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The Journal of Neuroscience, August 15, 2002, 22(16):7027-7044
Regulation of A-Kinase Anchoring Protein 79/150-cAMP-Dependent
Protein Kinase Postsynaptic Targeting by NMDA Receptor
Activation of Calcineurin and Remodeling of Dendritic Actin
Lisa. L.
Gomez1,
Shuvo
Alam1,
Karen E.
Smith1,
Eric
Horne1, and
Mark L.
Dell'Acqua1, 2
1 Department of Pharmacology, 2 Program in
Neuroscience, University of Colorado Health Sciences Center, Denver,
Colorado 80262
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ABSTRACT |
At the postsynaptic membrane of glutamatergic synapses, the
cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and calcineurin (CaN) anchoring protein AKAP79/150 is recruited to NMDA and
AMPA glutamate receptors by postsynaptic density (PSD)-95 family
membrane-associated guanylate kinase (MAGUK) scaffold proteins. These signaling scaffold complexes may function to regulate receptor phosphorylation in synaptic plasticity. Thus, it is important to
understand regulation of AKAP79/150 targeting to synapses and recruitment to PSD-MAGUK complexes. AKAP79 is targeted to the plasma
membrane by an N-terminal basic domain that binds
phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2)
and is regulated by PKC phosphorylation and calmodulin binding. Here we
demonstrate that this same domain also binds F-actin in a calmodulin-
and PKC-regulated manner, targets to membrane ruffles enriched in
F-actin and PI-4,5-P2 in COS7 cells, and localizes to
dendritic spines with F-actin and PSD-MAGUKs in hippocampal neurons.
Inhibition of actin polymerization disrupted AKAP79 targeting of
PKA and CaN to ruffles in COS7 cells and endogenous AKAP79/150
dendritic spine localization with PKA, CaN, and PSD-MAGUKs in neurons.
AKAP79/150 postsynaptic localization was rapidly regulated by NMDA
receptors through CaN activation and F-actin remodeling, further
suggesting that AKAP79/150 signaling scaffold targeting depends on
actin dynamics. NMDA receptor activation also regulated dendritic spine
localization of PKA and CaN and association of the AKAP79/150-PKA
complex with PSD-MAGUKs. Because AMPA receptor PKA phosphorylation and
synaptic localization are regulated by similar NMDA receptor-CaN
signaling pathways linked to hippocampal long-term depression,
this regulation of AKAP79/150 postsynaptic targeting might be important
for synaptic plasticity.
Key words:
postsynaptic density; actin; PSD-95 MAGUKs; AKAPs; PKA; calcineurin; NMDA and AMPA receptors
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INTRODUCTION |
NMDA and AMPA glutamate
receptors form complexes with cytoskeletal and scaffold proteins in the
postsynaptic density (PSD) (Kennedy, 1997
; Ziff, 1997). Prominent among
these scaffolds are PSD-95 family membrane-associated guanylate kinases
(PSD-MAGUKs) containing postsynaptic density-95, discs large, zona
occludens-1 (PDZ), Src homology-3 (SH3), and guanylate kinase (GK)
domains. Through binding MAGUKs and additional proteins, receptors are linked to intracellular signaling pathways and the actin cytoskeleton (Sheng and Scala, 2001). It is believed that regulation of this molecular architecture is essential for controlling glutamate receptors
in hippocampal long-term potentiation (LTP) and long-term depression
(LTD) synaptic plasticity (Luscher et al., 2000; Tomita et al.,
2001).
Recent studies have shown that AMPA receptors are rapidly recruited to
synapses by exocytosis during NMDA receptor-dependent LTP and rapidly
removed from synapses by endocytosis during LTD through pathways
involving PDZ scaffolds and F-actin (Carroll et al., 2001; Sheng and
Lee, 2001). AMPA receptor activity and phosphorylation are also
bi-directionally regulated during LTP and LTD by
calcium/calmodulin-dependent protein kinase II (CaMKII), cAMP-dependent
protein kinase (PKA), protein phosphatase 2B-calcineurin (CaN), and
protein phosphatase 1 (PP1) (Mulkey et al., 1994; Barria et al., 1997,
Lee et al., 1998, 2000; Morishita et al., 2001). Importantly, these
kinases and phosphatases also regulate receptor trafficking in
plasticity; for instance, synaptic delivery of AMPA receptors during
LTP requires CaMKII (Hayashi et al., 2000; Shi et al., 2001). In
addition, AMPA receptor endocytosis under LTD conditions requires CaN,
and receptor recycling requires PKA (Beattie et al., 2000; Ehlers,
2000).
Mechanisms regulating PKA and CaN localization that are likely to be
important for coordinating AMPA receptor phosphorylation and
trafficking have not been studied extensively. Human A-kinase anchoring
protein (AKAP) 79/rat AKAP150 (AKAP79/150) is a postsynaptic scaffold protein for PKA and CaN that may play an important role in
these processes (Bregman et al., 1989; Carr et al., 1992; Coghlan et
al., 1995). AKAP79/150 binds to PSD-95 and synapse-associated protein (SAP)97 MAGUKs in complexes with NMDA and AMPA
receptors, respectively (Colledge et al., 2000). AKAP-PKA anchoring
regulates AMPA-receptor activity in hippocampal neurons and GluR1
subunit phosphorylation through SAP97 in transfected cells (Rosenmund et al., 1994; Colledge et al., 2000; Tavalin et al., 2002). Thus, here
we seek to characterize AKAP79/150 targeting mechanisms that control
PKA and CaN signaling and association with PSD-MAGUKs.
AKAP79 is targeted to plasma membranes by three N-terminal basic
regions that bind acidic phospholipids, including
phosphatidy-linositol-4,5-bisphosphate (PI-4,5-P2) (Dell'Acqua et al., 1998). Each of
these basic regions is similar to the effector domain of myristoylated
alanine-rich C-kinase substrate (MARCKS) that binds membrane
PI-4,5-P2 and F-actin (Aderem, 1992; Tall et al.,
2000; Wang et al., 2001). Here we demonstrate that the AKAP79
N-terminal domain binds F-actin and targets to
PI-4,5-P2 and actin-rich membrane ruffles and
neuronal dendritic spines. We show that F-actin is required for
postsynaptic localization of AKAP79/150 with PKA, CaN, and PSD-MAGUKs.
Importantly, we demonstrate that association of the AKAP79/150-PKA
complex with PSD-MAGUKs is regulated by F-actin dynamics and NMDA
receptor-CaN signaling pathways that others have implicated in AMPA
receptor regulation during LTD.
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MATERIALS AND METHODS |
Primary culture and transfection of rat hippocampal
neurons. The hippocampus was dissected from brains of Sprague
Dawley neonatal rats (0-1 d) and dissociated by papain digestion.
Neurons were plated at low density (75-100,000 cells/ml) in MEM, 10%
FBS (Invitrogen, Rockville, MD) on poly-D-lysine,
laminin-coated glass coverslips and cocultured with glia previously
plated on a separate coverslip (in six-well plates) by the sandwich
culture method of Goslin et al. (1998) (see Fig. 1A).
After 1 d the media was replaced with Neurobasal supplemented with
B-27 (Invitrogen) and mitotic inhibitors [uridine+fluoro-deoxyuridine
(Ur+FdUr)]. The neurons were then fed with Neurobasal, B-27, Ur+FdUr
by replacing half the media on day 4 and then twice weekly for the
period of 1-4 weeks in culture. For the remaining experiments, neurons
were plated at medium-high density (200-300,000 cells/ml) in MEM, 10% FBS on coverslips in 12-well plates. After 1 d the media was
replaced with Neurobasal, B-27, Ur+FdUr. The neurons were then fed by
replacing half the media on day 4 and then weekly with feedings on days 11 and 18. Neurons were subjected to pharmacological treatments and
fixed for immunocytochemistry on days 16-20. For immunoblotting and
immunoprecipitation experiments, neurons were plated at high density
(300-400,000 cells/ml) on 6 cm Petri plates. For expression of
AKAP79-green fluorescent protein (GFP) fusion proteins (vectors described below), 12-14 d in vitro (DIV) neurons cultured
at medium-high density in 12-well plates were transfected by Helios
Gene Gun Biolistic methods (Bio-Rad, Hercules, CA) with ~1 µg cDNA
per well coated on 0.6-1.0 µm gold particles injected at 120-160
psi. Neurons were fixed and stained for immunofluorescence microscopy (described below) 36-48 hr after transfection.
COS7 cell culture and transfection. COS7 cells at 20-50%
confluency (24-48 hr after plating on glass coverslips in six-well plates) were transfected by calcium phosphate precipitation with the
appropriate cDNA expression constructs (1-2 µg each plasmid) for
4-6 hr at 5% CO2, 37°C. Cells were then
washed twice with PBS, fed with DMEM, 10% FBS, 1%
penicillin/streptomycin (Invitrogen), and grown for 24-48 hr before
fixation and immunochemical staining as described below. Construction
of pEGFPN1-AKAP79WT full-length and (1-153) basic targeting domain
vectors has been described previously (Dell'Acqua et al., 1998). The
AKAP79 (150-427) fragment lacking the basic targeting domains was
subcloned into PstI-BamHI-digested pEGFPC2
(Clontech, Palo Alto, CA) to express a GFP-(150-427) N-terminal fusion
protein in cells. The mouse PKA-RII
coding sequence was subcloned
from pET11-RII (Hausken et al., 1996) by PCR as a
HindIII-BamHI fragment into pEGFPN3 (Clontech).
pECFPN1-AKAP79 and (1-153) were made by
BamHI-NotI exchange of GFP with cyan fluorescent
protein (CFP). pEGFPN1-phospholipase C-
pleckstrin-homology
domain (PLC-PH) was provided by Ed Tall and Mario Rebecchi (State
University of New York, Stonybrook, NY) (Tall et al., 2000);
pEYFPN1-PLC-PH was then made by BamHI-NotI
exchange of GFP with yellow fluorescent protein (YFP).
Immunocytochemistry and digital fluorescence microscopy.
Cultured hippocampal neurons or COS7 cells on glass coverslips were washed in PBS, fixed in 3.7% formaldehyde/PBS (10 min), and
permeabilized with 0.2% Triton X-100 in PBS (10 min). Cells were then
washed with PBS and blocked in PBS + 10% BSA for 30-60 min. The
primary antibodies were incubated for 1-2 hr at room temperature in
PBS + BSA. Primary antibodies were used as follows: 1:500 rabbit
anti-AKAP150, 1:1000 rabbit anti-AKAP79 (Dr. Yvonne Lai, ICOS,
Bothel, WA); 1:100 mouse anti-PSD-95 (ABR, Golden, CO); 1:500 mouse
anti-synapsin (Chemicon, Temulca, CA); 1:100 mouse
anti-GABAA receptor (Chemicon); 1:200
anti-myc-9E10 (Santa Cruz Biotechnologies, Santa Cruz, CA); 1:100
rabbit anti-GluR1 (UBI, Lake Placid, NY); 1:200 mouse
pan-anti-PSD-MAGUK family (UBI); 1:200 mouse anti-PKA-RII
(Transduction Labs, Los Angeles, CA); 1:500 mouse anti-CaNB (UBI).
After incubation with primary antibodies the cells were washed
extensively in PBS + BSA and incubated for 1 hr with fluorescent
secondary antibody conjugates [goat anti-mouse- or goat
anti-rabbit-Texas Red 1:250, -FITC 1:500, or -Cy5 1:250 (Molecular
Probes, Eugene, OR, or Jackson ImmunoResearch, West Grove, PA) or Texas
Red-phalloidin 1:200 (Molecular Probes)]. Coverslips were then washed
in PBS and water and mounted on glass slides with Pro-long (Molecular
Probes). For the AKAP79-CFP/PLC-PH-YFP colocalization studies,
COS7 cells were placed in a chamber (Molecular Probes) for live cell
imaging. Specific indirect fluorescence and intrinsic GFP, CFP, or YFP fluorescence were detected with chroma filter sets using a Nikon TE-300
inverted microscope (100Xplan-apo, oil, 1.4 numerical aperture) equipped with Micromax or Sensicam digital CCD camera and Slidebook 3.0 software (Intelligent Imaging Innovations, Denver, CO). Images were
exported from Slidebook as TIFF files and assembled using Adobe
Photoshop 5.5.
Image quantitation was performed in Slidebook using Masks and
Segmentation. Briefly, for calculation of normalized mean fluorescence intensities for immunostaining, both red-Texas Red and green-FITC channels were segmented together to generate a total neuron mask (T)
including only areas of continuous pixels corresponding to the cell(s)
of interest with background removed. A separate mask was drawn for the
soma (S) and subtracted from the T mask to generate a dendrite (D)
mask. Mean fluorescence intensities were calculated from the D and S
masks for each channel and expressed as a ratio (%D/S) as well as
normalized to corresponding values for the T mask (%D/T or %S/T).
These normalized mean intensity ratios calculated from multiple images
are expressed as average percentages ± SEM in Results or in the
bar graphs in the figures. For AKAP79-GFP neuron expression studies,
separate masks were drawn manually for dendritic spine heads and
dendrite shafts in each image. Mean fluorescence intensities were then
calculated for these masks for each image to generate a normalized
ratio of spine/shaft GFP fluorescence. These normalized ratios for
multiple images were then averaged and are reported ± SEM in the
text. An average spine/shaft ratio of 1 was seen for untargeted GFP;
thus ratios >1 seen for the AKAP-GFPs reflect relative enrichment on
dendritic spines.
Dendritic punctate colocalization of immunostaining was measured by
generating segmented dendritic puncta masks showing discrete areas of
continuous pixels of fluorescence intensity >50% above the mean
dendritic fluorescence for AKAP150 and the marker of interest (i.e.,
PSD-95, F-actin, RII, CaN). Finally, a mask showing only areas of
punctate dendritic colocalization for AKAP150 and the marker was
generated using the "AND" function. Integrated intensity values
were then calculated separately for the marker from this AKAP150
colocalization mask and the marker-specific dendritic puncta mask. The
AKAP150 colocalization index for the marker was then generated by
dividing the integrated intensities. These integrated colocalization
indices (i.e., 150-RIIco,
150-CaNco, PSDco)
calculated from multiple images for each condition are expressed as
percentages ± SEM in Results and bar graphs in the figures. Note,
this method of quantitation, although more objective than manual
counting of numbers of puncta, actually underrepresents true
colocalization of puncta that are overlapping but have somewhat different sizes and shapes. Nonetheless, these colocalization indices
clearly give a measure of the reproducibility of the data (as shown by
small SEM values) and allow meaningful comparisons between different
treatment conditions. In all measurements described above, the impacts
of small variations in focus and volume in images are controlled for by
sampling large numbers of similar structures and the normalization
provided by using ratios.
Pharmacological treatments of cultured neurons and COS7
cells. Neurons or COS7 cells plated on coverslips (for
immunocytochemistry) or Petri plates (for biochemical analysis) were
incubated in normal growth media for all treatments. The various
treatments are described in detail in Results and in the figure
legends. Reagents were obtained from the following sources:
L-glutamate, NMDA, AP-V, S-AMPA, and CNQX
(Sigma/RBI, St. Louis, MO); latrunculin A and jasplakinolide (Molecular
Probes); cytochalasin D, H-89, bis-indomaleimide I,
chelerthyrine (CHE), KN-62, cyclosporin A (CsA), FK506, rapamycin, and
5,6-dichloro-l--ribofuranosylbenzimidazole (DRB) (Calbiochem, La Jolla, CA). Electrophoresis reagents were from Bio-Rad. Other general chemicals and reagents were obtained from Sigma or Fisher Scientific (Houston, TX).
F-actin binding assay. Recombinant AKAP79WT, (1-153),
and (154-427) were expressed as His6-tagged
fusion proteins [AKAP79 in pET16B; (1-153) and (154-427) in pET30a]
in Escherichia coli and purified by Ni-Agarose
chromatography as described previously (Dell'Acqua et al., 1998).
However, (1-153) was His-tagged in pET30 at the C terminus in the
current study, instead of the N terminus as in previous studies, to
remove the N-terminal T7 epitope tag that contains exogenous
phosphorylation sites. For actin-binding studies, N-His-AKAP79WT,
N-His(154-427), or (1-153)C-His was incubated with F-actin and then
assayed for binding by cosedimentation. Briefly, F-actin was
polymerized from purified G-actin (10 µM; Sigma) in vitro for 30 min in 125 mM
KCl, 2.5 mM MgCl2, 0.2 mM ATP, 2 mM Tris, pH 7.6 (F-Buffer). Actin (2 mg/ml) was stored at 4°C in the control G-buffer
(0.2 mM ATP, 0.2 mM
MgCl2, 2 mM Tris, pH 7.6).
F-actin filaments or unpolymerized G-actin was then incubated for 10 min with AKAP79WT (700 ng), (1-153) (250 ng), or (154-427) (500 ng)
followed by centrifugation (20 min, 100,000 × g). The
distributions of the AKAP fragments between the supernatant containing
G-actin (unbound fraction) and pellet containing F-actin (bound
fraction) were determined by immunoblotting with rabbit anti-AKAP79
918I, 1:2000 (ICOS), and these immunoblots are shown in Figures
3B and
5A,B,D.
Regulation of (1-153) F-actin binding by PKC phosphorylation and
CaM binding. For actin binding regulation studies, (1-153)C-His was unphosphorylated or PKC phosphorylated with unlabeled ATP as
described below or incubated 10 min with 1 µM
CaM (Calbiochem) with (+) or without (
)
Ca2+ (0.1 mM) or
EGTA (5 mM), incubated with F-actin, and then
assayed for actin binding by cosedimentation. For PKC phosphorylation order experiments, (1-153)C-His was either phosphorylated with PKC for
15 min as described above and then incubated with F-actin for 10 min or
incubated with F-actin for 10 min before the addition of an equivalent
amount of PKC for 15 min before centrifugation (note: F-buffer already
contains 0.2 mM ATP). For CaM-binding order
experiments, (1-153)C-His was either incubated with 1 µM CaM, 0.1 mM
Ca2+ for 10 min before incubation with
F-actin for 10 min or incubated with F-actin for 10 min before the
addition of 1 µM CaM, 0.1 mM Ca2+ for 10 min
before centrifugation.
In vitro phosphorylation of the (1-153) targeting
domain. Recombinant (1-153)C-His (1 µg) was PKC phosphorylated
essentially as described previously (Dell'Acqua et al., 1998) except
for use of a PKC constitutively active catalytic trypsin fragment
(PKC-M, 20 ng) (provided by Dr. Michael Browning, University of
Colorado Health Science Center). For direct visualization of PKC
phosphorylation of (1-153)C-His, 600 cpm/pmol
[
32P]ATP was included, and the
reaction was then analyzed by SDS-PAGE and imaged using a Molecular
Dynamics Storm Phosphor-imager. To look at competition between
Ca2+-CaM binding and PKC phosphorylation,
phosphorylation of (1-153) by PKC was done with or without 0.1 mM Ca2+, 5 mM
EGTA, and 1 µM CaM as indicated.
Immunoprecipitation of AKAP79/150 complexes from hippocampal
neuron cell extracts. Hippocampal neurons cultured at high density were left untreated, treated with 5 µM
latrunculin A for 4 hr, or treated with 50 µM
NMDA for 10 min followed by washing twice in PBS. The cells from two to
five (6 cm) dishes each for untreated control or treated conditions
were pooled and lysed in 1-2 ml of 4°C Triton lysis buffer (TLB)
(0.5% Triton X-100, 20 mM HEPES, pH 7.4, 0.1 M KCl, 30 mM NaPPi, 50 mM NaF, 1 mM EDTA, 1 mM EGTA, 2 µg/ml leupeptin/pepstatin, 1 mM benzamidine, 1 mM
4-(2-aminoethyl)benzenesulfonylfluoride) followed by dounce
homogenization to prepare whole-cell extracts. One hundred microliters
of these whole-cell extracts for both untreated control and treated
conditions were set aside. The remaining volumes of these extracts were
clarified by centrifugation (20,000 × g, 15 min,
4°C). The resulting supernatants were divided into two to four
separate tubes with equal protein contents, and then each tube was
incubated at 4°C overnight with 5 µg of rabbit anti-AKAP150 antibodies (ICOS). This incubation was followed by 1-2 hr
incubation with protein A-Sepharose (25 µl, 50% slurry equilibrated
in TLB; UBI). The immune complexes were then pelleted by
microcentrifugation (3000 × g, 1 min), and the beads were washed
in TLB 6 × 1 ml. The immune complexes were finally eluted with
SDS-sample buffer, separated by SDS-PAGE along with cell extracts
(12.5-25 µg), and analyzed by immunoblotting with rabbit
anti-AKAP150 (1:2000), mouse pan-anti-PSD-MAGUK family (1:1000, UBI),
or mouse anti-PKA-RII (1:2000; Transduction Labs).
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RESULTS |
Previous studies have demonstrated that AKAP79/150 is the major
AKAP in the PSD (Carr et al., 1992), coimmunoprecipitates with
PSD-MAGUKs, and binds directly to both SAP97 and PSD-95 MAGUKs in vitro (Colledge et al., 2000). Association of AKAP79 with
MAGUKs in vitro involves direct binding between an unmapped
central AKAP domain and the MAGUK SH3 and GK domains (Colledge et al.,
2000). However, proper membrane localization of these scaffolds clearly involves additional targeting determinants such as palmitoylation sites
in PSD-95 and N-terminal basic regions in AKAP79 (Lue et al., 1994,
1996; Li et al., 1996; Dell'Acqua et al., 1998; Wu et al., 1998;
Craven and Bredt, 2000; El-Husseini et al., 2000a). Consistent with
these independent targeting mechanisms, although AKAP79/150 shows
extensive punctate colocalization with PSD-95 on dendritic spines in
hippocampal neurons, it is also more widely distributed along dendrite
spine and shaft membranes (Fig. 1). Thus,
the ability of AKAP79/150 to form scaffolding complexes with
PSD-MAGUKs at synapses in vivo is likely to depend first on
the primary mechanisms that independently target the AKAP to plasma
membranes and dendritic spines. We have previously identified three
N-terminal basic regions that bind to PI-4,5-P2
and are necessary and sufficient for targeting GFP to the plasma
membrane in human embryonic kidney (HEK)-293 cells and cortical neurons (Dell'Acqua et al., 1998). Each of these basic regions is similar to
the basic effector domain of MARCKS that binds both membrane PI-4,5-P2 and cortical F-actin (Aderem, 1992;
Bubb et al., 1999; Wang et al., 2001); however, it is not known whether
the AKAP79 targeting domain also binds F-actin. Although F-actin is not
necessary for AKAP79 general membrane targeting in HEK-293 cells, it is quite possible that interactions with F-actin could further direct membrane-localized AKAP to more specific structures such as dendritic spines (Li et al., 1996; Dell'Acqua et al., 1998). To evaluate this
possibility, we conducted a developmental analysis of AKAP79/150 localization in low-density hippocampal neuron cultures across time
periods when synapses first form and then mature into spiny actin-rich
synapses.
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Figure 1.
Postsynaptic localization of
AKAP79/150 at excitatory synapses in hippocampal neurons.
A, Developmental regulation of AKAP79/150 colocalization
with the MAGUK PSD-95. Rat neonatal hippocampal neurons cultured at low
density for the indicated number of days were stained for AKAP150
(red) and the postsynaptic density MAGUK PSD-95
(green). The small panels are
magnifications of dendrites. B, Punctate colocalization
of AKAP79/150 with PSD-95 on dendritic spines: proximity of AKAP79/150
puncta to presynaptic terminals stained for synapsin but not inhibitory
postsynaptic elements stained for GABAA receptors. Neurons
(2-3 weeks old) cultured at medium-high density were stained with
anti-rat AKAP150 (red) and anti-PSD-95, anti-synapsin,
or anti-GABAA receptor (all in green).
Codistribution is seen as yellow-orange in composite
images. The red and green
arrowheads point to puncta for AKAP150
and PSD-95, respectively, that are not colocalized even in "mature"
dendrites. The images shown are representative of multiple neurons
imaged in more than three experiments for each condition.
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Postsynaptic localization of AKAP79/150 on dendritic spines in
hippocampal neurons
In neurons cultured for 8 DIV, AKAP79/150 showed nonuniform
membrane localization in the soma and dendrites, including fine filopodial structures (Fig. 1A). However, at 8 DIV
there was very little codistribution of AKAP79/150, with puncta for
PSD-95 located primarily on dendritic shafts. By 16 DIV there was much
more extensive colocalization of AKAP79/150 and PSD-95 puncta (Fig.
1A). This somatodendritic colocalization of
AKAP79/150 and PSD-95 developed more during the third week in culture,
and by 22-26 DIV most PSD-95 puncta overlapped with AKAP79/150 puncta,
especially on dendritic spines (Fig. 1A). However,
even at 22-26 DIV there were still sites of AKAP79/150 staining that
did not stain positive for PSD-95 and vice versa (Fig.
1A; see arrowheads on
magnification composite panels). Similar results were obtained in
medium-high-density neuron cultures that form more numerous synapses
and mature more rapidly than low-density cultures. Neurons cultured at
medium-high density for 2-3 weeks showed extensive but still not
complete dendritic colocalization of AKAP79/150 with PSD-95 (Fig.
1B, arrowheads). Costaining of
neurons for AKAP79/150 and synapsin reveals that most of the AKAP79/150
puncta seen in these dendrites are closely opposed to presynaptic
terminals (Fig. 1B). Specificity in this localization
of AKAP79/150 to excitatory over inhibitory synapses was seen in very
little overlap between dendritic AKAP79/150 and GABAA receptor puncta (Fig.
1B).
Postsynaptic localization of AKAP79/150 depends on the dendritic
actin cytoskeleton
This developmental progression of AKAP79/150 colocalization with
PSD-95 closely parallels time courses elucidated by others for the
formation of F-actin-rich dendritic spines and recruitment of glutamate
receptors and PSD-95 to spiny synapses (Rao et al., 1998; Pickard et
al., 2000; Okabe et al., 2001; Zhang and Benson, 2001).
Importantly, postsynaptic localization of AMPA receptors depends on
F-actin and is disrupted by the actin polymerization inhibitor
latrunculin A (Allison et al., 1998, 2000; Shen et al., 2000; Zhou et
al., 2001). In agreement with previous studies on AMPA receptors and as
a positive control, treatment with latrunculin A caused decreased
dendritic colocalization of GluR1-AMPA receptors with PSD-95 (Fig.
2A). Consistent with possible targeting
of AKAP79/150 to postsynaptic actin, in untreated neurons AKAP79/150
exhibited colocalization with phalloidin-stained F-actin and PSD-95 on
dendritic spines (Fig. 2B,C). After
latrunculin treatment, AKAP79/150 assumed a more diffuse pattern in
dendrites, with very little F-actin colocalization (Figs.
2B,C). Importantly, treatment with
latrunculin also disrupted AKAP79/150 codistribution with PSD-95 (Figs.
2B,C), which remained punctate, as
seen in previous studies (Allison et al., 1998, 2000). These effects of
latrunculin on AKAP79/150 colocalization with F-actin and PSD-95 on
dendritic spines were reversible after removal of the drug and a
recovery period to allow repolymerization of spine actin (Fig.
2B). These findings indicate that, like AMPA
receptors, the dendritic F-actin cytoskeleton is important for
maintaining postsynaptic localization of AKAP79/150.
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Figure 2.
AKAP79/150 colocalization with F-actin,
PSD-95, PKA, and calcineurin on dendritic spines depends on the actin
cytoskeleton: disruption with latrunculin A. A, AMPA
receptor GluR1 (red) dendritic colocalization with
PSD-95 (green) is disrupted by pretreatment with
latrunculin A (+LtrA; 5 µM, 2 hr).
B, Dendritic spine colocalization in hippocampal neurons
(seen as white in Composite panels) of
AKAP79/150 (red) with F-actin
(green) and PSD-95 (blue) is
disrupted by latrunculin A treatment (2 µM; 2 hr) and is
reversible after drug washout and recovery (16 hr). C,
Dendritic spine colocalization of AKAP79/150 (red) with
CaN and PKA as well as PSD-95 and F-actin (markers; all in
green) is disrupted by latrunculin A (5 µM, 2 hr). Magnifications of dendrites are shown. See
Results for quantitation.
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Quantitation of neurons from multiple experiments (n = 3-5) revealed AKAP150-integrated colocalization indices (see Materials and Methods) of 65 ± 9% for F-actin and 66 ± 3% for
PSD-95 (PSDco) in untreated neurons. After
latrunculin treatment, these indices fell to 22 ± 6 and 19 ± 6% for F-actin and PSDco, respectively. This
loss of AKAP79/150 from the PSD might be expected to result in a
corresponding change in localization of PKA and CaN anchored to it. In
control neurons, both PKA-RII and CaNB staining showed extensive
punctate codistribution with AKAP79/150 on dendritic spines with
colocalization indices of 150-RIIco = 73 ± 4% and 150-CaNco = 68 ± 3%, respectively
(Fig. 2D) (n = 3-5). However, after
treatment of neurons with latrunculin, punctate codistribution of PKA
and CaN with AKAP79/150 was lost, with all three proteins exhibiting
primarily diffuse dendritic localization (Fig. 2D). Without numerous AKAP150, PKA-RII, or CaNB puncta to quantitate after latrunculin treatment, accurate colocalization indices could not
be calculated. However, calculation of normalized mean fluorescence values (see Materials and Methods; D/T ratio) revealed corresponding 17-21% decreases in overall dendritic localization caused by
latrunculin treatment for AKAP150 [81 ± 3% Control; 63 ± 3% latrunculin A (LtrA)], PKA-RII (70 ± 3% Control; 53 ± 2% LtrA), and CaNB (76 ± 4% Control; 55 ± 3%
LtrA), suggesting that a significant amount of PKA and CaN might be
moving as a unit with the AKAP. Thus, the actin cytoskeleton is
necessary for maintaining proper dendritic localization of multiple
components of the AKAP79/150 scaffold.
The AKAP79 N-terminal basic domain binds F-actin and mediates
targeting to the cortical membrane cytoskeleton in COS7 cells
In light of the results of these latrunculin studies and the
similarities of the AKAP N-terminal basic domains with the MARCKS effector domain, we assayed purified recombinant AKAP79WT, an N-terminal targeting domain (1-153) fragment, and a C-terminal (154-427) control fragment (Fig.
3A) for direct binding to
F-actin using cosedimentation methods. We observed direct binding of
AKAP79 to F-actin as seen in cosedimentation of AKAP79 in the F- but not G-actin pellet fractions (Fig. 3B). An additional
control for nonspecific aggregation is that AKAP79 did not sediment in the absence of actin in either G- or F-buffer conditions. Importantly, a recombinant N-terminal (1-153) fragment corresponding to the previously mapped basic membrane targeting and
PI-4,5-P2 binding domain also exhibited specific
cosedimentation with F-actin (Fig. 3B). In contrast, a
C-terminal (154-427) control fragment lacking the basic domains but
containing the CaN and PKA anchoring domains (Fig. 3A) did
not cosediment with F-actin (Fig. 3B).
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Figure 3.
The AKAP79 N-terminal basic domain binds F-actin
in vitro and localizes with PI-4,5-P2 and
cortical F-Actin in COS7 cells: targeting of PKA and CaN to membrane
ruffles. A, Diagram showing the structures of
AKAP79WT(1-427), (1-153), and (154-427) proteins used for
actin-binding studies. Locations of the three (A,
B, C) basic membrane
targeting/phospholipid binding regions and the CaNA and PKA-RII
anchoring domains are indicated. B, AKAP79WT and an
N-terminal (1-153) fragment but not a C-terminal (154-427) fragment
bind to F-actin in vitro. Purified full-length AKAP79WT,
(1-153), or (154-427) fragments were incubated with (+) or
without () purified actin in buffers favoring monomeric G-actin
(G) or F-actin polymerization (F).
Binding to polymerized F-actin was detected as sedimentation of AKAP79
immunoreactivity in pellet fractions (P) after
centrifugation. The data shown are representative of results obtained
in multiple (more than three) independent experiments. C,
AKAP79-GFP colocalizes with cortical/membrane F-actin in COS7 cells.
In control untreated COS7 cells, overlap of AKAP79-GFP
(green)
and F-actin
(TxRd-Phalloidin; red) in plasma membrane
ruffles is seen as yellow-orange in the composite image.
Treatment of AKAP79-GFP-transfected COS7 cells with the actin
polymerization inhibitor cytochalasin D (5 µM,
4-5 hr) (+CHD) disrupts both the actin cytoskeleton and
AKAP79-GFP/F-actin colocalization in membrane ruffles. D,
The AKAP79(1-153)-GFP (green) N-terminal targeting
domain also colocalizes with cortical F-actin (red) ruffles
in a cytochalasin D (+CHD)-sensitive manner. E,
The GFP-AKAP79(150-427) (green) C-terminal fragment
containing CaNA and PKA anchoring domains but lacking the basic
targeting domains is localized to the cytoplasm and not colocalized
with cortical F-actin (red) in COS7 cells. PKA-RII-GFP
(F, green) and mycCaNA (anti-myc; G,
red) are found in the cytoplasm when expressed alone in COS7
cells. H, Coexpression of AKAP79 (anti-79, red)
targets PKA-RII-GFP (green) to plasma membrane
ruffles in a cytochalasin D (+CHD)-sensitive manner.
I, Coexpression of AKAP79-GFP (green)
targets mycCaNA (anti-myc; red) to plasma membrane ruffles
in a cytochalasin D (+CHD)-sensitive manner. J,
Colocalization (blue-green) of AKAP79-CFP (blue)
with PI-4,5-P2 detected by PLC-PH-YFP
(green) in membrane ruffles of living COS7 cells.
K, Colocalization (blue-green) of the
AKAP79(1-153)-CFP N-terminal targeting domain (blue) with
PI-4,5-P2 detected by PLC-PH-YFP
(green) in membrane ruffles of living COS7 cells. The
images shown in C-K are representative of
multiple cells imaged in three independent experiments for each
condition. TxRd, Texas Red.
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We next sought to determine whether this N-terminal basic domain that
binds F-actin in vitro might also be sufficient for targeting to the membrane actin cytoskeleton in a model cell system. We
transfected COS7 cells with full-length AKAP79 tagged with GFP (Tsien,
1998) and then costained for F-actin with phalloidin (Fig.
3C). In control cells, AKAP79-GFP exhibited plasma membrane localization characterized by prominent enrichment with F-actin in
membrane ruffles but not cytoplasmic stress fibers (Fig.
3C). After disruption of F-actin with the polymerization
inhibitor cytochalasin D, AKAP79-GFP colocalization with F-actin was
eliminated, and the AKAP assumed a diffuse plasma membrane localization
with significant accumulation in intracellular membrane structures and
aggregates (Fig. 3C). Importantly, an N-terminal
(1-153)-GFP protein also displayed specific localization to F-actin
ruffles, and this localization was sensitive to disruption with
cytochalasin D (Fig. 3D). In contrast, a GFP-(150-427)
control fusion protein lacking the N-terminal basic domains was
cytoplasmic and showed no localization with membranes or cortical actin
in COS7 cells (Fig. 3E). Thus, the N-terminal basic regions
of AKAP79 appear to be necessary and sufficient for targeting to
cortical/membrane actin structures in cells.
Our findings above suggest that inhibition of F-actin polymerization in
neurons may disrupt dendritic spine localization of PKA and CaN through
mislocalization of AKAP79/150. In support of this model, PKA-RII-GFP
and mycCaNA subunits are cytoplasmic when expressed alone in COS7 cells
(Figs. 3F,G) but are targeted to
the membrane and enriched in ruffles when coexpressed with AKAP79 (Fig.
3H,I). Importantly,
disruption of F-actin with cytochalasin D caused loss of membrane
ruffle localization of PKA or CaN with AKAP79, diffuse plasma membrane
localization, and the appearance of these proteins in intracellular
structures (Figs. 3H,I).
Thus, disrupting postsynaptic targeting of AKAP79/150, the predominant AKAP in the PSD, is likely to also alter dendritic spine localization of PKA and CaN in neurons as seen in Figure 2. In addition, from the
results of all of these COS7 studies, it seems reasonable that the
diffuse dendritic localizations seen for AKAP79/150, PKA, and CaN in
latrunculin-treated neurons is the product of both lateral membrane
diffusion away from synapses as well as some redistribution to
intracellular membranes.
Our previous studies showed that the AKAP79 basic targeting domains,
like the MARCKS effector domain, could bind through nonspecific electrostatic interactions to membranes containing acidic phospholipids such as PI-4,5-P2. Interestingly, cellular
imaging studies using PLC-PH, which binds with high affinity and
specificity to PI-4,5-P2, have shown enrichment
of PI-4,5-P2 with actin in plasma membrane ruffles (Tall et al., 2000). Thus, we wanted to examine whether the
AKAP79 N-terminal basic domain might also colocalize with PI-4,5-P2 in membrane ruffles. To make this
inquiry possible, we generated AKAP79 full length and (1-153) tagged
with cyan-CFP and the PLC-PH domain tagged with yellow-YFP for
simultaneous imaging in live cells. Coexpression of either AKAP79-CFP
(Fig. 3J) or (1-153)-CFP (Fig.
3K) with PLC-PH-YFP revealed extensive codistribution of CFP and YFP fluorescence in membrane ruffles of
living COS7 cells. Some intracellular vesicular membrane localization of AKAP79 and (1-153)-CFP that was independent of PH-PLC-YFP was
also seen (Fig. 3J,K).
However, the extensive overlap of AKAP79(1-153) with PLC-PH in
ruffles clearly shows significant enrichment of the AKAP targeting
domain and PI-4,5-P2 in the same plasma membrane structures. These findings suggest that multiple electrostatic interactions of the three AKAP79 N-terminal basic regions, like the
MARCKS basic domain, may allow the AKAP to link plasma membrane PI-4,5-P2 to cortical actin in ruffles.
The AKAP79 N-terminal basic domain mediates targeting to dendritic
spines in hippocampal neurons
We next wanted to establish that this same N-terminal basic region
that targeted to cortical actin and PI-4,5-P2
membrane structures in COS7 cells was also involved in targeting AKAP79 to dendritic spines in neurons. Previously, we demonstrated that this
region could target to somatodendritic plasma membranes in microinjected cortical neurons; however, specific colocalization with
F-actin or PSD-MAGUKs at synapses was not addressed (Dell'Acqua et
al., 1998). Expression of either full-length AKAP79-GFP or (1-153)-GFP containing just the N-terminal basic domain resulted in
targeting of GFP to plasma membrane sites in both the soma and
dendrites of transfected neurons (Fig.
4A). In contrast, GFP alone was clearly cytoplasmic and diffusely distributed throughout the
soma and dendrites (Fig. 4A). Dendritic localization
of both AKAP79 and (1-153) was characterized by punctate enrichment on dendritic spines (Fig. 4A), where colocalization with
F-actin or PSD-95 could be seen (Fig. 4B). Enrichment
on dendritic spines was quantitated by 30-50% higher average
spine/shaft normalized mean fluorescence ratios for AKAP79 (1.55 ± 0.1; n = 10) and (1-153)-GFP (1.33 ± 0.03;
n = 13) relative to GFP alone (1.05 ± 0.06;
n = 11), which showed equal distribution between
dendritic spines and shafts. These findings suggest that the N-terminal
basic domain is sufficient for targeting AKAP79 to dendritic spines in
hippocampal neurons.
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Figure 4.
The AKAP79 N-terminal basic domain
targets to dendritic spines in hippocampal neurons. A,
Targeting of AKAP79-GFP and the N-terminal basic domain (1-153)-GFP
but not GFP alone (all in green) to dendritic spines in
transfected neurons. B, Colocalization of AKAP79-GFP
and (1-153)-GFP (both green) with F-actin and PSD-95
(both red) on dendritic spines. See Results and
Materials and Methods for quantitation and details.
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Regulation of AKAP79 targeting domain binding to F-actin by
phosphorylation and calmodulin binding
The three basic regions of the AKAP79 targeting domain contain
serine/threonine phosphorylation sites and bind
Ca2+-CaM (Faux and Scott, 1997;
Dell'Acqua et al., 1998). We have shown previously that in
vitro binding of the AKAP79(1-153) targeting domain to
PI-4,5-P2 is regulated by PKC phosphorylation and
Ca2+-CaM binding (Dell'Acqua et al.,
1998). Thus, it is likely that binding of the AKAP79 targeting domain
to F-actin is also regulated by PKC and CaM. Consistent with these
expectations, preincubation of AKAP79(1-153) with CaM in the presence
of Ca2+ inhibited binding to F-actin in
the cosedimentation assay (Fig. 5A). This inhibitory effect of
CaM was Ca2+ dependent and not observed in
the absence of Ca2+ plus the chelator
EGTA. Also in agreement with earlier work, previous phosphorylation of
(1-153) with PKC (readily seen in electrophoretic mobility shifts)
also inhibited F-actin cosedimentation (Fig. 5B). For the
MARCKS protein, PKC phosphorylation of the effector domain and
Ca2+-CaM binding, both of which regulate
targeting function, are competitive with one another (Aderem, 1992). To
evaluate this possibility for AKAP79, we phosphorylated (1-153) with
PKC in the presence of Ca2+-CaM. PKC
phosphorylation was significantly inhibited by
Ca2+-CaM but not CaM and EGTA (Fig.
5C). These findings suggest that CaM binding to the AKAP
basic targeting domain blocks access to sites phosphorylated by PKC.
Thus, in cells it is possible that phosphorylation, multiple CaM
binding events, or some combination of both processes acting through
the three basic regions may regulate AKAP79 membrane/cytoskeletal
interactions in response to Ca2+
signals.
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Figure 5.
Regulation of AKAP79 targeting domain F-actin
binding by phosphorylation and Ca2+-calmodulin
binding. A, Regulation of AKAP79(1-153) F-actin binding
by Ca2+-CaM. AKAP79(1-153) was incubated for 10 min as indicated with (+) or without () CaM (1 µM), Ca2+ (100 µM), or EGTA (5 mM) before assaying for
F-actin binding by cosedimentation. B, Regulation of
AKAP79(1-153) F-actin binding by PKC phosphorylation. AKAP79(1-153)
was PKC phosphorylated before assaying for F-actin binding by
cosedimentation. Control incubations were incubated in the absence of
kinase. C, Competition between
Ca2+-CaM binding and PKC phosphorylation
of the AKAP79(1-153) targeting domain. AKAP79(1-153) was
phosphorylated with [32P]ATP by PKC
(Control), with
Ca2+-CaM or CaM and EGTA present.
D, Previous incubation with F-actin inhibits regulation of
AKAP79(1-153) F-actin binding by PKC phosphorylation and
Ca2+-CaM binding. In the top
panel, AKAP79(1-153) was either phosphorylated by PKC before
incubation with F-actin (Actin 2nd; as in B) or
incubated first with F-actin and then with PKC (Actin 1st)
followed by assaying for F-actin cosedimentation. In the bottom
panel, the AKAP79(1-153) targeting domain fragment either was
incubated with Ca2+-CaM before incubation
with F-actin (Actin 2nd; as in A) or was
incubated first with F-actin and then with
Ca2+-CaM (Actin 1st) followed
by assaying for F-actin cosedimentation. The data shown are
representative of results obtained in at least three independent
experiments. A, B, and D are
immunoblots (IB:); C is an autoradiogram
(P-32).
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F-actin competes with phosphorylation and calmodulin binding in
regulation of the AKAP79 targeting domain
Because F-actin binding, phosphorylation, and CaM binding all
converge on the same three basic regions, it is possible that when the
targeting domain is bound to cortical actin in cells the basic regions
may not be readily accessible for phosphorylation or CaM binding. Such
a scenario would explain why in previous studies activation of PKC or
CaM released very little AKAP79 from HEK-293 membrane fractions
(Dell'Acqua et al., 1998). To test this idea biochemically, we varied
the order of incubations of the (1-153) targeting domain with PKC or
Ca2+-CaM and F-actin. In agreement with
results presented above, under control conditions most of (1-153)
sedimented with F-actin, and phosphorylation of (1-153) with PKC or
incubation with Ca2-CaM before incubation
with F-actin strongly inhibited this sedimentation (Fig.
5D). In contrast, previous incubation of (1-153) with
F-actin significantly attenuated the ability of PKC or
Ca2+-CaM to then release the targeting
domain from F-actin, as seen in nearly equal distributions between the
supernatants and pellets (Fig. 5D). Overall, these findings
help confirm that the same basic regions that mediate membrane
targeting and PI-4,5-P2 binding also regulate
interactions with F-actin.
Glutamate regulation of dendritic F-actin and AKAP79/150
postsynaptic targeting
On the basis of observations that AKAP79 targeting may be subject
to regulation by Ca2+ signals as well as
actin polymerization, we wanted to see whether postsynaptic
localization of AKAP79/150 could be regulated by neuronal signaling
pathways that elevate Ca2+ and reorganize
the dendritic actin cytoskeleton. We treated cultured neurons with 50 µM glutamate for 10 min and then fixed and stained to
visualize localization of AKAP79/150 relative to F-actin, PSD-MAGUKs (including both PSD-95 and SAP97; pan-PSD-MAGUK antibody), and synapsin (Fig. 6). Similar glutamate
treatments of hippocampal neurons have been shown to stimulate rapid
AMPA receptor endocytosis and reorganization of dendritic F-actin
(Halpain et al., 1998; Carroll et al., 1999b; Lissin et al., 1999;
Beattie et al., 2000; Zhou et al., 2001). Consistent with earlier
studies, exposure to glutamate led to a reorganization of F-actin in
the dendrites manifested by loss of F-actin puncta on dendritic spines
and increased actin filament staining along dendrite shafts (Fig.
6A) (Halpain et al., 1998). Also in agreement with
previous work, treatment with glutamate did not drastically alter
punctate distribution of the postsynaptic MAGUKs (Fig.
6B) or presynaptic terminals marked by synapsin (Fig.
6C) (Halpain et al., 1998). Glutamate treatment also caused
a dramatic redistribution of AKAP79/150 seen as loss of
somatodendritic puncta, weak and diffuse staining in dendrite shafts,
and increased fluorescence in the cytoplasm of the soma (Fig.
6A). This shift in localization of AKAP79/150 from dendrites to the soma was reflected in decreased dendritic fluorescence (D/T = 89 ± 2% control, n = 6;
D/T = 56 ± 4% glutamate, n = 5) and
increased somatic fluorescence (S/T = 131 ± 6% control; S/T = 175 ± 7% glutamate) that can be expressed together as
a decreased ratio of mean dendritic to somatic fluorescence (D/S = 69 ± 4% control; D/S = 32 ± 2% glutamate).
Importantly, this glutamate-stimulated redistribution of
AKAP79/150 resulted in loss of colocalization with PSD-MAGUKs (Fig.
6B) (see below) and synapsin (Fig.
6C).
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Figure 6.
Glutamate regulation of AKAP79/150
postsynaptic localization and dendritic spine F-actin in hippocampal
neurons. A, Redistribution of AKAP79/150 and
reorganization of dendritic F-actin in response to glutamate in
hippocampal neurons. B, Loss of AKAP79/150
colocalization with postsynaptic PSD-95 family MAGUK scaffolds after
glutamate treatment. C, Glutamate regulation of
AKAP79/150 localization near presynaptic terminals marked by synapsin.
Neurons were treated for 10 min with (+) or without () glutamate (50 µM) before staining for AKAP150 (red),
F-actin-phalloidin, PSD-95 family members (including PSD-95 and
SAP97), or synapsin (all in green). The images shown are
representative of neurons imaged in multiple (>3) experiments for each
condition.
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Regulation of AKAP79/150 postsynaptic localization by NMDA
receptor activation
Both reorganization of dendritic spine F-actin and endocytosis of
AMPA receptors can be triggered by activation of either NMDA or AMPA
receptors alone (Halpain et al., 1998; Carroll et al., 1999b; Beattie
et al., 2000; Lin et al., 2000; Man et al., 2000; Zhou et al., 2001).
However, for regulation of some important forms of hippocampal LTD,
Ca2+ influx through NMDA receptors is
thought to be the key signaling event. In agreement with previous
studies and as a positive control for NMDA receptor regulation of AMPA
receptors, NMDA treatment clearly disrupted dendritic colocalization of
GluR1 with PSD-95 (Fig. 7A).
As shown above, in untreated neurons, AKAP79/150 showed significant
codistribution with PSD-95 (PSDco = 64 ± 3%; n = 6), and treatment with glutamate led to rapid
redistribution of AKAP79/150 from dendrites (D/S = 32 ± 2%)
manifested in loss of dendritic colocalization with PSD-95
(PSDco = 8 ± 3%; n = 5)
(Fig. 7B,C). Brief preincubation of
neurons in the presence of EGTA to chelate extracellular
Ca2+ mostly prevented glutamate-stimulated
redistribution of AKAP79/150 from dendrites (D/S = 69 ± 4%)
and maintained colocalization with PSD-95 (PSDco = 56 ± 5%; n = 5) (Fig. 7C).
Consistent with a predominant role for
Ca2+ influx through NMDA receptors in this
response, glutamate-stimulated redistribution of AKAP79/150 was blocked
by the NMDA receptor antagonist AP-V (100 µM;
D/S = 65 ± 6%; PSDco = 62 ± 4%; n = 6), with no obvious additional effect seen
with addition of the AMPA receptor antagonist CNQX (50 µM; D/S = 63 ± 2%;
PSDco = 67 ± 8%; n = 3)
(Fig. 7B,C). Furthermore, selective
activation of NMDA receptors with 50 µM NMDA
was sufficient to cause redistribution of AKAP79/150 away from
postsynaptic specializations (D/S = 40 ± 4%;
PSDco = 7 ± 2%; n = 7) in
an AP-V-sensitive manner (D/S = 64 ± 4%;
PSDco = 58 ± 3%; n = 12)
(Fig. 7B,C). Thus, our conclusion is that NMDA receptor activation is both necessary and sufficient for
activation of Ca2+-dependent signaling
pathways regulating localization of AKAP79/150 to postsynaptic
sites.
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Figure 7.
Regulation of AKAP79/150 postsynaptic localization
by NMDA receptor activation. A, Loss of GluR1
AMPA-receptor (red) dendritic colocalization with PSD-95
(green) in NMDA-treated neurons.
B, Loss of AKAP79/150 (red)
colocalization with PSD-95 (green) in glutamate
or NMDA-treated neurons. Neurons were treated for 10 min with glutamate
(50 µM) or NMDA (50 µM). Glutamate
regulation of AKAP79/150 targeting was blocked by the NMDAR antagonist
AP-V (100 µM, 15 min pretreatment). Large
panels show AKAP150 and PSD-95 localization
separately in both dendrites and soma. The Magnification
panels show AKAP150, PSD-95, and Composite
images for localization in single dendrites to show details better.
C, Quantitation of AKAP79/150 mean fluorescence
intensity distributions between dendrites and somata
(%D/S, red) and integrated
colocalization with PSD-95 (PSDco,
blue) from multiple experiments for the treatment
conditions in B as well additional conditions described
below. See Results and Materials and Methods for additional details.
Glutamate regulation of AKAP79/150 targeting was also blocked by
chelation of extracellular Ca2+ with EGTA (5 mM). In the presence of glutamate and AP-V, no
additional effect of the AMPAR antagonist CNQX (50 µM, 15 min pretreatment) was seen. Selective
activation of AMPA receptors with 50 µM AMPA (in the
presence of 100 µM AP-V), although having some
effect on AKAP79/150 postsynaptic localization, did not result in a
dramatic redistribution as seen with NMDA or glutamate (data not
shown). NMDA regulation of AKAP79/150 targeting was also blocked by the
NMDAR antagonist AP-V (100 µM, 15 min
pretreatment).
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Activation of the phosphatase calcineurin but not protein kinases
is necessary for NMDA receptor regulation of AKAP79/150 targeting
One potentially important target of
Ca2+ influx that has been implicated in
NMDA receptor regulation of dendritic spine F-actin and AMPA receptor
localization is the Ca2+-activated
phosphatase CaN-PP2B (Halpain et al., 1998; Beattie et al., 2000;
Ehlers, 2000). It is possible that CaN stimulation of dendritic spine
F-actin remodeling could have an active role in regulating the
AKAP79/150 cytoskeletal linkage that maintains it at synapses. In
agreement with this hypothesis, inhibition of CaN with either CsA
(D/S = 47 ± 4%; PSDco = 46 ± 4%; n = 8) or FK506 (D/S = 50 ± 3%;
PSDco = 55 ± 3%; n = 9)
mostly prevented NMDA-regulated redistribution of AKAP79/150 when
compared with untreated (control D/S = 57 ± 3%;
PSDco = 61 ± 2%; n = 14)
and NMDA-treated cells (NMDA D/S = 37 ± 3%;
PSDco = 4 ± 1%; n = 10) (Fig.
8A,B).
No effects were seen in NMDA-treated cells with rapamycin, a drug that
binds to immunophilins like CsA and FK506 but does not inhibit CaN
(data not shown). Importantly, CaN is directly anchored to AKAP79/150
and thus may be optimally positioned to participate in regulation of
the postsynaptic actin cytoskeleton in response to NMDA receptor
activation. However, NMDA receptor Ca2+
signaling can also activate kinases such as PKC, CaMKII, and PKA
(through Ca2+-regulated adenylyl cyclase).
Furthermore, previous work and studies presented above show that PKC
phosphorylation of the AKAP79 targeting domain can regulate
PI-4,5-P2 and F-actin binding in vitro
(Fig. 5) (Dell'Acqua et al., 1998). However, consistent with a
dependence on phosphatases and not kinases, NMDA-mediated
AKAP79/150 redistribution was insensitive to inhibition of PKC
with chelerythrine-CHE (D/S = 34 ± 3%;
PSDco = 5 ± 2%; n = 5),
CaMKII with KN-62 (D/S = 29 ± 2%;
PSDco = 5 ± 1%, n = 3), or
PKA with H-89 (D/S = 33 ± 3%; PSDco = 5 ± 2%; n = 4)
(Fig. 8B).
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Figure 8.
Activation of the protein
phosphatase-2B calcineurin and reorganization of F-actin are necessary
for NMDA receptor regulation of AKAP79/150 postsynaptic localization.
A, Activation of CaN and reorganization of F-actin are
necessary for NMDA regulation of AKAP79/150 localization. Neurons were
left untreated or pretreated with the indicated CaN-PP2B phosphatase
inhibitors CsA (1 µM, 30 min) or FK506 (1 µM, 30 min), the actin-stabilizing drug jasplakinolide (2 µM, 2 hr), or the NMDA receptor antagonist AP-V (100 µM, 30 min) before NMDA treatment (50 µM,
10 min). The neurons were then fixed and stained for AKAP79/150
(red) and PSD-95 (green).
Large panels show AKAP150 and PSD-95 localization
separately in both dendrites and soma. The Magnification
panels show AKAP150, PSD-95, and Composite
images for localization in single dendrites to show details better.
B, Calcineurin protein phosphatase but not protein
kinase activity is necessary for NMDA regulation of AKAP79/150
localization. Quantitation of AKAP79/150 mean fluorescence intensity
distributions between dendrites and somata (%D/S,
red) and integrated colocalization with PSD-95
(PSDco, blue) from
multiple experiments for the treatment conditions in A
as well additional conditions described below are shown. See
Results and Materials and Methods for additional details. Neurons were
also pretreated for 30 min with the indicated kinase inhibitors for
PKA, H-89 (500 nM); PKC, CHE (10 µM); or CaMKII, KN-62 (5 µM)
before NMDA treatment (50 µM, 10 min). Pretreaments
with casein kinase II inhibitor (DRB, 50-100 µM) or
multiple kinase inhibitors (H-89, CHE, KN-62, DRB) had no effects on
AKAP150 redistribution in NMDA-treated cells (data not
shown).
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Reorganization of dendritic spine F-actin is necessary for NMDA
receptor regulation of AKAP79/150 postsynaptic localization
NMDA receptor activation of CaN-regulated F-actin remodeling could
certainly control localization of AKAP79/150, as shown above by
inhibition of actin polymerization with latrunculin disrupting AKAP79/150 postsynaptic targeting (Fig. 2). However, although F-actin
reorganization accompanies redistribution of AKAP79/150 after glutamate
or NMDA treatment (Fig. 6A) (data not shown), the
AKAP does not appear to move directly with F-actin in these experiments. Nonetheless, it is possible that actin remodeling in
response to NMDA receptor activation is a necessary first step allowing
AKAP release from the PSD and lateral membrane diffusion followed by
secondary redistribution (see below) away from the membrane. To test
whether reorganization of F-actin is in fact necessary for NMDA
receptor-regulated redistribution of AKAP79/150, we pretreated neurons
with jasplakinolide, a drug that stabilizes F-actin filaments and
prevents NMDA-induced reorganization of dendritic spine F-actin
(Halpain et al., 1998). Pretreatment with jasplakinolide for 2 hr
prevented (D/S = 59 ± 3%; PSDco = 46 ± 4%; n = 7) NMDA-mediated redistribution of
AKAP79/150 away from PSD-95 (D/S = 37 ± 3%;
PSDco = 4 ± 1%; n = 10),
similar to inhibition of CaN (see above), and nearly as well receptor
block with AP-V (D/S = 64 ± 4%; PSDco = 58 ± 3%; n = 12) (Fig.
8A,B). Thus, remodeling of F-actin
in response to CaN activation is likely to untether the AKAP79/150
targeting domain from the postsynaptic cytoskeleton. These results
taken together with the insensitivity to kinase inhibition are most
consistent with a model in which F-actin reorganization makes the
targeting domain accessible for subsequent
Ca2+-CaM binding (Fig. 5D).
CaM binding could then mediate secondary redistribution of AKAP79/150
away from the plasma membrane as well as prevent reassociation with
dendritic F-actin after NMDA receptor activation. Unfortunately, this
predicted secondary involvement of CaM binding in AKAP79/150
translocation cannot be investigated with CaM inhibitors because they
also inhibit CaN activation.
Inhibition of actin polymerization and NMDA receptor activation
both regulate association of the AKAP79/150-PKA complex with
PSD-MAGUKs
Immunocytochemistry presented above shows that inhibition of actin
polymerization and NMDA receptor activation both cause loss of
AKAP79/150 from synapses, suggesting dissociation from PSD-MAGUK
complexes. To independently confirm disassembly of AKAP79/150-MAGUK complexes, we immunoprecipitated AKAP79/150 from cell extracts prepared
from neurons treated with latrunculin or NMDA and analyzed the
immunoprecipitates by immunoblotting for AKAP150, PSD-MAGUKs (pan-PSD-MAGUK antibody), and PKA-RII subunits. The levels of AKAP79/150, PSD-MAGUKs, and PKA-RII were essentially the same in
cell extracts prepared from untreated control neurons and latrunculin- or NMDA-treated neurons, indicating that there was no significant proteolytic loss of these proteins resulting from the treatments (Fig.
9A,B). Roughly equal amounts
of AKAP150 and PKA-RII were detected in AKAP150 immunoprecipitates
from control and latrunculin- or NMDA-treated neurons, indicating that
the AKAP-PKA complex is still intact after these treatments (Fig.
9A,B). In contrast, PSD-MAGUK
proteins, which are readily seen coprecipitating with AKAP150 from
control neurons, are not detectable in AKAP150 precipitates from
latrunculin- or NMDA-treated neurons (Fig.
9A,B), even after long exposures
(data not shown). Thus, reorganization of F-actin caused by either
latrunculin treatment or NMDA receptor activation leads not only to
altered subcellular targeting of AKAP79/150 seen by immunocytochemistry
but also to disruption of AKAP-MAGUK complexes detected biochemically.
Disruption of AKAP-MAGUK complexes should in turn break AKAP79/150
linkage to glutamate receptors. Although we were unable to address this
question directly by coprecipitation because of the small amounts of
extract obtained from cultured neurons, our immunocytochemistry
supports this prediction. Latrunculin A and NMDA both trigger loss of
AKAP79/150 and GluR1 colocalization with PSD-95, but GluR1 remains
punctate, consistent with trafficking to endosomes (Ehlers, 2000),
whereas AKAP79/150 becomes more diffuse and untargeted (Figs. 2,
7).
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Figure 9.
Regulation of AKAP79/150-PKA association with
PSD-MAGUKs by inhibition of actin polymerization and NMDA receptor
activation. A, Inhibiting F-actin polymerization with
latrunculin A disrupts association of AKAP79/150 with PSD-MAGUKs but
not PKA in complexes isolated from hippocampal neuron cell extracts.
B, NMDA receptor activation disrupts association of
AKAP79/150 with PSD-MAGUKs but not PKA in complexes isolated from
hippocampal neuron cell extracts. Neurons grown at high density on
Petri plates were either untreated for control conditions or treated
with latrunculin A (+LtrA; 5 µM, 4 hr)
(A) or NMDA (50 µM, 10 min)
(B) before harvesting and lysis to prepare
whole-cell extracts. AKAP79/150 was then immunoprecipitated (IP:
AKAP150) from Triton X-100-soluble fractions prepared from
these extracts. The immunoprecipitates and whole-cell extracts were
then analyzed by immunoblotting (IB:) for AKAP150,
PSD-MAGUK family members, and the PKA-RII regulatory subunit as
indicated. The data shown are representative of three experiments done
on two to four samples for each condition.
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NMDA receptor regulation of AKAP79/150 postsynaptic targeting and
redistribution of PKA and CaN
An additional prediction that is supported by our results showing
equal coprecipitation of AKAP150 and PKA-RII after latrunculin or
NMDA treatment is that anchored PKA may be redistributing from dendritic spines in a complex with AKAP79/150. As shown above (Fig. 2),
in untreated cells PKA-RII showed extensive punctate codistribution
with AKAP79/150 on dendritic spines (150-RIIco = 68 ± 6%; n = 7) (Fig.
10A). PKA-RII also
displayed independent localization in cell bodies and proximal
dendritic shafts, probably reflecting anchoring to other AKAPs in those
locations (Fig. 10A). After treatment with glutamate,
RII and AKAP79/150 became diffuse and punctate dendritic spine
colocalization was lost, with insufficient puncta for either protein
remaining for quantitation (Fig. 10A). However, in
response to glutamate, dendritic fluorescence decreased for both
AKAP79/150 (control D/T = 83 ± 2%, n = 6;
glutamate D/T = 51 ± 2%, n = 5) and RII
(control D/T = 67 ± 2%; glutamate D/T = 54 ± 2%) (Fig. 10A). After all treatments, the prominent
localization of RII in the soma and proximal dendritic shafts was
unaltered, suggesting that NMDA receptor activation selectively
regulated localization of a pool of PKA bound to AKAP79/150.
Importantly, the NMDA receptor antagonist AP-V inhibited the effects of
glutamate on RII and AKAP79/150 localization (RII D/T = 77 ± 4%; AKAP150 D/T = 86 ± 2%) and spine colocalization
(150-RIIco = 50 ± 7%; n = 4) (Fig. 10A).
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Figure 10.
NMDA receptor activation regulates
dendritic spine localization of PKA and CaN with AKAP79/150.
A, NMDA receptor regulation of PKA-RII and AKAP79/150
localization. B, Disruption of dendritic spine
colocalization of CaNB and AKAP79/150 in response to NMDA receptor
activation. Neurons (2-3 weeks old) were untreated, pretreated with
antagonist AP-V (100 µM, 30 min), or treated with
glutamate (50 µM, 10 min) as indicated. Control untreated
and treated neurons were stained for AKAP79/150 (red)
and either PKA-RII regulatory subunits (green)
in A or CaNB (green) in
B. The smaller panels are magnifications
of dendrites. See Results for quantitation.
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From the results presented in Figure 8, redistribution of AKAP79/150
involves activation of CaN. Anchoring of the CaN A, B holoenzyme to
AKAP79/150 is mediated through binding of catalytic CaNA subunit in a
manner that noncompetitively inhibits phosphatase activity (Coghlan et
al., 1995; Dell'Acqua et al., 1998; Kashishian et al., 1999). Thus, we
wanted to see whether the AKAP-CaN interaction might be regulated by
the NMDA receptor. Unfortunately, our efforts to look for release of
CaN from AKAP79/150 by immunoprecipitation were unsuccessful because of
limitations of CaN antibody sensitivities and small amounts of extracts
obtained from cultured neurons (data not shown). Nonetheless,
regulation of the CaN-AKAP79/150 interaction by NMDA receptor
activation might be manifested in changes in the dendritic localization
of CaN relative to the AKAP. Others have observed changes in the
pattern of CaNA subunit staining in hippocampal neurons after NMDA and
glutamate treatments similar to those used here (Halpain et al., 1998).
In untreated neurons, CaNB and AKAP79/150 showed punctate
colocalization (150-CaNco = 58 ± 2%;
n = 6) on dendritic spines (Fig.
10B); however, CaN was distributed more extensively
throughout the cytoplasm of the cell body. Treatment of neurons with
glutamate disrupted punctate dendritic spine colocalization of
AKAP79/150 and CaNB (150-CaNco = 3 ± 1%;
n = 8), with the AKAP redistributing to a diffuse
localization in dendrites and the soma as observed above (control
D/T = 74 ± 3%, D/S = 50 ± 3%, S/T = 149 ± 7%; glutamate D/T = 42 ± 2%, D/S = 22 ± 1%, S/T = 190 ± 8%) (Fig.
10B). AP-V pretreatment inhibited the
glutamate-regulated redistribution of AKAP79/150 (D/T = 66 ± 3%, D/S = 40 ± 3%, S/T = 168 ± 6%) relative to
CaNB (150-CaNco = 44 ± 3%;
n = 6), thus confirming involvement of NMDA receptors (Fig. 10B). In response to glutamate, punctate CaNB
staining certainly decreased (~45%); however, unlike AKAP and PKA,
overall CaNB staining remained distributed in dendrites and did not
concentrate any more in soma in glutamate-treated (D/T = 64 ± 4%, D/S = 41 ± 4%, S/T = 155 ± 7%) versus
untreated neurons (D/T = 67 ± 4%, D/S = 43 ± 3%, S/T = 159 ± 8%) (Fig. 10B).
Disruption of AKAP79/150-CaN colocalization on dendritic spines
without coincident CaN movement from dendrites to the soma could be
indicative of CaN release from the AKAP. However, an alternative
explanation is that CaN is simply in excess of AKAP in dendrites.
Nonetheless, NMDA receptor regulation of AKAP79/150-PKA localization
(Fig. 10A) is also accompanied by distinct changes in
colocalization with CaN (Fig. 10B).
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DISCUSSION |
TOP
ABSTRACT
INTRODUCTION
