Casein Kinase 2 Regulates the NR2 Subunit
Composition of Synaptic NMDA Receptors
Antonio Sanz-Clemente,1Jose A. Matta,2John T.R. Isaac,2and Katherine W. Roche1,*
1Receptor Biology Section
2Synaptic Plasticity Section
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA
N-methyl-D-aspartate (NMDA) receptors (NMDARs)
and neurological disease. NMDAR subunit composi-
tion defines their biophysical properties and down-
stream signaling. Casein kinase 2 (CK2) phosphory-
lates the NR2B subunit within its PDZ-binding
domain; however, the consequences for NMDAR
localization and function are unclear. Here we show
NR2B and NR2A in response to activity. We find that
CK2 phosphorylates NR2B, but not NR2A, to drive
resulting in an increase in synaptic NR2A expression.
During development there is an activity-dependent
switch from NR2B to NR2A at cortical synapses. We
observe an increase in CK2 expression and NR2B
phosphorylation over this same critical period and
show that the acute activity-dependent switch in
synapses requires CK2 activity. Thus, CK2 plays
a central role in determining the NR2 subunit content
of synaptic NMDARs.
N-methyl-D-aspartate receptors (NMDARs) are a subtype of
ionotropic glutamate receptors, which are widely expressed
throughout the nervous system. NMDARs play important roles
in development, learning and memory, as well as in some neuro-
psychiatric disorders (Lau and Zukin, 2007). The NMDAR
subunits (NR1, NR2A-D, and NR3A-B) assemble as tetramers
containing two NR1 subunits and two NR2 (or NR3) subunits to
form functional NMDARs (Cull-Candy and Leszkiewicz, 2004;
Furukawa et al., 2005). In particular, NMDARs in cerebral cortex
are primarily composed of two NR1 subunits, and two NR2A or
NR2B subunits (Al-Hallaq et al., 2007; Kohr, 2006; Tovar and
Protein composition and receptor density at synapses are
strongly regulated by several mechanisms including phosphory-
lation and interactions with PDZ domain-containing proteins
(Chen and Roche, 2007; Kimand Sheng, 2004;Kim and Huganir,
1999). For example, phosphorylation of the PDZ-binding domain
oftheinwardly rectifying K+channels Kir2.3andKir5.1bycAMP-
dependent protein kinase disrupts their association with PSD-95
and modulates their function (Cohen et al., 1996; Tanemoto
et al., 2002). In addition, AMPA receptor internalization is regu-
lated by protein kinase C, which directly phosphorylates the
PDZ binding domain of GluR2 (S880) and prevents its associa-
tion with GRIP, but not with PICK1 (Chung et al., 2003; Perez
et al., 2001; Seidenman et al., 2003). The NR2B subunit of
NMDARs is also phosphorylated within its PDZ binding domain,
on serine 1480 (S1480). Casein kinase 2 (CK2) phosphorylates
S1480, disrupts the interaction of NR2B with PSD-95 and
SAP102, and leads to a decrease in NR2B surface expression.
Interestingly, phosphorylation of NR2B on S1480 is regulated
by synaptic activity and CaMKII (Chung et al., 2004), but the
impact of CK2 phosphorylation on synaptic NMDARs has not
CK2 is a highly conserved serine/threonine kinase, organized
as a tetramer composed of two catalytic subunits (a and a0)
and two regulatory b subunits (Litchfield, 2003; Pinna and
Meggio, 1997). Although it is constitutively active, CK2 activity
can be modulated by a diverse array of stimuli, and a number
of mechanisms contribute to CK2 regulation in vivo, including
interaction with proteins and small molecules, phosphorylation
and regulated expression and assembly (Blanquet, 2000; Faust
and Montenarh, 2000; Litchfield, 2003; Litchfield et al., 1994).
Although ubiquitous, the activity of CK2 is 3-8 fold higher in
brain than in nonneuronal tissues, and, in particular, cortex and
hippocampus express high levels of CK2 (Blanquet, 2000; Gir-
ault et al., 1990; Martin et al., 1990). Little is known about the
a role in learning and memory (Blanquet, 2000). For example,
long-term potentiation (LTP) transiently increases CK2 activity
in hippocampus (Charriaut-Marlangue et al., 1991).
The subunit composition of synaptic NMDARs in forebrain
changes during development and this switch is activity-depen-
dent (Barria and Malinow, 2002; Bellone and Nicoll, 2007; Car-
mignoto and Vicini, 1992; Quinlan et al., 1999). During early
development, NR2B-containing NMDARs are predominant,
hood. However, the precise molecular mechanisms mediating
the switch remain obscure. We now show that CK2 differentially
phosphorylates NR2A and NR2B. In addition, CK2 modulates
the synaptic expression of NR2 subunits, increasing the level
984 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
of synaptic NR2A and decreasing synaptic NR2B via increased
endocytosis. Finally, CK2 regulates NR2B phosphorylation
induced by activity in young animals, showing that CK2 is a key
regulator of NR2 subunit composition of synaptic NMDARs.
which disrupts its association with PDZ domain-containing
proteins (Chung et al., 2004). The extreme C termini of NR2A
and NR2B share a high degree of homology and, in particular,
their six last amino acids (aa), including the PDZ binding domain
(-ESDV), are identical (Figure 1A). Therefore, we tested if NR2A
was phosphorylated by CK2 on S1462, the analogous serine
within its PDZ binding domain. We carried out in vitro phosphor-
ylation assays, using GST fusion proteins containing the last
175 aa of NR2A and NR2B, both wild-type (WT) and mutants
to disrupt the phosphorylation (GST-NR2A S1462A and GST-
NR2B S1480A). GST-proteins were incubated with [g-32P]ATP
and CK2 ±25 mM 4,5,6,7-tetrabromobenzotriazole (TBB),
a selective CK2 inhibitor (Sarno et al., 2001, 2005), as described
inExperimental Procedures.NR2B wasrobustly phosphorylated
by CK2, and the S1480A mutation completely abolished the
Figure 1. CK2 Phosphorylates NR2B Much
More Efficiently than NR2A
(A) Alignment of the extreme C termini of rodent
NR2A and NR2B. The PDZ binding domain
(-ESDV) is shown in bold and the tyrosine-based
endocytic motif in NR2B is underlined in gray.
(B) In vitro phosphorylation of the last 175 aa of
NR2A (1289–1464) or NR2B (1307–1482) by CK2.
GST-NR2A, GST-NR2A S1462A, GST-NR2B, or
GST-NR2B S1480A was incubated with CK2 and
[g-32P]ATP for 20 min at 30?C. When indicated,
TBB (25 mM) was added to the sample.
(C) Cortical cultures (DIV10) were incubated
with 25 mM TBB overnight to reduce endogenous
phosphorylation of CK2 substrates. Cells were
lysed and receptors recovered using specific
NR2A or NR2B antibodies. Immunoprecipitates
were subjected to an in vitro phosphorylation
assay (as in B) with recombinant CK2 for 20 min
at 30?C; n = 3.
(D) HEK293 cells were cotransfected with PSD-95,
NR1, and GFP-NR2A or GFP-NR2B. After treat-
ment with TBB for 4 hr, cells were lysed in PBS
with 1% TX-100. The amount of PSD-95 bound
to NMDAR subunits was analyzed by coimmuno-
precipitation with anti-NR2A or anti-NR2B anti-
bodies. Graph represents means ± SEM (n = 4).
*p < 0.05.
phosphorylation. Furthermore, incuba-
tion with TBB inhibited32P incorporation.
However, strikingly, NR2A was not effi-
ciently phosphorylated by CK2 in vitro
To evaluate the phosphorylation of
endogenousNR2 subunitsby CK2
in vivo, we carried out a back phosphorylation assay. We first
treated cortical neurons ±25 mM TBB to inhibit CK2, which
should reduce the endogenous phosphorylation of CK2
substrates. After cell lysis, NR2A or NR2B was immunoprecipi-
tated using specific antibodies and subjected to an in vitro
phosphorylation assay with CK2 as described above.32P incor-
poration into NR2B was more robust when isolated from
TBB-treated neurons than when isolated from control cultures
(Figure 1C), consistent with the reduced CK2 phosphorylation
of endogenous NR2B after TBB-treatment. In contrast we
observed no specific signal in NR2A immunoprecipitates, either
control or TBB-treated, showing that endogenous NR2A is not
efficiently phosphorylated by CK2.
An important consequence of NR2B phosphorylation on
Ser1480 is the disruption of PDZ interactions. To determine if
CK2 activity modulates the association of NR2A and NR2B
with PDZ-containing proteins, we performed coimmunoprecipi-
tation experiments in HEK293 cells transfected with PSD-95,
NR1, and NR2A or NR2B. After treatment with 25 mM TBB for
4 hr, we observed an increase in the association of PSD-95
with NR2B, as predicted. However, TBB treatment did not
increase the binding of PSD-95 to NR2A (Figure 1D). The associ-
ation of NR2A or NR2B with NR1, evaluated as control, was not
affected by TBB (data not shown). Taken together, these data
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 985
show that, despite its high homology with NR2B, the NR2A C
terminus is a poor substrate for CK2 and, accordingly, CK2
activity regulates NR2B, but not NR2A, binding to PDZ
Our data showing that CK2 differentially regulates NR2A and
effect of inhibiting CK2 activity on the surface expression of the
NR2A and NR2B. We treated cortical neurons (DIV 10) with
25 mM TBB and carried out cell surface biotinylation assays as
described in the Experimental Procedures. Consistent with
previous studies (Chung et al., 2004), we observed an increase
in the surface expression of NR2B in the cultures treated with
TBB (Figure 2A). In contrast, the level of NR2A expressed on
the cell surface was dramatically reduced with the same treat-
ment (Figure 2A). CK2 activity did not modify AMPA receptor
surface expression. In addition, we used a fluorescence-based
assay and confocal microscopy to visualize receptor surface
expression. Hippocampal neurons expressing GFP-NR2A or
Surface and Synaptic NR2A Expression,
but Increases Surface and Synaptic NR2B
(A) Cortical cultures (DIV 10) were treated over-
night with 20 mM TBB or vehicle. Surface proteins
were biotinylated and isolated with streptavidin-
agarose beads as described in Experimental
Procedures. Proteins were resolved by SDS-
PAGE and blotted for NR2A, NR2B, NR1, GluR1,
or synaptophysin (as control). Graph represents
means ± SEM. *p < 0.05; **p < 0.01 (n = 5).
(B) Hippocampal neurons expressing GFP-NR2A
or GFP-NR2B were treated ±TBB (25 mM). Surface
receptors were labeled with anti-GFP antibody
and Alexa-568 conjugated anti-rabbit secondary
antibody (shown in white). After permeabiliza-
tion, the internal pool of receptors was visualized
by labeling with anti-GFP and Alexa-633 conju-
gated anti-rabbit secondary antibody (shown in
green). n for NR2A (-/+ TBB) = 21, 16; n for NR2B
(±TBB) = 17, 22. Data represent means ± SEM.
**p < 0.01.
(C) Colocalization of endogenous PSD-95 and
NR2B or NR2A was evaluated in hippocampal
neurons treated with 25 mM TBB, after transient
transfection with GFP-NR2A or GFP-NR2B. n for
NR2A(±TBB) = 15, 18; n for NR2B (±TBB) = 21,
28. Data represent means ± SEM. *p < 0.05. See
also Figure S1.
2. Inhibitionof CK2 Decreases
GFP-NR2B were treated with TBB and
with anti-GFP antibody. Consistent with
our biochemical results, neurons treated
with TBB showed a significant reduction
in the ratio of surface NR2A compared to
an increase in NR2B surface expression
We next investigated if CK2 activity
also regulates the synaptic expression
of NR2 subunits. Thus, we analyzed the synaptic localization of
GFP-NR2A and GFP-NR2B after TBB-treatment using immuno-
fluorescence microscopy to measure the colocalization with
PSD-95, a classical postsynaptic marker. We observed a
decrease in the colocalization of NR2A with PSD-95 in TBB-
treated hippocampal neurons, as well as an increase in the co-
localization of PSD-95 with NR2B (Figure 2C).
Based on these findings, we conclude that CK2 differentially
regulates the surface expression and synaptic localization of
NR2 subunits in an inverse manner, increasing the level of
NR2A-containing and decreasing NR2B-containing NMDARs.
Similar results were observed using DRB, an additional CK2
inhibitor (see Figure S1 available online), confirming the role of
CK2 in NR2 regulation.
CK2 is a pleiotropic kinase, which phosphorylates several
proteins present at synapses that could potentially modulate
synaptic expression of NMDAR subunits (Blanquet, 2000). To
investigate whether the effects observed after CK2 inhibition
are caused by the direct phosphorylation of NR2B within its
Regulation of Synaptic NMDARs by CK2
986 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
PDZ binding domain, we generated a nonphosphorylatable
mutant of NR2B. Mutation of S1480 to Ala prevents phosphory-
lation, but also disrupts the interaction of NR2B with PSD-95
family members (Lim et al., 2002; Prybylowski et al., 2005)
making results obtained from this mutant difficult to interpret.
However, we found that mutating E1479 to Gln (position ?4 in
the PDZ binding domain) eliminates CK2 phosphorylation,
whereas binding to PDZ domain-containing proteins is retained.
As shown in Figure 3A, NR2B E1479Q is not phosphorylated by
CK2 in an in vitro phosphorylation assay. However, pull-down
experiments carried out incubating the last 175 aa of NR2B
attached to GST with lysates of HEK293 cells expressing
PSD-95or SAP102showedthatNR2B E1479Qretains theability
to bind to MAGUK proteins. As expected, two mutants with a
defective PDZ-domain, either containing a phosphomimetic
mutation (NR2B S1480E) or truncation (NR2B V1482Stop),
showed no binding to MAGUK proteins, confirming the speci-
ficity of our assay (Figure 3B; data for SAP102 not shown).
Therefore, we used the phosphorylation deficient mutant
NR2B E1479Q to study whether S1480 phosphorylation of
NR2B regulates the surface and synaptic expression of NR2B.
We quantified surface-expressed receptors and the colocaliza-
tion with PSD-95 in hippocampal neurons transfected with
GFP-NR2B WT or GFP-NR2B E1479Q as described for Figure 2.
Figure 3. NR2B E1479Q, Which Is Not Phos-
phorylated by CK2, Shows Increased Surface
Expression and Synaptic
Compared to NR2B WT
(A) In vitro CK2 phosphorylation (as described in
Figure 1B) of the last 175 aa of NR2B attached to
GST (WT, E1479Q, S1480E, and V1482Stop).
(B) Pull-down experiments of GST-NR2B (WT,
E1479Q, S1480E, and V1482Stop). Beads were
incubated with lysate of HEK293 cells expressing
PSD-95 for 2 hr at 4?C. After washes, the recovered
material was analyzed by immunoblotting with an
(C) Surface expression analysis (as described
in Figure 2B) was carried out with hippocampal
neurons expressing GFP-NR2A (WT or E1461Q) or
GFP-NR2B (WT or E1479Q). Graph represents
mean ± SEM. ***p < 0.001. n for NR2A (WT, E/Q) =
25, 21. n for NR2B (WT, E/Q) = 20, 26.
(D) Colocalization of endogenous PSD-95 with GFP-
NR2A (WT or E1461Q) and GFP-NR2B (WT or
Graph indicates mean ± SEM. **p < 0.01. n for NR2A
(WT, E/Q) = 17, 17. n for NR2B (WT, E/Q) = 16, 14.
Strikingly, we found that the level of the
present at thecell surface was dramatically
elevated (Figure 3C). Furthermore, it ex-
hibited an increased colocalization with
PSD-95 (Figure 3D).
In addition, we generated and analyzed
the analogous mutant for NR2A, NR2A
E1461Q. In contrast to NR2B, NR2A
E1479Q was very similar to wild-type
NR2A in both the level of surface expression and its colocaliza-
tion with PSD-95 (Figures 3C and 3D). These results demon-
strate that the ratio of synaptic NR2B is regulated via S1480
phosphorylation and that the analogous residue of NR2A does
not regulate synaptic NR2A expression.
We next investigated the mechanisms underlying the regula-
tion of NR2B trafficking by CK2. One likely possibility is that
the observed decrease in surface and synaptic expression is
due to increased NMDAR endocytosis. This hypothesis is sup-
ported by the fact that CK2 activity disrupts the binding of
NR2B with MAGUK proteins (Figure 1D and (Chung et al.,
2004) and it has been demonstrated that PSD-95 stabilizes
NMDARs at the surface and inhibits endocytosis (Lavezzari
et al., 2003). Therefore, we analyzed the effect of CK2 on
NMDAR endocytosis using a fluorescence-based trafficking
assayin GFP-NR2B-or GFP-NR2B
neurons (Suh et al., 2008). Surface receptors were labeled with
anti-GFP antibody and the cells returned to 37?C for 30 min to
allow protein internalization (±TBB). After incubating with
Alexa-568 conjugated secondary antibody (shown in green),
cells were permeabilized and the internalized pool of receptors
was visualized with Alexa-633 conjugated secondary antibody
(white). The endocytosis of NR2B is strongly reduced by the
inhibition of CK2 (Figure 4A). Interestingly, we also observed
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 987
a decrease in the endocytosis of the phosphorylation deficient
mutant NR2B E1479Q that was not further decreased by the
presence of TBB. These findings show that CK2 reduces
NR2B internalization via S1480 phosphorylation (Figure 4A).
Consistent with our previous findings, endocytosis of NR2A
E1461Q was the same as NR2A WT (Figure S2).
Several motifs have been identified in NMDAR subunits that
regulate endocytosis (Lau and Zukin, 2007). Among them, the
tyrosine-based endocytic motif (YEKL) present in the extreme
C terminus of NR2B is a prime candidate to be affected by
CK2 activity because of its close proximity to S1480 (Figure 1A).
In addition, there is an interplay between the binding of
MAGUK proteins with NR2B via the PDZ-domain and the phos-
phorylation of the YEKL motif (Y1472) that results in the stabi-
lization of NMDARs in the membrane (Lavezzari et al., 2003;
Prybylowski et al., 2005). Therefore, we tested whether phos-
phorylation in the PDZ domain of NR2B regulates its surface
Figure 4. Phosphorylation of NR2B S1480
Increases Endocytosis via a Coordinated
Dephosphorylation of Y1472 in the YEKL
(A) An endocytosis assay of hippocampal neurons
transfected with GFP-NR2B (WT or E1479Q) was
carried out as described in Experimental Proce-
dures. At DIV10, neurons were labeled with
anti-GFP antibody, washed, and returned to
conditioned media (±25 mM TBB) for 30 min at
37?C to allow receptor internalization. Cells were
labeled with Alexa 568-conjugated secondary
antibody (shown in green). After permeabilization
with 0.25% TX-100, internalized receptors were
labeled with Alexa 633-conjugated secondary
antibody (shown as white). n for NR2B WT
(±TBB) = 15, 25. n for NR2B E1479Q (±TBB) =
25, 21. Graph represents means ± SEM. ***p <
0.001; n.s. denotes not significant differences.
(B) Endocytosis assay with NR2B constructs with
mutations in the YEKL and/or PDZ-domain carried
sents means ± SEM. **p < 0.01 ***p < 0.001.
(C) Surface expression of NR2B constructs with
mutations in the YEKL and/or PDZ-domain was
analyzed as in Figure 2B. n = 38, 32, 22, 32, 17,
30. Graph represents means ± SEM. **p < 0.01;
***p < 0.001.
(D) Levels of NR2B phosphorylation (Y1472 and
S1480) were analyzed in HEK293T cells trans-
fected with PSD-95, NR1, and GFP-NR2B and
incubated ±25 mM TBB for 4 hr. n = 3. Graph
represents means ± SEM. *p < 0.05; **p < 0.01.
See also Figure S2.
expression and endocytosis via phos-
phorylation of Y1472. We generated
both the PDZ-binding domain (E1479Q
or S1480E) and in the YEKL endocytic
observed reduced internalization for the
NR2B Y1472A mutant (Lavezzari et al.,
2004; Prybylowski et al., 2005). Strikingly the level of endocy-
tosis of NR2B E1479Q was almost indistinguishable from
NR2B Y1472A. Most important, the double mutant, NR2B
Y1472A;E1479Q, did not show any additional decrease in
endocytosis, indicating that the phosphorylation in the PDZ
domain and in the YEKL motif share a common molecular
mechanism to reduce NR2B endocytosis. Consistently, the
increased endocytosis of NR2B S1480E was decreased to
the levels of NR2B Y1472A by introducing an additional muta-
tion in the YEKL motif (NR2B Y1472A;S1480E). Similar levels of
endocytosis were observed with NR2B E1479Q and NR2B
Y1472A;E1479Q (Figure 4B). Accordingly, we found that the
mutation in the YEKL motif did not modify the surface expres-
sion of NR2B E1479Q, whereas it did significantly increase the
surface levels of the phosphomimetic S1480E to the levels
obtained by the nonphosphorylatable NR2B E1479Q mutant
Regulation of Synaptic NMDARs by CK2
988 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
The findings of these immunofluorescence experiments
suggest that phosphorylation in the PDZ binding domain of
NR2B affects the phosphorylation of the YEKL endocytic motif,
which increases NMDARs internalization. To examine this
hypothesis, we investigated the effect of CK2 inhibition on
Y1472 phosphorylation. We analyzed the levels of NR2B
pY1472 and pS1480 in HEK293 transfected with PSD-95, NR1
and NR2B after incubation ±25 mM TBB for 4 hr. As expected,
we observed a reduction in NR2B pS1480 and a concomitant
increase in Y1472 phosphorylation (Figure 4D).
It is well known that the subunit composition of synaptic
NMDARs changes during development. During early postnatal
whereas NR2A-containing NMDARs are the major synaptic
subtype in the adult central nervous system (Groc et al., 2009;
Lau and Zukin, 2007). Thus far our data demonstrate that CK2
can modify the NR2A/2B ratio of synaptic NMDARs, so we
hypothesized that CK2 might be important in the developmental
switch of NR2 subunit composition. The fact that CK2 activity is
developmentally regulated (Blanquet, 2000) and ismodulated by
synaptic activity (Charriaut-Marlangue et al., 1991) makes it
a good candidate. Therefore, we evaluated the expression of
CK2, NR2A, NR2B, and NR2B S1480 phosphorylation during
development by immunoblotting extracts of cortical synapto-
somes from mice at different ages. As previously reported
(Petralia et al., 2005; van Zundert et al., 2004) we observed
a substantial increase in NR2A and a slight decrease in NR2B
expression throughout development. Remarkably, we found
that S1480 phosphorylation (evaluated as a ratio to total NR2B)
is elevated in the second postnatal week, the critical period for
replacement of NR2B by NR2A (Figures 5A and 5B). We also
analyzed the levels of CK2 catalytic and regulatory subunits
highest level of expression during the critical period for the NR2
subunit switch (P11-15) (Figures 5A and 5C). The accessibility of
CK2 to the substrate can play a major role in regulation of CK2
phosphorylation (Faust and Montenarh, 2000). Therefore, we
used subcellular fractionation and immunoblotting to see if CK2
Figure 5. Phosphorylation of NR2B S1480 and Total Expression of CK2 Increase during the Second Postnatal Week and the Association
of CK2 with Synaptic Plasma Membranes Is Elevated at P13
(A) Protein expression was analyzed in cortical synaptosomes from mice of different stages of development by immunoblotting.
Graphsshownin(B)and(C)summarize thedata ofsixexperimentsanalyzing thelevel ofNR2BSer1480phosphorylationandexpressionofNR2AandCK2 (aand
b subunits) throughout development (the gray area indicates the critical period of time for the NMDAR subunit switch).
(D) Synaptic plasma membranes (SPMs) were isolated from P7, P13, or adult animals, using a standard purification protocol (Hallett et al., 2008). The level of
phosphorylated NR2B (Y1472 and S1480), CK2 (a and b), EEA1 (as negative control) and actin (as loading control) present in the SPMs was analyzed by
(E and F) Graph represents means ± SEM. ***p < 0.001; n = 6.
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 989
is enriched at SPMs along with NR2 subunits. We found that the
association of CK2 a and b subunits with the SPM at P13 was
5D and 5F). Consistent with our previous data, the level of NR2B
pS1480 was elevated in P13 in SPM and pY1472 was reduced in
comparison with the adult levels (Figures 5D and 5E).
The synaptic incorporation of NR2A requires synaptic activity
and can be blocked by NMDAR inhibitors (Barria and Malinow,
2002; Bellone and Nicoll, 2007). As previously reported (Chung
et al., 2004), we find that phosphorylation of NR2B on S1480 is
induced by activity that is dependent on synaptic NMDAR
activation, because we observe a substantial reduction in
NR2B S1480 phosphorylation in cultures treated for 8 hr with
tetrodotoxin (2 mM) or overnight with NMDAR inhibitors
(100 mM APV; 40 mM MK801) (Figures 6A and 6B). Conversely,
increasing synaptic activity with bicuculline (40 mM for 8 hr) or
KCl (20 mM for 5 min) results in an elevated level of NR2B
S1480 phosphorylation compared with control cultures (Figures
6A). In addition, NR2A expression modulates synaptic NR2A by
promoting its insertion into synaptic membranes (Barria and Ma-
linow, 2002). Therefore, to investigate whether the level of NR2A
expression can also modify NR2BpS1480, we analyzed NR2A
knockout mice by immunoblotting cortical synaptosomes from
P11.Interestingly, wefound asignificant reduction inthe amount
of NR2B phosphorylated on S1480 in mice lacking NR2A (Fig-
ure 6C). Therefore, the same stimuli that are required for the
NMDAR switch also regulate NR2B S1480 phosphorylation.
We have substantial evidence that CK2 activity affects
synaptic expression of NR2 subunits and that NR2B S1480 is
a critical residue. However, it is possible that NR2B is phosphor-
ylated on S1480 by another kinase. To investigate this possi-
bility, we carried out an in vitro phosphorylation assay with the
C terminus NR2B attached to GST, using 10 mg of brain lysate
as source of endogenous kinases. We found that NR2B was
phosphorylated in vitro on incubation with the brain lysate, but
the phosphorylation was blocked in the presence of 25 mM
TBB (Figure 6D) consistent with the phosphorylation of NR2B
S1480 being mediated exclusively by CK2.
LTP induction rapidly induces a switch in NMDA receptor
subunit composition from NR2B- to NR2A-containing NMDARs
at synapses on hippocampal CA1 pyramidal neurons in young
(2- to 9-day-old) rats (Bellone and Nicoll, 2007). We next studied
a role for CK2 in this activity-driven switch using whole-cell
patch-clamp recordings from CA1 pyramidal neurons in acute
hippocampal slices prepared from 4- to 9-day-old rats. Consis-
tent with previous work (Bellone and Nicoll, 2007), an LTP induc-
tion protocol caused the speeding of NMDA receptor excitatory
postsynaptic current (EPSC) decay and a decrease in ifenprodil
(5 mM) block that was specific to the pathway in which the
LTP protocol was applied (Figures 7A–7C, 7G, and 7H). These
findings confirm that activity causes a rapid switch from
NR2B-containing NMDA receptors, which exhibit slow decay
kinetics and are blocked by ifenprodil, to NR2A-containing
NMDA receptors that exhibit faster kinetics and a lack of ifenpro-
dil sensitivity. To determine if CK2 is involved in this subunit
composition switch, in experiments interleaved with the controls
(described above) we incubated slices in TBB (10 mM) for at least
2 hr and then tested the ability of activity to drive the subunit
switch. In TBB-treated slices, the LTP induction protocol failed
Figure 6. Phosphorylation of NR2B on S1480 Increases in Response to NMDAR Activity and Is Regulated by NR2A Expression
(A) Cortical cultures (DIV10) were incubated for 8 hr with tetrodoxin (TTX; 2 mM) or bicuculline (Bicuc.; 40 mM) to block or stimulate neuronal activity respectively.
Treatment with KCl (20 mM for 5 min) was used to induce neuronal depolarization. The level of phosphorylated NR2B was analyzed by immunoblotting after
isolation of cellular membranes. The same membrane was reblotted for NR2B or phospho-specific NR2B S1480 antibodies. Graph represents means ± SEM.
**p < 0.01; n = 5.
(B) Cortical cultures were incubated overnight ±NMDAR antagonists (100 mM APV; 40 mM MK-801). Graph represents means ± SEM. *p < 0.05; n = 3.
(C) Corticalsynaptosomeswereisolated fromP11 mice (WT orNR2Aknockout)and analyzed by immunoblotting withtheindicatedantibodies.Graph represents
means ± SEM. *p < 0.05; n = 4.
(D) GST-NR2B (last 175 aa) was phosphorylated in vitro using 10 mg of brain lysate as source of kinases. TBB (25 mM) was added to the sample when indicated.
Regulation of Synaptic NMDARs by CK2
990 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
to cause a speeding of NMDA EPSC kinetics or reduce
sensitivity to ifenprodil (Figures 7D–7H). Thus CK2 is involved
in the activity-dependent switch in NR2 subunit composition of
synaptic NMDA receptors.
Many functional properties of NMDARs are determined by NR2
subunits, including affinity for glutamate, sensitivity to Mg2+,
single channel conductance, open probability, and deactivation
time (Furukawa et al., 2005; Groc et al., 2009). Not surprisingly,
NR2 subunits are subject to strict control mechanisms and the
NR2A and NR2B subunits are differentially regulated. For
instance, these subunits show distinct patterns of expression
during the development and, in adult brain, NR2B is restricted
to forebrain whereas NR2A is ubiquitously expressed (Kohr,
2006; Monyer et al., 1994; Wenzel et al., 1997). NR2A and
NR2B also exhibit a different subcellular localization. In cortex
and hippocampal pyramidal cells NR2A is highly localized
Activity-Dependent Switch in the Subunit
Composition of Synaptic NMDA Receptors
(A) Pooled data (n = 10) for NMDA EPSC amplitude
versus time from control experiments showing
block (red is test pathway to which the LTP induc-
tion protocol was applied; black is the control
pathway; these colors are consistent throughout
(B) NMDA EPSCs from example control experi-
ment showing the speeding of kinetics in test
path after induction (lower panel).
(C) NMDA EPSCs from example control experi-
ment showing the reduced block by ifenprodil
(5 mM) in test path after induction (lower panel).
incubated with TBB (10 mM) for at least 2 hr (n = 7).
(G) Summary data of weighted decay time con-
stant (control [?TBB] n = 10; +TBB n = 8). *Indi-
cates p < 0.05 between pre- and postinduction.
(H) Summary data of NMDA EPSC amplitude in
ifenprodil (% of EPSC amplitude in absence of
ifenprodil; control [?TBB] n = 10; +TBB n = 7).
*Indicates p < 0.05 between control and test
7. CK2ActivityRegulates the
to postsynaptic membranes whereas
there is a high amount of NR2B at extra-
synaptic sites (Kew et al., 1998; Scimemi
et al., 2004; Tovar and Westbrook, 1999).
Trafficking of NR2A and NR2B is also
differentially modulated, as NR2B-con-
taining receptors are more dynamic
than NR2A, with a higher rate of endocy-
tosis (Lavezzari et al., 2004), lateral diffu-
sion (Groc and Choquet, 2006) and, a
higher association with recycling endo-
somes (Lavezzari et al., 2004) compared
NMDARs. Both subunits are substrates for a number of kinases,
although differences between subunits also exist (Chen and
Roche, 2007). In this study, we show differential regulation of
synaptic NR2A and NR2B by CK2. It has been reported that
CK2 phosphorylates NR2B within the PDZ binding domain
(Chung et al., 2004). We now show that CK2 phosphorylates
NR2B much more efficiently than NR2A, which leads to a
decrease in synaptic expression of NR2B. In addition, we have
characterized a NR2B mutant that cannot be phosphorylated
by CK2, but maintains the binding with MAGUK proteins
(NR2B E1479Q). We found that this mutant mimics the effect
of CK2 inhibition in altering both NR2B surface expression
and colocalization with PSD-95, indicating that the phosphoryla-
tion in the PDZ binding domain of NR2B by CK2 regulates
receptor trafficking. Furthermore, we have identified an interplay
between S1480 phosphorylation and the YEKL endocytic motif,
providing a molecular mechanism for the observed effects on
NMDAR trafficking. Our findings support a model in which the
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 991
coordinated phosphorylation of two different residues of NR2B:
S1480 within the PDZ domain binding domain and Y1472 within
the tyrosine-based YEKL endocytic motif regulates receptor
endocytosis, and ultimately the surface expression of NR2B
(Figure 8). Specifically, the association of NR2B with MAGUK
proteins, such as PSD-95, is disrupted when NR2B is phosphor-
ylated by CK2 within its PDZ binding domain (S1480). The
disruption of this interaction triggers a decrease in Y1472 phos-
phorylation of NR2B within the YEKL endocytic domain and,
ultimately, an increase in NR2B internalization by association
with AP-2 (Blanpied et al., 2002; Chung et al., 2004; Prybylowski
et al., 2005; Roche et al., 2001). This model is based on our anal-
yses of NR2B constructs with mutations in the YEKL and/or PDZ
binding domain and on the quantification of Y1472 and S1480
phosphorylation on NR2B after pharmacological inhibition of
CK2. In addition, this model is entirely consistent with previous
studies showing that increased PSD-95 binding to NR2B leads
to increased Y1472 phosphorylation and NR2B surface expres-
sion (Song et al., 2003; Zhang et al., 2008). In addition, it has
been recently reported that chronicethanol treatment in cultured
neurons results in a decrease in NR2B S1480 phosphorylation
and leads to a NR2B translocation from synaptic to extrasynap-
tic sites (Clapp et al., 2010). Although a very similar treatment
with ethanol also decreases NR2B Y1472 phosphorylation
(Alvestad et al., 2003) this is likely due to a pathologically
elevated tyrosine-phosphatase activity (Xu et al., 2003; Zhao
the YEKL and PDZ binding domain.
We also show that NR2B Y1472A, which is unable to bind to
the clathrin adaptor protein AP-2, exhibits reduced endocytosis,
and accordingly, increased surface expression. These results
are consistent with previous studies in which different stimuli
that reduce Y1472 phosphorylation also decrease NR2B surface
expression (Snyder et al., 2005; Zhang et al., 2008). However,
Prybylowski et al. (2005) reported that NR2B Y1472A shows
increased synaptic but not total surface expression, based on
their current density recording in response to applied NMDA.
This discrepancy with our current findings could be due to the
use of cerebellar granular cells (CGCs) for the electrophysiolog-
authors found a significant increase of NR2B Y1472A surface
expression when analyzed in hippocampal cultures by immuno-
cytochemistry in the same study.
We find that NR2A, like NR2B, is strongly regulated by CK2
activity. However, NR2B is an excellent substrate for CK2,
whereas NR2A is not (Figure 1). We mutated E1479Q on
NR2B to block CK2 phosphorylation and found this eliminated
the effect of CK2 inhibition on NR2B internalization. However,
the analogous mutation on NR2A (NR2A E1461Q) showed the
same ratio of endocytosis as wild-type NR2A. These data all
suggest that CK2 activity affects NR2A trafficking via an indirect
modulation by phosphorylating other synaptic proteins. This
mechanism would include an increase in NR2A stabilization in
postsynaptic membranes, the delivery to synapses or even
modulation of NR2A expression (Groc et al., 2009; Perez-Otano
and Ehlers, 2005). MAGUK proteins such as PSD-95 (Soto et al.,
2004), kinases such as PKC (Allende and Allende, 1995) or PKA
(Carmichael et al., 1982; Hemmings et al., 1982), phosphatases
such as PP2A (Heriche et al., 1997), PP2C (Pinna and Meggio,
1997) or PTP1B (Jung et al., 1998) and a large number of
transcription factors (Blanquet, 2000; Meggio and Pinna, 2003)
are some of the candidate proteins phosphorylated by CK2
that might affect NR2A synaptic expression. An alternative and
appealing explanation for our observations is that the NMDAR
2A/2B subunit switch is a mechanism with two sequential and
coupled stages, in which the synaptic removal of NR2B is
required to allow NR2A synaptic incorporation. CK2, therefore,
might facilitate NR2A insertion by removing NR2B from the
synaptic sites via S1480 phosphorylation (Figure 8). All our
data are consistent with such a model.
Our findings reveal an important role for CK2 in regulating
synaptic NMDARs. CK2 is a ubiquitous serine/threonine kinase
that is highly expressed in the brain, where it is widely distributed
in both neuronal and nonneuronal cells (Allende and Allende,
1995; Blanquet, 2000; Pinna, 1990). CK2 activity is high in
cortex and, interestingly, 25%–30% of its total activity is local-
ized in the synaptosomal fraction (Girault et al., 1990). It is not
clear what function CK2 is performing at synapses, but
increasing evidence suggests a role in learning and memory.
For example, LTP can be blocked by CK2 inhibitors (5,6-di-
chloro-1-b-D-ribofuranosyl-benzimidazole [DRB] and TBB) by
reducing NMDAR activity (Kimura and Matsuki, 2008). Further-
more, DRB is able to reduce fear-motivated learning (Igaz
et al., 2002). In addition to NR2B, a large number of proteins
associated with synaptic plasticity has been reported to be
phosphorylated by CK2 (Blanquet, 2000). Consistently, extracts
Figure 8. Model of CK2 Regulation of
(A) Early in development, the association of NR2B
membranes via phosphorylation of Y1472 by
Fyn. Phosphorylation of the Y1472 within the tyro-
sine-based endocytic motif blocks AP-2 binding.
(B) During the critical period, NMDAR activity
induces NR2B S1480 phosphorylation by CK2,
which results in the disruption of NR2B associa-
tion with MAGUKs. NR2B Y1472 is now dephos-
phorylated and AP-2 can bind to the YEKL motif
and promote NR2B endocytosis.
(C) NR2A expression increases and NR2A-
containing receptors replace NR2B-containing
NMDARs at synaptic sites.
Regulation of Synaptic NMDARs by CK2
992 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
of the frontal cortex of Alzheimer’s disease patients show
a recent study reported an increase on spatial learning after
inhibition of CK2 using dominant-negative mutants (Chao
et al., 2007). Notably, CK2 is developmentally regulated. Its
activity is high at embryonic day 16 in cortex, remains elevated
during the early postnatal period and decreases slightly in the
adult. However, in liver, CK2 activity decreases around birth
(Girault et al., 1990). Those data and our findings that the highest
expression levels of CK2 and its association with synaptic
plasma membranes are reached at P11-15 suggest a develop-
mental role for CK2.
The NMDAR subunit switch is one of the more studied events
during synaptic maturation. It is well established that synaptic
NR2B-containing NMDARs are replaced by NR2A-containing
during the second postnatal week in rodents, a process that
has been studied extensively in visual cortex (Carmignoto and
Vicini, 1992; Hestrin, 1992; Philpot et al., 2001; Quinlan et al.,
1999; van Zundert et al., 2004). It is known that NR2A incorpora-
tion into postsynaptic membranes is dependent on synaptic
activity and the level of NR2A expression (Barria and Malinow,
2002), but the molecular mechanisms controlling the NMDAR
switch remain unclear. Remarkably, it has been reported
recently that induction of LTP in young animals (2–9 days old)
results in a rapid replacement of synaptic NR2B subunits by
NR2A, as demonstrated by faster kinetics and a lower inhibition
by ifenprodil obtained in the LTP path compared to the control
(Bellone and Nicoll, 2007). However, no molecular mechanism
has been implicated in mediating these effects. We now find
strated that CK2 differentially regulates synaptic NMDAR
subunits, increasing NR2A and reducing NR2B, and, in addition,
CK2 activity is known to increase rapidly during the induction of
LTP in hippocampus (Charriaut-Marlangue et al., 1991). Impor-
tantly, we have shown that the NMDAR subunit switch induced
by LTP is blocked in the presence of a CK2 inhibitor. Consistent
with this hypothesis, both the switch and NR2B S1480 phos-
phorylation by CK2 are dependent on synaptic activity and
NMDAR activation. How activity modulates NR2B S1480 phos-
on CaMKII (Chung et al., 2004 and data not shown). Although
CaMKII can phosphorylate the CK2 b subunit directly, this phos-
phorylation does not modify NR2B S1480 phosphorylation in an
in vitro phosphorylation assay (Chung et al., 2004). One possi-
bility is that CaMKII phosphorylation of CK2 targets CK2 to the
synaptic plasma membrane, allowing the interaction with
NR2B (see the coincident increased of NR2B S1480 phosphory-
lation and association of CK2 with SPMs at P13 in Figures
5D–5F). Identifying the molecular mechanisms underlying the
spatiotemporal regulation of synaptic CK2 activity is an impor-
tant topic for future investigation as our data demonstrate that
CK2 strongly regulates synaptic NMDARs composition.
Neuronal Cultures, Antibodies, and Reagents
Primary cultured neurons were prepared from E18 Sprague-Dawley rats as
we used cortical neurons because the cortex yields enough material for
biochemical analyses, whereas hippocampal cultures are the preferred for
immunocytochemistry. Theuseandcareofanimals usedinthisstudyfollowed
the guidelines of the NIH Animal Research Advisory Committee. C-terminal
NR2B, synaptophysin, and CK2 subunit b antibodies were purchased from
Sigma (St. Louis, MO). We obtained phosphorylation state-specific S1480
NR2B antibody from Pierce (Rockford, IL), NR2A from Upstate (Lake Placid,
NY), actin from Applied Biological Materials (Richmond, BC, Canada), and
EEA1 from BD Biosciences (San Jose, CA). GluR1 and phosphorylation
state-specific Y1472 NR2B antibody were from Chemicon (Billerica, MA).
NR1, PSD-95 and CK2 subunit a antibodies were purchased from Affinity
BioReagents (Golden, CO). Anti-GFP and all secondary antibodies for immu-
nofluorescence were obtained from Invitrogen (Molecular Probes, Eugene,
OR). All the drugs and inhibitors used in this study were purchased from Tocris
Cookson (Ellisville, MO) except APV and picrotoxin (Sigma) and DRB (Calbio-
chem, San Diego, CA). GFP-NR2A and GFP-NR2B constructs were kindly
provided by Dr. Stefano Vicini (Georgetown University).
Isolation of the Neuronal Membrane Fraction and Biotinylation
Assay of Surface-Expressed Receptors
For the preparation of the crude membrane fraction, cortical neurons were
harvested in cold phosphate buffer solution (PBS)/Ca2+(PBS, 0.1 mM
CaCl2) and collected by centrifugation. The pellet was resuspended in
hypotonic buffer (20 mM Tris pH 8.8; 5 mM ethylenediaminetetraacetic
acid (EDTA); 1 mM Na3VO4) with protease and phosphatase inhibitors (Roche
resis (SDS-PAGE), the samples were immunoblotted with the indicated
antibodies. The same membrane was reblotted for the analysis of NR2B phos-
pho/total ratio.Forthebiotinylation assay, cultureswerewashed three times in
cold PBS+ (PBS; 1 mM MgCl2; 0.1 mM CaCl2) and incubated with 1 mg/ml
EZ-Link Sulfo-NHS-SS-Biotin(Pierce) inPBS+for15minat4?C.Afterquench-
ing the reaction with 100 mM glycine in PBS+ at 4?C for 10 min, the total
membrane fraction was isolated as described above. Membranes were solu-
bilized in 1% SDS for 15 min at 37?C, diluted with 10 volumes of cold PBS/1%
TX-100 and centrifuged for 45 min at 100,000 x g. The supernatant was then
incubated with Streptavidin beads (Thermo Scientific) for 2 hr at 4?C and, after
four washes, bound proteins were immunoblotted with the indicated anti-
bodies. Data are presented as mean ± standard error of mean (SEM) and
significance was analyzed using the Student’s t test. Experiments were
repeated at least three times, independently.
Subcellular Fractionation of Brain Tissue
Biochemical fractionation was carried out following standard methods (Hallett
et al., 2008). Briefly, cortex from mice of different developmental stages was
homogenized in cold TEVP buffer (10 mM Tris pH 7.5; 1 mM EDTA; 1 mM
ethylene glycol tetraacetic acid [EGTA]; 1 mM Na3VO4) containing 0.32 M
sucrose and phosphatase and protease inhibitors. Homogenate was centri-
fuged 10 min to 1000 x g at 4?C to remove nuclei and large debris. The super-
natant (SN1) was centrifuged to 10,000 x g to obtain the crude synaptosome
(P2) fraction. P2 was incubated in hypoosmotic solution (20 mM Tris pH 8.8;
5 mM EDTA; 1 mM Na3VO4) for 20 min on ice and centrifuged 30 min at
25,000 x g to obtain synaptic plasma membranes (SPMs). Tissue was
analyzed twice for each tissue collection.
Coimmunoprecipitation and Pull-Down Assays
HEK293T cells were cotransfected with PSD-95, NR1 and GFP-NR2A, or
facturer’s instructions (Clontech). After 24 hr of expression, cells were lysed in
anti-NR2A or anti-NR2B antibodies and protein A-Sepharose beads overnight
at 4?C, washed and immunoblotted. For pull-down experiments, HEK293T
cells were transfected with PSD-95 or SAP102 and processed as before.
Lysates were incubated with GST-NR2B C-terminal (wild-type or mutant) for
2 hr at 4?C. After three washes, bound proteins were immunoblotted for
PSD-95 or SAP102 to confirm the protein interaction.
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 993
In Vitro Phosphorylation and Back Phosphorylation Analysis
The last 175 aa of NR2A or NR2B were fused to GST and purified as previously
described (Chen et al., 2006). Mutants were generated by polymerase chain
reaction using the QuikChange site-directed mutagenesis kit (Stratagene)
following the manufacturer’s instructions. In vitro phosphorylation was carried
out by incubating GST-proteins with 50 units of CK2 (New England BioLabs)
and 2 pmol [g-32P]ATP (3000 Ci/mmol) for 20 min at 30?C in 50 ml of 20 mM
Tris pH 7.5; 50 mM KCl; 10 mM MgCl2; 0.1 mM ATP. For brain lysate phos-
phorylation, SN1 fraction from adult mice cortex was dissolved in 2%
TX-100 with protease inhibitors for 5 min at RT. GST-proteins were incubated
with 10 mg of the dissolved SN1 in 20 mM HEPES, pH 7.0, 1.67 mM CaCl2,
1 mM dithiothreitol, 10 mM MgCl2, 0.1 mM ATP, and phosphatase inhibitors
(Sigma) for 20 min at 30?C. When indicated, 25 mM TBB was added to the
reaction. For back phosphorylation, cortical cultures (DIV10) were incubated
with 25 mM TBB overnight to reduce endogenous phosphorylation of CK2
substrates. Cells were harvested and lysed with 1% DOC. After the addition
of equal volume of RIPA buffer, receptors were recovered using specific
of CK2 as above.
Receptor endocytosis was analyzed using a fluorescence-based antibody
uptake assay, as previously reported (Lavezzari et al., 2004; Suh et al.,
2008). Briefly, hippocampal neurons were transfected with GFP-NR2A or
GFP-NR2B at DIV7 and maintained with 25 mM TBB. At DIV10, surface
receptors were labeled with anti-GFP antibody for 15 min at room tempera-
ture, and returned at 37?C for 30 min to allow protein internalization. Surface
proteins were labeled with Alexa 568-conjugated secondary antibody (shown
in green). After permeabilization, the internalized pool of receptor was labeled
with Alexa 633-conjugated secondary antibody (shown in white). To compare
surface-expressed protein versus the intracellular pool, hippocampal neurons
were transfected with GFP-NR2A or GFP-NR2B and treated with TBB as
above. Surface-expressed receptors were labeled with anti-GFP antibody
and Alexa 568-conjugated secondary antibody (shown in white). Cells were
permeabilized with 0.25% TX-100 in PBS and internal pool was labeled with
anti-GFP antibody and Alexa 633-conjugated secondary antibody (shown in
green). Quantification was performed analyzing the fluorescence intensity of
three independent areas per neuron using MetaMorph 6.0 software (Universal
Imaging Corp), and is presented as mean ± SEM. For surface-expression
analysis the ratio in the intensity surface/intracellular is shown. Data from
endocytosis are presented as ratio internalized/surface intensities.Colocaliza-
tion of NR2 subunits and PSD-95 was analyzed in hippocampal neurons
(DIV14) transfected as above and maintained with 25 mM TBB. Colocalization
level was analyzed using ImageJ and presented as mean ± SEM. Cells were
imaged on a Zeiss LSM 510 confocal microscope. Serial optical sections
collected at 0.35-mm intervals were used to create maximum projection
images. Experiments were repeated at least, three times independently and
significance analyzed using Student’s t test (n = number of cells).
Wistar rats (4-to 9-day-old) were anesthetized withisoflurane and decapitated
in accordance with NIH animal care and use guidelines. Transverse hippo-
campal slices (400-mm thick) were cut in ice-cold artificial cerebrospinal fluid
(ACSF) containing (mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 9 MgSO4, 1 NaH2PO4,
26.2 NaHCO3, 11 glucose equilibrated with 95% O2and 5% CO2. Slices were
allowed to recover for at least 1 hr in ACSF at room temperature (composition
as above except for 1.3 mM MgSO4). Whole cell patch clamp recordings were
made from visually identified CA1 pyramidal neurons in the presence of 50 mM
picrotoxin at room temperature. The whole-cell solution contained (mM) 115
CsMeSO4, 20 CsCl2, 10 HEPES, 2.5 MgCl2, 4 NaATP, 0.4 NaGTP, 10 NaCrea-
tine, and 0.6 EGTA (pH 7.2).
EPSCs were evoked by electrical stimulation of two independent sets of
Schaffer collateral/commissural axons using two bipolar stimulating elec-
trodes placed in stratum radiatum of CA1 (0.1 Hz stimulation frequency for
each pathway). The stimulating electrodes were placed on opposite sides of
the recorded cell. NMDA receptor EPSCs were obtained in the presence of
NBQX (5 mM) and picrotoxin (50 mM) while cells were voltage-clamped at
+40 mV. Recordings were carried out using a Multiclamp 700B patch-clamp
amplifier (Axon Instruments, Foster City, CA); signals were filtered at 4 kHz,
digitized at 10 Hz and displayed and analyzed on-line using pClamp 9.2
(Axon Instruments). For induction of the activity-dependent switch in the
subunit composition of synaptic NMDA receptors, an LTP induction protocol
were voltage clamped at 0 mV while Schaffer collateral/commissural axons
were stimulated at 1 Hz for 120 s. Cells were then voltage-clamped
at ?70 mV for 5 min after LTP induction. After these 5 min, NMDA receptor
EPSCs were once again recorded at +40 mV. The EPSC decay is fit with
a double exponential function using OriginLab software (Northampton, MA)
and decay kinetics are expressed as a weighted decay time constant.
Statistical significance was tested using a Student’s t test.
Supplemental Information includes two figures and can be found with this
article online at doi:10.1016/j.neuron.2010.08.011.
We thank John D. Badger II for technical assistance. We also thank the NINDS
sequencing facility and light imaging facility for expertise and advice. This
research was supported by the NINDS Intramural Research Program
(A.S-C.; K.W.R.; J.T.R.I) and the Pharmacology Research Associate (PRAT)
Program, NIGMS (J.A.M).
Accepted: August 10, 2010
Published: September 22, 2010
Aksenova, M.V., Burbaeva, G.S., Kandror, K.V., Kapkov, D.V., and Stepanov,
A.S. (1991). The decreased level of casein kinase 2 in brain cortex of
schizophrenic and Alzheimer’s disease patients. FEBS Lett. 279, 55–57.
Al-Hallaq, R.A., Conrads, T.P., Veenstra, T.D., and Wenthold, R.J. (2007).
NMDA di-heteromeric receptor populations and associated proteins in rat
hippocampus. J. Neurosci. 27, 8334–8343.
Allende, J.E., and Allende, C.C. (1995). Protein kinases. 4. Protein kinase CK2:
an enzyme with multiple substrates and a puzzling regulation. FASEB J. 9,
Alvestad, R.M., Grosshans, D.R., Coultrap, S.J., Nakazawa, T., Yamamoto, T.,
and Browning, M.D. (2003). Tyrosine dephosphorylation and ethanol inhibition
of N-Methyl-D-aspartate receptor function. J. Biol. Chem. 278, 11020–11025.
Barria, A., and Malinow, R. (2002). Subunit-specific NMDA receptor trafficking
to synapses. Neuron 35, 345–353.
Bellone, C., and Nicoll, R.A. (2007). Rapid Bidirectional Switching of Synaptic
NMDA Receptors. Neuron 55, 779–785.
Blanpied, T.A., Scott, D.B., and Ehlers, M.D. (2002). Dynamics and regulation
of clathrin coats at specialized endocytic zones of dendrites and spines.
Neuron 36, 435–449.
Blanquet, P.R. (2000). Casein kinase 2 as a potentially important enzyme in the
nervous system. Prog. Neurobiol. 60, 211–246.
Carmichael, D.F., Geahlen, R.L., Allen, S.M., and Krebs, E.G. (1982). Type II
regulatory subunit of cAMP-dependent protein kinase. Phosphorylation by
casein kinase II at a site that is also phosphorylated in vivo. J. Biol. Chem.
Carmignoto, G., and Vicini, S. (1992). Activity-dependent decrease in NMDA
receptor responses during development of the visual cortex. Science 258,
Chao, C.C., Ma, Y.L., and Lee, E.H. (2007). Protein kinase CK2 impairs spatial
memory formation through differential cross talk with PI-3 kinase signaling:
activation of Akt and inactivation of SGK1. J. Neurosci. 27, 6243–6248.
Regulation of Synaptic NMDARs by CK2
994 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.
Charriaut-Marlangue, C., Otani, S., Creuzet, C., Ben-Ari, Y., and Loeb, J.
(1991). Rapid activation of hippocampal casein kinase II during long-term
potentiation. Proc. Natl. Acad. Sci. USA 88, 10232–10236.
Chen, B.S., and Roche, K.W. (2007). Regulation of NMDA receptors by
phosphorylation. Neuropharmacology 53, 362–368.
Chen, B.S., Braud, S., Badger, J.D., 2nd, Isaac, J.T., and Roche, K.W. (2006).
Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phos-
phorylation. J. Biol. Chem. 281, 16583–16590.
Chung, H.J., Steinberg, J.P., Huganir, R.L., and Linden, D.J. (2003).
Requirement of AMPA receptor GluR2 phosphorylation for cerebellar long-
term depression. Science 300, 1751–1755.
Chung,H.J.,Huang,Y.H., Lau,L.F.,andHuganir, R.L.(2004).Regulation ofthe
NMDA receptor complex and trafficking by activity-dependent phosphoryla-
tion of the NR2B subunit PDZ ligand. J. Neurosci. 24, 10248–10259.
Clapp, P.,Gibson,E.S.,Dell’acqua, M.L., andHoffman,P.L.(2010).Phosphor-
ylation regulates removal of synaptic N-methyl-D-aspartate receptors after
withdrawal from chronic ethanol exposure. J. Pharmacol. Exp. Ther. 332,
Cohen, N.A., Brenman, J.E., Snyder, S.H., and Bredt, D.S. (1996). Binding of
the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase
A phosphorylation. Neuron 17, 759–767.
Cull-Candy, S.G., and Leszkiewicz, D.N. (2004). Role of distinct NMDA
receptor subtypes at central synapses. Sci. STKE 2004, re16.
Faust, M., and Montenarh, M. (2000). Subcellular localization of protein kinase
CK2. A key to its function? Cell Tissue Res. 301, 329–340.
Furukawa, H., Singh, S.K., Mancusso, R., and Gouaux, E. (2005). Subunit
arrangement and function in NMDA receptors. Nature 438, 185–192.
Girault, J.A., Hemmings, H.C., Jr., Zorn,S.H., Gustafson, E.L., and Greengard,
P. (1990). Characterization in mammalian brain of a DARPP-32 serine kinase
identical to casein kinase II. J. Neurochem. 55, 1772–1783.
Groc, L., and Choquet, D. (2006). AMPA and NMDA glutamate receptor
trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue
Res. 326, 423–438.
Groc, L., Bard, L., and Choquet, D. (2009). Surface trafficking of N-methyl-D-
aspartate receptors: physiological and pathological perspectives. Neurosci-
ence 158, 4–18.
Hallett, P.J., Collins, T.L., Standaert, D.G., and Dunah, A.W. (2008). Biochem-
ical fractionation of brain tissue for studies of receptor distribution and
trafficking. Curr Protoc Neurosci., Chapter 1, Unit 1.16.
Hemmings, B.A., Aitken, A., Cohen, P., Rymond, M., and Hofmann, F. (1982).
Phosphorylation of the type-II regulatory subunit of cyclic-AMP-dependent
protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase
5. Eur J Biochem. 127, 473–481.
Heriche, J.K., Lebrin, F., Rabilloud, T., Leroy, D., Chambaz, E.M., and
Goldberg, Y. (1997). Regulation of protein phosphatase 2A by direct interac-
tion with casein kinase 2alpha. Science 276, 952–955.
Hestrin, S. (1992). Developmental regulation of NMDA receptor-mediated
synaptic currents at a central synapse. Nature 357, 686–689.
Igaz, L.M., Vianna, M.R., Medina, J.H., and Izquierdo, I. (2002). Two time
periods of hippocampal mRNA synthesis are required for memory consolida-
tion of fear-motivated learning. J. Neurosci. 22, 6781–6789.
Jung, E.J., Kang, Y.S., and Kim, C.W. (1998). Multiple phosphorylation of
chicken protein tyrosine phosphatase 1 and human protein tyrosine phospha-
tase 1B by casein kinase II and p60c-src in vitro. Biochem. Biophys. Res.
Commun. 246, 238–242.
Kew, J.N., Richards, J.G., Mutel, V., and Kemp, J.A. (1998). Developmental
changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal
three distinct populations of NMDA receptors in individual rat cortical neurons.
J. Neurosci. 18, 1935–1943.
Kim, J.H., and Huganir, R.L. (1999). Organization and regulation of proteins at
synapses. Curr. Opin. Cell Biol. 11, 248–254.
Kim, E., and Sheng, M. (2004). PDZ domain proteins of synapses. Nat Rev
Neurosci. 5, 771–781.
Kimura, R., and Matsuki, N. (2008). Protein kinase CK2 modulates synaptic
plasticity by modification of synaptic NMDA receptors in the hippocampus.
J. Physiol. 586, 3195–3206.
Kohr, G. (2006). NMDA receptor function: subunit composition versus spatial
distribution. Cell Tissue Res. 326, 439–446.
Lau, C.G., and Zukin, R.S. (2007). NMDA receptor trafficking in synaptic
plasticity and neuropsychiatric disorders. Nat. Rev. Neurosci. 8, 413–426.
Lavezzari, G., McCallum, J., Lee, R., and Roche, K.W. (2003). Differential
binding of the AP-2 adaptor complex and PSD-95 to the C-terminus of the
NMDA receptor subunit NR2B regulates surface expression. Neuropharma-
cology 45, 729–737.
Lavezzari, G., McCallum, J., Dewey, C.M., and Roche, K.W. (2004). Subunit-
specific regulation of NMDA receptor endocytosis. J. Neurosci. 24, 6383–
Lim, I.A., Hall, D.D., and Hell, J.W. (2002). Selectivity and promiscuity of the
first and second PDZ domains of PSD-95 and synapse-associated protein
102. J. Biol. Chem. 277, 21697–21711.
Litchfield, D.W. (2003). Protein kinase CK2: structure, regulation and role in
cellular decisions of life and death. Biochem. J. 369, 1–15.
Litchfield, D.W., Dobrowolska, G., and Krebs, E.G. (1994). Regulation of
casein kinase II by growth factors: a reevaluation. Cell. Mol. Biol. Res. 40,
Martin, M.E., Alcazar, A., and Salinas, M. (1990). Subcellular and regional
distribution of casein kinase II and initiation factor 2 activities during rat brain
development. Int. J. Dev. Neurosci. 8, 47–54.
Meggio, F., and Pinna, L.A. (2003). One-thousand-and-one substrates of
protein kinase CK2? FASEB J. 17, 349–368.
Monyer, H., Burnashev, N., Laurie, D.J., Sakmann, B., and Seeburg, P.H.
(1994). Developmental and regional expression in the rat brain and functional
properties of four NMDA receptors. Neuron 12, 529–540.
PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in
spines of hippocampal neurons and reduces surface levels of the AMPA-
type glutamate receptor subunit 2. J. Neurosci. 21, 5417–5428.
Perez-Otano, I., and Ehlers, M.D. (2005). Homeostatic plasticity and NMDA
receptor trafficking. Trends Neurosci. 28, 229–238.
Petralia, R.S., Sans, N., Wang, Y.X., and Wenthold, R.J. (2005). Ontogeny of
postsynaptic density proteins at glutamatergic synapses. Mol. Cell. Neurosci.
Philpot, B.D., Sekhar, A.K., Shouval, H.Z., and Bear, M.F. (2001). Visual
experience and deprivation bidirectionally modify the composition and
function of NMDA receptors in visual cortex. Neuron 29, 157–169.
Pinna, L.A. (1990). Casein kinase 2: an ‘eminence grise’ in cellular regulation?
Biochim. Biophys. Acta 1054, 267–284.
Pinna, L.A., and Meggio, F. (1997). Protein kinase CK2 (‘‘casein kinase-2’’)
and its implication in cell division and proliferation. Prog. Cell Cycle Res. 3,
Prybylowski, K., Chang, K., Sans, N., Kan, L., Vicini, S., and Wenthold, R.J.
(2005). The synaptic localization of NR2B-containing NMDA receptors is
controlled by interactions with PDZ proteins and AP-2. Neuron 47, 845–857.
Quinlan, E.M., Olstein, D.H., and Bear, M.F. (1999). Bidirectional, experience-
dependent regulation of N-methyl-D-aspartate receptor subunit composition
in the rat visual cortex during postnatal development. Proc. Natl. Acad. Sci.
USA 96, 12876–12880.
Roche, K.W., and Huganir, R.L. (1995). Synaptic expression of the high-affinity
kainate receptor subunit KA2 in hippocampal cultures. Neuroscience 69,
Roche, K.W., Standley, S., McCallum, J., Dune Ly, C., Ehlers, M.D., and
Wenthold, R.J. (2001). Molecular determinants of NMDA receptor internaliza-
tion. Nat. Neurosci. 4, 794–802.
Regulation of Synaptic NMDARs by CK2
Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc. 995
Sarno, S., Reddy, H., Meggio, F., Ruzzene, M., Davies, S.P., Donella-Deana, Download full-text
A., Shugar, D., and Pinna, L.A. (2001). Selectivity of 4,5,6,7-tetrabromobenzo-
triazole, an ATP site-directed inhibitor of protein kinase CK2 (‘casein kinase-
2’). FEBS Lett. 496, 44–48.
Sarno, S., Ruzzene, M., Frascella, P., Pagano, M.A., Meggio, F., Zambon, A.,
Mazzorana,M., DiMaira,G.,Lucchini, V.,and Pinna,L.A. (2005). Development
and exploitation of CK2 inhibitors. Mol. Cell. Biochem. 274, 69–76.
Scimemi, A., Fine, A., Kullmann, D.M., and Rusakov, D.A. (2004). NR2B-
containing receptors mediate cross talk among hippocampal synapses.
J. Neurosci. 24, 4767–4777.
Seidenman, K.J., Steinberg, J.P., Huganir, R., and Malinow, R. (2003).
Glutamate receptor subunit 2 Serine 880 phosphorylation modulates synaptic
transmission and mediates plasticity in CA1 pyramidal cells. J. Neurosci. 23,
Snyder, E.M., Nong, Y., Almeida, C.G., Paul, S., Moran, T., Choi, E.Y., Nairn,
A.C., Salter, M.W., Lombroso, P.J., Gouras, G.K., and Greengard, P. (2005).
Regulation of NMDA receptor trafficking by amyloid-beta. Nat. Neurosci. 8,
Song, C., Zhang, Y., Parsons, C.G., and Liu, Y.F. (2003). Expression of
polyglutamine-expanded huntingtin induces tyrosine phosphorylation of
N-methyl-D-aspartate receptors. J. Biol. Chem. 278, 33364–33369.
Soto, D., Pancetti, F., Marengo, J.J., Sandoval, M., Sandoval, R., Orrego, F.,
and Wyneken, U. (2004). Protein kinase CK2 in postsynaptic densities:
phosphorylation of PSD-95/SAP90 and NMDA receptor regulation. Biochem.
Biophys. Res. Commun. 322, 542–550.
Suh, Y.H., Pelkey, K.A., Lavezzari, G., Roche, P.A., Huganir, R.L., McBain,
C.J., and Roche, K.W. (2008). Corequirement of PICK1 binding and PKC
phosphorylation for stable surface expression of the metabotropic glutamate
receptor mGluR7. Neuron 58, 736–748.
Tanemoto, M., Fujita, A., Higashi, K., and Kurachi, Y. (2002). PSD-95 mediates
formation of a functional homomeric Kir5.1 channel in the brain. Neuron 34,
with a distinct subunit composition at nascent hippocampal synapses in vitro.
J. Neurosci. 19, 4180–4188.
van Zundert, B., Yoshii, A., and Constantine-Paton, M. (2004). Receptor
compartmentalization and trafficking at glutamate synapses: a developmental
proposal. Trends Neurosci. 27, 428–437.
Wenzel, A., Fritschy, J.M., Mohler, H., and Benke, D. (1997). NMDA receptor
heterogeneity during postnatal development of the rat brain: differential
expression of the NR2A, NR2B, and NR2C subunit proteins. J. Neurochem.
Xu, J., Yeon, J.E., Chang, H., Tison, G., Chen, G.J., Wands, J., and de la
Monte, S. (2003). Ethanol impairs insulin-stimulated neuronal survival in the
developing brain: role of PTEN phosphatase. J. Biol. Chem. 278, 26929–
Zhang, S., Edelmann, L., Liu, J., Crandall, J.E., and Morabito, M.A. (2008).
Cdk5 regulates the phosphorylation of tyrosine 1472 NR2B and the surface
expression of NMDA receptors. J. Neurosci. 28, 415–424.
Zhao, Y., and Zhang, Z.Y. (1996). Reactivity of alcohols toward the phospho
enzyme intermediate in the protein-tyrosine phosphatase-catalyzed reaction:
probing the transition state of the dephosphorylation step. Biochemistry 35,
Regulation of Synaptic NMDARs by CK2
996 Neuron 67, 984–996, September 23, 2010 ª2010 Elsevier Inc.