin binding site offers an

explanation for the mechanism of their competitive interaction. These proteins may require

at least overlapping if not identical sets of binding target residues for interaction and

simultaneous binding may, therefore, not be possible.

When the Ca2+ concentration is low, i.e. under resting conditions, α-actinin interacts

with the NMDA receptor and keeps its activity level high; upon Ca2+ influx,

Ca2+/calmodulin can displace α-actinin and decrease the receptor activity (25-28) (Fig. 6). If the

Ca2+/calmodulin-mediated reduction in NMDA receptor activity is mainly due to the

displacement of α-actinin, the competition between CaMKII and α-actinin for NR1

interaction (Fig. 7) would predict that CaMKII binding down-regulates the receptor activity

by a similar mechanism, i.e. displacement of α-actinin. However, CaMKII binding per se

could change the NMDA receptor conformation in a way that is at variance with a

conformational change induced by Ca2+/calmodulin. Accordingly, it is possible that

CaMKII and Ca2+/calmodulin affect the NMDA receptor activity differently. In any case,

the observation that CaMKII associates with the same segment of NR1 as Ca2+/calmodulin

and α-actinin will stimulate a careful analysis of CaMKII’s effect on NMDA receptor

activity. We have started to perform electrophysiological experiments along these lines.

CaMKII binding to the NMDA receptor may be critical for several forms of synaptic

plasticity, especially LTP. Ca2+ influx through the NMDA receptor and subsequent

activation of CaMKII are critical steps in the induction of LTP (2-4). Docking of CaMKII

on NMDA receptors puts CaMKII at a location that is strategically ideal for activation by

Ca2+ influx through NMDA receptors and subsequent phosphorylation of neighboring AMPA

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receptors, thereby contributing to LTP (3,7,8). We started to evaluate the relevance of

CaMKII association with the NMDA receptor for the phosphorylation of AMPA receptors

and for the induction of LTP. In addition to upregulation of AMPA receptor activity by

CaMKII-mediated phosphorylation, an increased number of AMPA receptors accumulate

during LTP at the postsynaptic site (36). A similar accumulation was induced by

overexpression of a constitutively active form of CaMKII (37). It is currently unclear

whether this CaMKII-induced AMPA receptor accumulation depends on anchoring of

CaMKII by the NMDA receptor as recently suggested by Lisman et al. (3,38). Defining the

exact binding sites for CaMKII on the NMDA receptor provides the molecular basis for

future studies, which will test the hypothesis that CaMKII anchoring at the NMDA receptor

helps to control the number of AMPA receptors by specifically disturbing the CaMKII-

NMDA receptor interactions.

Ca2+ influx through NMDA receptors plays a crucial role not only in LTP but also in

stroke-related neuronal damage. Uncontrolled release of glutamate under anoxic conditions

leads to overstimulation of the NMDA receptor and ultimately neuronal damage and death

(39,40). In rodents, administration of NMDA receptor antagonists prevent neuronal loss to a

large extent even when given more than one hour after the anoxic conditions occurred.

Unfortunately, these drugs did not alleviate neuronal damage in clinical trials, perhaps

because humans, unlike rodents, may not tolerate a strong block of NMDA receptor activity

(39). CaMKII has also been implicated in stroke-induced neuronal damage (41).

Disrupting the interaction between CaMKII and the NMDA receptor may specifically inhibit

CaMKII signaling downstream of the NMDA receptor-mediated Ca2+ influx as occurring

during hypoxia and thereby prevent neuronal dysfunction following stroke without affecting

other vital signaling pathways involving CaMKII or the NMDA receptor.

In summary, our results reveal several layers of complexity for CaMKII binding to

the NMDA receptor. It involves multiple interactions, which are likely to form

simultaneously between the NMDA receptor and CaMKII complexes, thereby stabilizing

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their association. We also identified multiple regulatory mechanisms of this interaction.

Defining the precise interaction sites and how these interactions are regulated provides the

basis for future studies that will delineate the role of CaMKII binding to the NMDA receptors

under physiological conditions such as synaptic plasticity and pathological conditions such as

stroke. Because excessive Ca2+ influx through the NMDA receptor as well as subsequent

activation of CaMKII have been implicated in stroke-related neuronal damage, unraveling

the molecular basis for these interactions will provide the necessary basis for developing

drugs that can disrupt these interactions and thereby inhibit the NMDA receptor - CaMKII

signaling pathway with high specificity. Such drugs may be able to effectively alleviate

neuronal damage following stroke.

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ACKNOWLEDGEMENTS

We thank Dr. Mary C. Horne, University of Iowa (Iowa City, IA) for many useful

discussions and comments and much help and advice on molecular biological techniques, Dr.

Stefan Strack, University of Iowa (Iowa City, IA), for critically reading the manuscript, Dr.

T. R. Soderling, Vollum Institute (Portland, OR), for the baculovirus CaMKIIα expression

construct, Dr. M. Sievert, University of Wisconsin (Madison, WI), for help with the CaMKII

expression in Sf9 cells, Dr. B. B. Kay, Argonne National Laboratory (Argonne, IL), for

consulting with F-moc peptide synthesis, Drs. F. Zheng and P. J. Conn, Emory University

(Atlanta, GA) for the expression construct for GST-NR2A(1222-1464), and Dr. Morgan

Sheng, MIT (Cambridge, MA), for the α-actinin expression construct. The work described

in this publication was supported by the National Institute of Health research grant R01-

NS35563 and the American Heart Association Established Investigator Award 0040151N (to

J.W.H.) and the National Institute of Health research grant GM30179 (to H.S.). A.S.L. was

in part supported by an American Heart Association pre-doctoral fellowship 9910185Z.

I.A.L. was in part supported by training grant AG00213. The content of this publication is

solely the responsibility of the authors and do not necessarily represent the official views of

the National Institute of Health and its divisions.

ABBREVIATIONS

AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CaMKII,

Ca2+/calmodulin-dependent protein kinase II; NMDA, N-methyl-D-aspartate; O.D., optical

density; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecylsulphate; wt, wild

type.

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FIGURE LEGENDS

Fig. 1. Ca2+/calmodulin and T286-phosphorylation differentially regulates

CaMKIIα association with its NMDA receptor binding sites NR1, NR2B/P and NR2B/C. GST fusion

proteins of NR1 (residues 725-938, which include the C0, C1, and C2 region), NR2B/P

(839-1120), and NR2B/C (1120-1482) were immobilized on glutathione Sepharose and

incubated with recombinant wt CaMKIIα, which had been pretreated without or with Ca2+,

calmodulin, and ATP as indicated. Some samples (lane 5) were further preincubated for 15

min at 30ÚC with EGTA added after initial prephosphorylation of T286 in the presence of

Ca2+/calmodulin and ATP; the latter procedure results in additional phosphorylation of T305

and T306. After washing, the amount of bound CaMKII was determined by immunoblotting

(lower panels) and subsequent densitometry (n=3; error bars: SD). Probing of the blots with

anti-GST antibodies indicated that comparable amounts of the GST fusion proteins were

present in all samples (not shown; see (11)).

Fig. 2. T286-phosphorylation is necessary and sufficient for high affinity CaMKIIα association

with NR1. Recombinant wt CaMKIIα or its T286A or T286D mutants were pretreated

without or with Ca2+/calmodulin (Ca2+/CaM ante) and ATP (ATP ante; to induce T286

phosphorylation in wt), when indicated at the bottom. Binding to the fusion protein and

subsequent washing was performed either in the presence or absence of Ca2+/calmodulin

(Ca2+/CaM wash) or EGTA (EGTA), when indicated. However, the temperature was never

raised above 4ÚC to avoid hyperphosphorylation. The GST fusion protein containing the C-

terminal C0, C1, and C2 regions of NR1 (residues 725-938) was immobilized on 96-well

plates coated with antibodies against GST and incubated with pretreated CaMKII. After

washing, the amount of bound CaMKII was detected by immunoblotting (upper panels).

Probing of the blots with anti-GST antibodies indicated that comparable amounts of the GST

fusion protein were present in all samples (lower panels).

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Fig 3. T286 phosphorylation decreases koff of CaMKIIα for its NR1 interaction.

About 10 µg GST-NR1C0-C1-C2 (residues 725-938) were adsorbed on glutathione

Sepharose (20µl) and incubated with recombinant CaMKIIα (1 µM final concentration) in

100 µl phosphorylation buffer with 0.5 mM CaCl2 , 1µM calmodulin, and with (squares) or

without (triangles) 0.5 mM ATP for 1.5 h at 4°C until partition equilibrium had been

established (initial experiments revealed that equilibrium was reached within 1 h). After 3

fast washing steps taking less than 3 min to remove unbound CaMKII, samples were diluted

50 fold (in some experiments 500 fold; not shown) with buffers and cofactors that were

identical to those used during the phosphorylation and binding step. After dilution, matrix

bound CaMKII was obtained by two quick washing steps taking less than 1 min at the

indicated time points and quantified by immunoblotting and densitometry. No

electrophoretic mobility shifts of CaMKII were visible in any sample, thus indicating that no

hyperphosphorylation had occurred under any of these conditions. The koff values were

obtained from single-exponential best-fit curves of experimental data. All data fits were

performed with PRISM software (Ver. 3.02, GraphPad Software, Inc. San Diego, CA)

(n=3+SD).

Fig. 4. CaMKIIα binding to the C0 region of NR1. A. GST alone and GST fusion

proteins of the C-termini of NR2A (1222-1464), NR2B (1120-1482) and the four different

splice isoforms of the C-terminal region of NR1 (NR11: C0-C1-C2; NR12: C0-C2; NR13:

C0-C1-C2; NR14: C0-C2), were immobilized on glutathione Sepharose. Recombinant wt

CaMKIIα was pretreated with Ca2+/calmodulin and ATP to induce T286 phosphorylation

and incubated with the various resin samples. After washing, the amount of bound CaMKII

was determined by immunoblotting with antibodies against CaMKIIα (upper panel).

Stripping the blots with SDS and dithiothreitol (42,43) and reprobing with antibodies against

GST indicated that equivalent amounts of each full length fusion protein were present (lower

panel).

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Fig. 5. Identification of the CaMKIIα, calmodulin, and α-actinin binding site on

NR1. A library of 12 residue-long peptides was synthesized on polystyrene lanterns via a

noncleavable seryl-glycyl linker at their carboxy-termini. The first peptide corresponded to

the membrane-proximal 12 residues of the C0 region (residues 834-845 of NR1).

Subsequent peptide sequences were shifted by one position towards the C-terminus, and

covered the C0, C1, C2, and C2’ regions and all possible connections between these regions

(not all peptides are shown). After deprotection of the peptides, which remained covalently

linked to the pins, successive binding assays with recombinant CaMKIIα (preincubated with

Ca2+, 100 nM calmodulin and ATP to induce T286 phosphorylation for high affinity

binding), Ca2+/calmodulin, and α-actinin were performed in a modified ELISA fashion with

respective primary and secondary antibodies. After each assay, bound proteins were

removed by sonication in 1% SDS; mock assays with antibodies alone confirmed that the

corresponding proteins had completely been removed by the sonication. Shown are

consecutive peptides covering the C0 region, which specifically binds CaMKII, calmodulin,

and α-actinin. Peptides further downstream only exhibited background levels of binding

similar to the first nine peptides of the panel (not shown).

Fig. 6. Competition of calmodulin and α-Actinin for NR1 binding. NR14-GST

(encoding the C0-C2 regions) or GST alone were incubated with purified His6-tagged α-

actinin-2 fusion protein (residues 344-894) in the absence or presence of Ca2+ and various

amounts of calmodulin as given at the bottom. After washing of the resins, α-actinin-2 was

visualized by immunoblotting with the anti-His6 antibody. α-actinin binding to NR1 is

Ca2+ independent (upper pane, lanes 1-3) but inhibited by calmodulin at and above

concentrations of 0.5 µM (upper panel, lanes 4-7). Stripping the blots with SDS and

dithiothreitol (42,43) and probing with the antibody against GST demonstrates that similar

amounts of NR1-GST and higher amounts of GST had been loaded (lower panel).

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Fig. 7. CaMKII and α-Actinin compete for binding to NR1. NR14-GST

(containing the C0-C2 regions) was immobilized on glutathione Sepharose and incubated

without and with increasing amounts of His6-tagged α-actinin-2 fusion protein (residues

344-894) for 1 h to reach binding equilibrium. 5 µg CaMKII was prephosphorylated in the

presence of 0.5 mM CaCl2, 0.1µM CaM, and 0.5mM ATP and then added to the incubation

mixture, which was then allowed to reach a second equilibrium for another 1 hr. After

washing of the complex we detected a strong specific signal for CaMKII by immunoblotting

with anti-CaMKII in the absence of α-actinin (lower panel, lane 1). CaMKII binding to

NR1-GST was progressively more antagonized by increasing amounts of α-actinin with

half-maximal inhibition occurring between 0.5 and 1.0 µM α-actinin (lanes 3 and 4).

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Howard Schulman and Johannes W. HellA. Soren Leonard, K.-Ulrich Bayer, Michelle A. Merrill, Indra A. Lim, Madeline A. Shea,

-actininαRegulation of CaMKII docking to NMDA receptors by calcium/calmodulin and

published online October 13, 2002J. Biol. Chem.

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