From the Department of Molecular Physiology and Biophysics and Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232-0615
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ABSTRACT |
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Activation and Thr286 autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx via N-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.
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INTRODUCTION |
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CaMKII is a multifunctional, calcium-activated kinase (1, 2),
whose and
isoforms are particularly abundant in brain cytosol
and in postsynaptic densities
(PSDs),1 submembranous
scaffolds for receptors, ion channels, and signal transducers (3, 4).
Postsynaptic calcium influx triggers autophosphorylation of CaMKII at a
threonine residue in the autoinhibitory domain (Thr286 in
CaMKII
) (5), which renders the kinase persistently active and causes
a translocation of soluble CaMKII to the PSD (6). Multiple lines of
evidence indicate Thr286 autophosphorylation of
postsynaptic CaMKII is necessary for NMDA receptor-dependent LTP (7-11), a cellular model of
learning and memory. PSD-associated CaMKII phosphorylates ionotropic
glutamate receptors (6, 12-14), providing a mechanism for increased
synaptic strength during LTP (15).
Mechanisms by which CaMKII is targeted to its postsynaptic substrates
are poorly understood. Previous gel overlay analyses revealed a
candidate PSD-associated CaMKII-anchoring protein, p190, that binds
selectively to the Thr286-autophosphorylated kinase
([P-T286]CaMKII) (16). 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
profiles2 (17). Here, we
demonstrate a direct and specific interaction between
[P-T286]CaMKII
and NR2B and show that NR2B targets CaMKII in
intact cells.
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EXPERIMENTAL PROCEDURES |
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Immunoprecipitations-- PSD isolation and immunoprecipitation of sodium dodecyl sulfate (SDS)-solubilized PSD proteins were carried out as described (6) using 2 µg/ml NR2A/B antibodies (Chemicon) and protein phosphatase 1 antibodies (18). 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 mM phenylmethylsulfonyl 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 (16) or preimmune IgG (19). The cross-linker was cleaved and proteins eluted from the beads by boiling in reducing SDS sample buffer.
CaMKII Gel Overlays--
Purified recombinant CaMKII was
autophosphorylated with [
-32P]ATP (8,000-40,000
cpm/pmol) in the presence of calcium/calmodulin or EGTA at
Thr286 or Thr305/306, respectively, and
desalted (16). Stoichiometries ranged between 0.17 and 0.39 (Thr286) and 0.24 and 0.47 (Thr305/306).
Protein blots to be analyzed for CaMKII binding were blocked and
incubated with 100-200 nM [32P]CaMKII in 5%
milk for 3 h, washed extensively, and autoradiographed.
Immunofluorescence-- 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 (16), rabbit anti-NR1 (20), 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.
Generation and Analysis of NMDA Receptor Fusion Proteins-- 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.
Microtiter Plate Solution
Binding--
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 -mercaptoethanol). After extensive
washes, [32P-T286]CaMKII
diluted in blocking buffer
(200 µl) was allowed to bind to the tethered fusion protein for
2 h, followed by 10-12 more washes. Bound CaMKII was solubilized
in 1% SDS, 0.2 N NaOH, 50 mM EDTA, and
quantified by liquid scintillation counting. Nonspecific binding to
control bacterial extracts was subtracted from total binding to obtain
specific binding. No specific binding was observed using
[32P-T306]CaMKII
.
GST Pull-down Analysis--
GST fusion proteins were incubated
(1 h, 4 °C) with either purified CaMKII (Fig. 2D, 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 Cell Colocalization--
HEK293 cells were seeded on
coverslips in 35-mm dishes, transfected with a total of 3 µg/dish DNA
(1 µg of SR promotor-CaMKII
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 (21). Robust expression of NMDA currents was verified by
patch-clamp recording of parallel cultures.3 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 (16), 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, CaMKII
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 (22). 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 to
bottom).
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RESULTS AND DISCUSSION |
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To determine whether NR2 subunits contribute to the previously
characterized "p190" overlay binding activity (16), we analyzed immunoprecipitated NR2A/B by gel overlay with
[32P-T286]CaMKII (Fig.
1A). 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.
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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. 1B). Higher magnification revealed a mostly postsynaptic localization of CaMKII in dendritic spines (Fig. 1C). Moreover, a complex of CaMKII with NMDA receptor subunits can be immunoprecipitated from PSDs using CaMKII antibodies, but not preimmune IgG (Fig. 1D). 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. 1D).
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.
2A, diagram). Bacterial
lysates expressing the cytoplasmic domains of the predominant forebrain
NMDA receptor subunits, NR1, NR2A, and NR2B, as His6 tag
fusion proteins were screened for [32P]CaMKII binding
by overlay (Fig. 2A). The NR2B cytoplasmic domain bound
about six times more [32P-T286]CaMKII
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 CaMKII
phosphorylated in the absence of calcium/calmodulin at
Thr305/306 ([P-T306]CaMKII
) 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. 2B).
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A solution interaction assay was employed to examine binding of CaMKII
to NR2B that had not undergone denaturation/renaturation for gel
overlay analysis. [32P-T286]CaMKII 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. 2C). Scatchard analysis
indicated that binding involves a simple bimolecular interaction with a
Kd of 138 ± 60 nM
(n = 3) (Fig. 2C, inset). This
Kd is ~100 times lower than the average concentration of CaMKII
in forebrain (16, 23), suggesting that the
interaction can readily occur in neurons.
The CaMKII-binding domain in NR2B contains a high-affinity
phosphorylation site, Ser1303, which is phosphorylated by
CaMKII in vitro and is also phosphorylated in
vivo (13). However, three lines of evidence indicate that the
binding of CaMKII to NR2B-(1260-1309) is not dependent on a substrate
interaction. First, the model peptide substrate syntide-2 only weakly
inhibits CaMKII binding (~30%) at concentrations of ~100-fold the
Km for phosphorylation (not shown). Second, even
though NR2A residues 1255-1298 are 36% identical to
NR2B-(1260-1309), and sequences surrounding the phosphorylation site
are almost perfectly conserved (NR2B, LRRQHSYD; NR2A,
INRQHSYD) (13), CaMKII binding to NR2A-(1255-1298) is
~10-fold weaker under our overlay conditions (10.7 ± 1.8%,
n = 3, Fig. 2B), suggesting that
nonconserved residues in NR2B-(1260-1309) are important for
high-affinity CaMKII binding. Third, "pull-down" experiments, in
which GST-NR2B fusion protein was purified with
glutathione-agarose, showed that calcium/calmodulin alone did not
promote CaMKII interaction with NR2B, but that stoichiometric interaction was instead strictly dependent on CaMKII
autophosphorylation at Thr286 (Fig. 2D). On the
other hand, calcium/calmodulin binding is sufficient for full CaMKII
activation, and Thr286 autophosphorylation stabilizes the
active conformation of the kinase in the absence of calcium/calmodulin
(1, 2). Thus, CaMKII residues outside the substrate binding site are
involved in the interaction with NR2B.
Further evidence for specific association of CaMKII with NR2B was
obtained by performing GST-NR2B pull-downs from brain cytosolic extracts. and
isoforms of CaMKII were isolated following
incubation with GST-NR2B-(1260-1309), but not GST alone.
Affinity-purified CaMKII
displayed an upward electrophoretic
mobility shift characteristic of autophosphorylation (Fig.
2E). CaM kinase IV, a related kinase with a similar
phosphorylation site preference (24), as well as other kinases and
phosphatases tested, were not detected in the precipitated material,
strongly indicating that NR2B-(1260-1309) binds selectively to
CaMKII.
The NR2B subunit of the NMDA receptor was shown to target
Thr286 autophosphorylated CaMKII in HEK293 cells. CaMKII
was coexpressed with various NMDA receptor subunit combinations, and
their distributions were compared by immunofluorescence (Fig.
3). Whereas NR1 alone does not form
functional NMDA receptors in HEK293 cells, activation of both NR1/NR2A
and NR1/NR2B receptors leads to massive calcium influx (25).
Coexpression of CaMKII
and NR1 alone resulted in low colocalization
scores that were unaffected by acute treatment with the receptor
coagonists NMDA/glycine (Fig. 3A). Perhaps reflecting the
low but detectable CaMKII binding activity of NR2A (Fig. 2, A and B), additional expression of the NR2A
subunit led to a small increase in CaMKII
and NR1/NR2A
colocalization, which was not significantly increased by NMDA/glycine
treatment (Fig. 3B). In cells expressing NR2B with CaMKII
and NR1, we observed a similarly modest increase in colocalization in
the absence of agonist treatment compared with CaMKII
and NR1 alone
(Fig. 3, C and D). In contrast to NR2A-containing
NMDA receptors, activation of NR1/NR2B receptors with NMDA/glycine
caused a highly significant redistribution of CaMKII
into
receptor-positive patches (Fig. 3, C and D),
strongly suggesting that receptor activation induced the formation of a CaMKII·NR2B complex. Replacing extracellular calcium with barium, which is receptor-permeable but binds only poorly to calmodulin, completely blocked the effect of NMDA (Fig. 3D). Thus,
opening of NMDA receptors is not sufficient for complex formation, but calcium influx is essential, presumably to stimulate
calcium/calmodulin-dependent autophosphorylation of CaMKII.
Consistent with this interpretation, an autophosphorylation-incompetent
form of the kinase, T286A-CaMKII
(26, 27), expressed at similar
levels of wild-type CaMKII
failed to show activity-induced
colocalization with NR1/NR2B containing NMDA receptors (Fig.
3D). Thus, NR2B mediates targeting of CaMKII to NMDA
receptors in a calcium- and Thr286
autophosphorylation-dependent manner in intact cells.
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Our data support a model in which dendritic calcium influx induced by synaptic activity triggers CaMKII autophosphorylation at Thr286 and subsequent binding to residues 1260-1309 in the NR2B subunit of the NMDA receptor. What are the functional consequences of this interaction? Autonomous CaMKII in the PSD is inactivated by PSD-associated serine/threonine phosphatases (18, 28, 29). Once dephosphorylated at Thr286, CaMKII positioned near the mouth of the NMDA receptor calcium channel is likely to undergo rapid re-autophosphorylation even during periods of low level NMDA receptor activation. Thus, an interaction of CaMKII with NMDA receptors is predicted to boosts autonomous kinase activity, leading to enhanced phosphorylation of nearby downstream effectors of synaptic plasticity (15). Furthermore, recruitment of CaMKII into the PSD structure (6), possibly via association with NR2B, may play a role in the rapid ultrastructural changes of synapses that undergo LTP (30, 31). The developmental appearance of NR2A and down-regulation of NR2B in the mammalian visual system correlate with the end of the "critical period" of synapse maturation (32, 33). Preferential association of CaMKII with NR2B over NR2A may therefore provide a mechanism by which NMDA receptor subunit composition can impact developmental plasticity.
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ACKNOWLEDGEMENTS |
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We thank L. MacMillan for scoring cells; M. Bass for invaluable technical assistance; V. Rema, F. Ebner, and M. Maguire (Vanderbilt) for NR1 antibodies and cortical cultures; D. Lynch (Penn State) for NR expression plasmids; T. Soderling (Vollum Institute) for CaMKII expression plasmids; DNAX, Inc. (Palo Alto, CA) for use of the pME18S expression vector; L. Popp, S. Sessoms, and D. Lovinger (Vanderbilt) for help with HEK cell transfections; and R. Blakely, F. Ebner, J. Exton, L. Limbird, J. Lisman, D. Lovinger, and B. Wadzinski for helpful suggestions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM47973 (to R. J. C.) and American Heart Association Grant-in-aid 96010040 (to R. J. C., an Established Investigator of the American Heart Association). Confocal microscopy was performed using the Vanderbilt University Medical Center Cell Imaging Resource (supported by National Institutes of Health Grants CA68485 and DK20593).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. Molecular
Physiology & Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232-0615. Tel.: 615-936-1630; Fax: 615-322-7236; E-mail: roger.colbran{at}mcmail.vanderbilt.edu.
The abbreviations used are:
PSD, postsynaptic
density; CaMKII, calcium/calmodulin-dependent protein
kinase II; CaMKII/
,
/
isoform of CaMKII; [P-T286]CaMKII
, CaMKII
autophosphorylated at threonine 286; [P-T306]CaMKII
, CaMKII
autophosphorylated at threonine 305 and/or threonine 306; NMDA, N-methyl-D-aspartateAPV, 2-amino-5-phosphonovaleric acidGST, glutathione
S-transferaseLTP, long term potentiation.
2 S. Strack, R. B. McNeill, and R. J. Colbran, unpublished data.
3 R. L. Popp and D. M. Lovinger, personal communication.
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REFERENCES |
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