 |
 Previous Article | Next Article 
The Journal of Neuroscience, January 15, 1998, 18(2):634-640
Region-Specific Phosphorylation of Rabphilin in Mossy Fiber Nerve
Terminals of the Hippocampus
György
Lonart and
Thomas C.
Südhof
Department of Molecular Genetics and Howard Hughes Medical
Institute, The University of Texas Southwestern Medical Center, Dallas,
Texas 75235
 |
ABSTRACT |
In mossy fiber synapses of the CA3 region of the hippocampus,
long-term potentiation (LTP) is induced presynaptically by activation of cAMP-dependent protein kinase A (PKA). Rab3A is a synaptic vesicle
protein that regulates vesicle fusion and is essential for mossy fiber
LTP. Rab3A probably acts via two effector proteins, rabphilin and RIM,
of which rabphilin is an in vitro substrate for PKA. To
test if rabphilin is phosphorylated in nerve terminals and if its
PKA-dependent phosphorylation correlates with the PKA-dependent induction of LTP in mossy fiber terminals, we have studied the phosphorylation of rabphilin in synaptosomes isolated from the CA1 and
CA3 regions of the hippocampus. Rabphilin was phosphorylated in both
CA1 and CA3 synaptosomes. However, when we treated the CA1 and CA3
synaptosomes with forskolin (an agent that enhances PKA activity) or
induced Ca2+ influx into synaptosomes with high
K+, rabphilin phosphorylation was increased
selectively in mossy fiber CA3 synaptosomes, but not in CA1
synaptosomes. In contrast, the phosphorylation of synapsin, studied as
a control for the specificity of the region-specific phosphorylation of
rabphilin, was augmented similarly by both treatments in CA1 and CA3
synaptosomes. These results reveal that the phosphorylation states of
two synaptic substrates for PKA and CaM KII, rabphilin and synapsin,
are regulated differentially in a region-specific manner, an unexpected
finding because rabphilin and synapsin are similarly present in CA1 and CA3 synaptosomes and are colocalized on the same synaptic vesicles. The
region-specific phosphorylation of rabphilin agrees well with the
restricted induction of LTP by presynaptic PKA activation in mossy
fiber, but not CA1, nerve terminals.
Key words:
rab3; rabphilin; exocytosis; neurotransmitter release; synaptic transmission; synaptic vesicles; long-term potentiation; protein kinase A; CaM kinase II
| |
INTRODUCTION |
Synapses transmit signals between
neurons and constitute fundamental units of information processing in
brain (Sheperd, 1990 ). The strength of synaptic transmission varies and
is modulated by processes of synaptic plasticity, such as long-term
potentiation (LTP). At least two forms of LTP are expressed in the
hippocampus (for review, see Stevens, 1993; Nicoll and Malenka, 1995):
(1) NMDA receptor-dependent LTP (CA1 LTP), most often studied in
synapses of Schaffer collateral/commissural fibers on pyramidal CA1
neurons, and (2) NMDA receptor-independent LTP (CA3 LTP), expressed by mossy fiber synapses onto CA3 neurons. Both forms of LTP involve long-lasting increases in synaptic strength but are mechanistically distinct. CA1 LTP (NMDA receptor-dependent LTP) is induced
postsynaptically by Ca2+ influx and activation of
Ca2+, calmodulin-dependent protein kinase II (CaM
KII) (for review, see Lisman et al., 1997). In contrast, CA3 LTP (NMDA
receptor-independent LTP) is initiated presynaptically by activation of
cAMP-dependent protein kinase A (PKA) (Huang et al., 1994; Weisskopf et
al., 1994; Salin et al., 1996). Induction of CA3 LTP must involve
changes in the Ca2+-triggered exocytosis of synaptic
vesicles, but no presynaptic phosphoproteins that might be involved in
LTP induction are known.
Considerable progress was made recently in the biochemical
dissection and genetic analysis of the proteins that govern presynaptic function (for review, see Ferro-Novick and Jahn, 1994; Südhof, 1995; Martin, 1997). Among these proteins, rab3A and synapsins Ia and
Ib are synaptic vesicle proteins with important functions in regulating
exocytosis. Knock-out experiments showed that rab3A is required for
limiting the amount of vesicle exocytosis per Ca2+
signal (Geppert et al., 1994, 1997) and is essential for CA3 LTP
(Castillo et al., 1997). The requirement for rab3A in CA3 LTP was
unexpected because rab3A is a ubiquitous synaptic protein that is not
enriched in CA3 mossy fibers (Li et al., 1994). Rab3A acts by binding
in a GTP-dependent manner to at least two putative effector proteins,
rabphilin and RIM (Shirataki et al., 1993; Li et al., 1994; Wang et
al., 1997). Rabphilin is an in vitro substrate for PKA
(Fykse et al., 1995) and for CaM KII (Kato et al., 1994; Fykse et al.,
1995), suggesting it might be involved in the rab3A-dependent
generation of CA3 LTP.
Similar to rabphilin, synapsins are also substrates for PKA and
CaM KII (for review, see Greengard, 1987). Synapsins are required for
normal short-term synaptic plasticity and synaptic vesicle stability
(Rosahl et al., 1993, 1995) but are not essential for CA1 and CA3 LTP
(Spillane et al., 1995). The exact functions of synapsins are unknown;
a morphometric analysis of synapses from mice lacking synapsins I and
II showed no changes in synapse number or vesicle clustering (Rosahl et
al., 1995), whereas major changes in these parameters were seen in a
second study on a different mouse line lacking only synapsin I (Li et
al., 1995).
Despite the progress in the electrophysiological and biochemical
analysis of synapses, little is known about the mechanisms involved in
presynaptic regulation. One problem is that most preparations used to
study presynaptic function electrophysiologically are inaccessible
biochemically. A second problem is that it is unclear which synaptic
proteins are phosphorylated in nerve terminals in correlation with
functional changes. This gap in understanding is most striking for
synapsins and rabphilin, which are the only currently known presynaptic
PKA and CaM KII substrates, but for which the phosphorylation in nerve
terminals either has not been studied yet (rabphilin) or has not been
associated directly with a synaptic plasticity (synapsins). In the
current study we have investigated the phosphorylation of rabphilin and
synapsins in synaptosomes from hippocampal synapses with distinct
properties, regular synapses from the CA1 region and large mossy fiber
synapses from the CA3 region. Our results demonstrate that rabphilin is phosphorylated in a stimulation-dependent and region-specific manner
that correlates with mossy fiber LTP. This suggests a mechanism of
presynaptic regulation that uses region-specific changes in the
phosphorylation states of generally distributed synaptic proteins.
| |
MATERIALS AND METHODS |
Preparation of synaptosomes. CA1 and CA3 regions were
dissected from rat hippocampus, using the minor and the hippocampal fissures as landmarks. Large mossy fiber synaptosomes were prepared by
homogenizing the CA3 region manually in 1 mM
MgSO4, 0.3 M sucrose, and 15 mM HEPES-NaOH, pH 7.4, to preserve the complex structure and large size of the mossy fiber terminals (Hajos et al., 1975; Terrian et al., 1988). The homogenate was passed through a series of
nylon filters (100, 60, and 40 µm mesh size) and centrifuged at
900 × g for 10 min to selectively pellet large
synaptosomes. The pellet was resuspended in 18% (w/v) Ficoll and 0.3 M sucrose and centrifuged at 16,000 × g
for 20 min to separate the synaptosomes that remained in suspension
from contaminating nuclei. Synaptosomes were diluted with 4 vol of 0.3 M sucrose and sedimented by centrifugation at 15,000 × g for 20 min to remove the Ficoll before use. Small synaptosomes were prepared from the CA1 region by gradient
centrifugation by a method modified from Nagy and Delgado-Escueta
(1984). The crude mitochondrial fraction (P2) was
resuspended in 8.5% (v/v) Percoll suspended in 0.25 M
sucrose and 5 mM HEPES-NaOH, pH 7.4, and layered on top of
an 12%/20% Percoll step gradient in the same buffer. After
centrifugation at 18,000 × g for 30 min, synaptosomes were recovered from the 12%/20% Percoll interface. Percoll was removed by the addition of 30 vol of 0.32 M sucrose and
centrifugation at 18,000 × g for 20 min. In a typical
experiment 2 mg of CA1 and 4 mg of CA3 synaptosomal protein were
isolated from seven rat brains.
Phosphorylation measurements. Synaptosomes (1 mg
protein) were incubated with 32P-orthophosphate (1 mCi) for
1 hr at 37°C in phosphate-free aerated (95%
O2/5% CO2)
Krebs-Henseleit-HEPES buffer [KHH buffer; composition (in
mM): NaCl 118, KCl 3.5, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, HEPES-NaOH 5 at pH 7.4, and glucose 11.5]
containing 0.1 mM CaCl2. The tubes were flushed
with 95% O2/5% CO2 every 15 min. Free
32P-orthophosphate was removed by pelleting the
synaptosomes at 1000 × g for 5 min at 4°C. After
washing, synaptosomes were resuspended in cold aerated KHH buffer
(final concentration 0.2 gm/l protein) and equilibrated for 10 min at
37°C. Protein phosphorylation was studied under four treatment
conditions. For controls and treatment with forskolin alone,
synaptosomes (225 µl) were added to 25 µl of KHH buffer containing
1% DMSO without or with 0.5 mM forskolin, and the
reactions were stopped after 5 min. For treatment with elevated
K+ alone or in combination with forskolin,
synaptosomes (225 µl) were added to 12.5 µl of KHH buffer
containing 2% DMSO without or with 1 mM forskolin,
respectively. Reactions were incubated for 4 min; then 12.5 µl of 0.4 M KCl in KHH buffer was added, and reactions were stopped
after an additional 1 min. Reactions were stopped by the addition of
100% TCA (7.5% final concentration) for analysis of total protein
phosphorylation, or by the addition of 1% SDS with (in mM)
1 EGTA, 1 EDTA, 50 NaF, and 10 Na4P2O7 (final concentrations) for
immunoprecipitations. For analysis of total protein phosphorylation,
TCA precipitates were washed with 90% ethanol and acetone, resuspended
in 1× SDS-PAGE sample buffer, and analyzed by linear gradient SDS-PAGE
and autoradiography. For immunoprecipitations, samples were adjusted to
0.17% SDS, 1% Triton X-100, plus (in mM) 10 HEPES-NaOH,
pH 7.4, 100 NaCl, 50 NaF, 1 EGTA, 1 EDTA, and 1 PMSF with 1 mg/l
pepstatin, 10 mg/l leupeptin, and 10 mg/l antipain. Particulate matter
was removed by centrifugation (20,000 × g for 10 min,
twice), and serum (1% v/v final concentration) was added. Rabphilin
was immunoprecipitated with antibodies raised against a recombinant
GST-rabphilin fusion protein (I734; Li et al., 1994), and synapsins
were raised with an antibody against the N-terminal sequence of
synapsins (E028; Rosahl et al., 1995). Antigen-antibody complexes were
recovered by incubation with 50 µl of a 50% slurry of protein
A-Sephadex beads for 45 min and subsequent centrifugation at 1000 × g for 5 min. The beads were washed four times in 10 mM HEPES-NaOH, pH 7.4, 0.15 M NaCl, 1 mM EDTA, and 0.5% Triton X-100; resuspended in SDS sample
buffer; and analyzed by SDS-PAGE. After staining and destaining,
phosphoproteins were visualized by autoradiography. 32P
incorporation was quantified with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). Data from multiple experiments performed in duplicate
were pooled, and the significance of changes was evaluated
statistically with the Kruskal-Wallis one-way ANOVA, followed by
Dunnett's test for pairwise multiple comparisons, with a significance
level of p < 0.05.
| |
RESULTS |
Purification of CA1 and CA3 synaptosomes from the hippocampus
To study the differential regulation of hippocampal synapses
biochemically, we purified synaptosomes from the CA1 and CA3 regions of
the hippocampus. We dissected the CA1 and CA3 regions from rat brains,
isolating regular "small" synaptosomes from the CA1 region by
standard techniques and large mossy fiber synaptosomes from the CA3
region by making use of their large size (see Materials and Methods).
To determine whether the protein composition of the two types of
synaptosomes is similar, we analyzed them by immunoblotting with
antibodies to several presynaptic proteins (Fig.
1). No major differences in the relative
levels of different proteins were found.
View larger version (30K):
[in this window]
[in a new window]
|
Figure 1.
Immunoblot analysis of synaptic proteins in CA1
and CA3 synaptosomes. Equivalent amounts of protein (10 µg/lane) from
the indicated fractions were analyzed by SDS-PAGE and immunoblotting, with antibodies to the proteins shown. Signals were visualized by
enhanced chemiluminescence. An asterisk in the
synaptotagmin 1 immunoblot marks the position of a major proteolytic
breakdown product commonly observed in immunoblots for
synaptotagmin.
|
|
Protein phosphorylation in CA1 and CA3 synaptosomes
To gain insight into potential differences between CA1 and
CA3 synaptosomes in the phosphorylation of proteins by PKA, we first
analyzed the overall pattern of protein phosphorylation in
32P-labeled synaptosomes. Synaptosomes were analyzed by
SDS-PAGE and autoradiography either under control conditions or after
treatment with forskolin, elevated K+, or a
combination of forskolin and elevated K+. Forskolin
was used because it activates adenylate cyclase, thereby increasing
cAMP levels and PKA activity. Elevated K+ was used
because it causes Ca2+ influx into synaptosomes,
thereby activating CaM KII and other kinases. Thus the two treatments
activate two different but overlapping signal transduction pathways in
synaptosomes, one of which (the PKA pathway) is required for CA3 LTP,
but not CA1 LTP.
No major differences in the phosphoprotein pattern of CA1 and CA3
synaptosomes were observed under resting conditions, providing additional evidence that they are similar in composition (Fig. 2). Forskolin did not induce major
alterations in the phosphorylation patterns of either CA1 or CA3
synaptosomal proteins. K+ elevation increased
phosphorylation of a protein in the 10 kDa molecular weight range in
both types of synaptosomes but otherwise caused only minor changes
(arrow in Fig. 2). We also analyzed synaptosomal protein
phosphorylation by two-dimensional gel electrophoresis after various
stimulation conditions to confirm these findings (Garrels, 1979).
Although the two-dimensional gels improved resolution, we again
observed no differences between CA1 and CA3 synaptosomes (data not
shown). These results suggest that CA1 and CA3 synaptosomes are similar
in composition and that forskolin treatment and K+
depolarization do not cause major changes in the phosphorylation of
abundant proteins.
View larger version (106K):
[in this window]
[in a new window]
|
Figure 2.
Protein phosphorylation in CA1 and CA3
synaptosomes. Analysis of total protein phosphorylation in synaptosomes
from the CA1 (lanes 1, 3, 5, 7) and the CA3
(lanes 2, 4, 6, 8) region. Synaptosomes were treated as
follows: lanes 1, 2, control; lanes 3, 4,
50 µM forskolin (5 min); lanes 5, 6, 20 mM K+ (1 min); lanes 7, 8, 50 µM forskolin (5 min) and 20 mM
K+ (1 min). Numbers on the
left indicate positions of molecular weight markers (in
kDa). The top part of the autoradiogram was exposed for
13 hr and the bottom part for 60 hr to visualize the 10 kDa band, the phosphorylation for which is induced by
K+ (arrow).
|
|
Phosphorylation of rabphilin and synapsins in CA1 and
CA3 synaptosomes
We next studied whether rabphilin is phosphorylated in the two
types of synaptosomes as a system resembling in vivo
conditions. For these and the following experiments, we used synapsins
as well characterized synaptic phosphoproteins as positive controls. We
immunoprecipitated rabphilin and synapsins from resting
32P-labeled synaptosomes and from synaptosomes that were
treated with okadaic acid. Okadaic acid was applied at a concentration of 50 µM at which it is an inhibitor of serine/threonine
protein phosphatases 1, 2A, and 2B. At this concentration okadaic acid nonspecifically increases protein phosphorylation. The experiments with
okadaic acid revealed that rabphilin and synapsins were phosphorylated in both CA1 and CA3 synaptosomes and that okadaic acid greatly increased the phosphorylation states of both proteins in the two types
of synaptosomes (Fig. 3). Thus rabphilin
is phosphorylated in synaptosomes similar to synapsins. However,
phosphorylation and immunoprecipitation experiments can be quite
variable because they involve multiple procedures, and single
experiments are insufficient for evaluating magnitudes of changes.
Therefore, we quantitated the degree of rabphilin and synapsin
phosphorylation in multiple independent experiments with a
phosphoimager. The increase in phosphorylation induced by okadaic acid
was calculated for each experiment relative to baseline
phosphorylation.
View larger version (40K):
[in this window]
[in a new window]
|
Figure 3.
Okadaic acid increases synapsin and rabphilin
phosphorylation in CA1 and CA3 synaptosomes. 32P-labeled
CA1 and CA3 synaptosomes were incubated for 5 min in control buffer or
buffer containing okadaic acid (50 µM) and then subjected
to immunoprecipitations with antibodies to synapsins (top) and rabphilin (bottom). Shown is a
representative autoradiogram.
|
|
Using this analysis, we found that okadaic acid increased both
rabphilin and synapsin phosphorylation three- to fourfold in both types
of synaptosomes (Fig. 4). These data
quantitatively confirm the qualitative impression from the experiment
in Figure 3, namely that rabphilin and synapsins are phosphoproteins in synaptosomes, the phosphorylation of which constantly turns over and
therefore is enhanced by the addition of a phosphatase inhibitor. The
overall phosphorylation signal is higher for synapsins than for
rabphilin, presumably because synapsins are more abundant. However, the
increase in phosphorylation induced by okadaic acid is similar for
rabphilin and synapsins and not dramatically different between CA1 and
CA3 synaptosomes. This indicates that at resting conditions the
turnover rate of phosphorylation is comparable for the two proteins and
the two types of synaptosomes.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 4.
Quantitative analysis of enhancement of rabphilin
and synapsin phosphorylation by okadaic acid. The phosphorylation of
synapsins and rabphilin in control synaptosomes and in synaptosomes
treated with okadaic acid (50 µM) was quantified by
phosphoimager detection after immunoprecipitation and SDS-PAGE, as
described in Figure 3. Data shown are mean ± SEM from four
independent experiments. Asterisks indicate that in both
CA1 and CA3 synaptosomes the increase in the phosphorylation of
synapsins and rabphilin is statistically significant.
|
|
Forskolin and high K+ enhance rabphilin
phosphorylation in CA3, but not in CA1, synaptosomes
Next we investigated the regulation of rabphilin phosphorylation
by PKA (activated by forskolin treatment) and by
Ca2+-dependent protein kinases (activated by
Ca2+ influx triggered by membrane depolarization in
solutions with 20 mM K+).
32P-labeled synaptosomes from the CA1 and CA3 regions were
treated with four conditions: (1) control, (2) forskolin, (3)
K+ depolarization, and (4) forskolin in combination
with K+ depolarization. We then immunoprecipitated
rabphilin from the treated synaptosomes and quantified its
phosphorylation by SDS-PAGE and phosphoimager detection. To ensure that
the immunoprecipitations were equally effective under various treatment
conditions, we confirmed the presence of rabphilin in the
immunoprecipitates by immunoblotting (data not shown). Because these
experiments involved multiple procedures that might fluctuate randomly
between experiments, we quantified phosphorylations in multiple
independent determinations. In each experiment the phosphorylation of
rabphilin was measured relative to baseline phosphorylation, and data
from multiple experiments were analyzed statistically.
Forskolin induced a dramatic increase in rabphilin phosphorylation in
CA3 synaptosomes (260-400% increase), suggesting that it is a
physiological substrate for PKA (Fig. 5).
K+ depolarization also enhanced rabphilin
phosphorylation in CA3 synaptosomes (Fig. 5). K+
depolarization only enhanced rabphilin phosphorylation in the presence
of Ca2+ in the incubation medium, but not in its
absence, suggesting that Ca2+ influx triggered by
K+ depolarization is essential for stimulating
rabphilin phosphorylation. In contrast, forskolin-stimulated
phosphorylation of rabphilin was not dependent on
Ca2+ (data not shown). The combination of forskolin
with K+ depolarization caused an additive increase
in phosphorylation in the CA3 synaptosomes but only a small effect in
CA1 synaptosomes. These observations suggest that rabphilin is a
substrate for PKA and CaM KII not only in vitro but also in
synaptosomes, a system resembling nerve terminals of living
neurons.
View larger version (26K):
[in this window]
[in a new window]
|
Figure 5.
Modulation of rabphilin phosphorylation in CA1 and
CA3 synaptosomes by forskolin and K+-induced
Ca2+ influx. Rabphilin was immunoprecipitated from
32P-labeled CA1 or CA3 synaptosomes incubated under four
treatment conditions: 1, control (5 min);
2, forskolin (50 µM for 5 min); 3, control (4 min), followed by 20 mM
K+ (1 min) to induce membrane depolarization and
Ca2+ influx; 4, forskolin (4 min),
followed by forskolin combined with 20 mM
K+ (1 min). After immunoprecipitations, samples were
analyzed on an SDS-polyacrylamide gel, and the phosphorylation of
immunoprecipitated rabphilin was quantitated on a phosphoimager. Data
shown are mean ± SEM from seven independent experiments performed
in duplicate. Statistically significant changes are marked by
asterisks.
|
|
Strikingly, forskolin had no significant effect on rabphilin
phosphorylation in CA1 synaptosomes. The stimulatory effect of K+ on rabphilin phosphorylation also was restricted
to synaptosomes from the CA3 region, and no effect was observed in CA1
region synaptosomes (Fig. 5). These results reveal that the
phosphorylation of rabphilin is regulated differentially in different
types of nerve terminals (CA1 vs CA3).
Forskolin and depolarization-induced Ca2+ influx
stimulate phosphorylation of synapsins
The fact that forskolin and Ca2+ influx
increase rabphilin phosphorylation in a region-specific manner is
striking because okadaic acid increased phosphorylation of rabphilin in
both types of synaptosomes (Fig. 4). This raises the question of
whether for an unknown reason forskolin and Ca2+
influx may have been unable to activate kinases in the CA1
synaptosomes. To evaluate this concern, we analyzed the effect of
forskolin and Ca2+ influx on the phosphorylation
state of synapsins in both synaptosomes under conditions identical to
those used for rabphilin.
Our results show that both forskolin and K+
elevation stimulated synapsin phosphorylation and stimulated it equally
in CA1 and CA3 synaptosomes (Fig. 6). The
increase in synapsin phosphorylation was relatively small (35-50%),
as compared with the increase in rabphilin phosphorylation observed in
CA3 synaptosomes (260-400%). However, the increase in synapsin
phosphorylation under these mild stimulation conditions is
statistically significant (p < 0.05) and
similar to that observed by others previously (Wang et al., 1988). The
fact that synapsin phosphorylation is increased equally in CA1 and CA3
synaptosomes demonstrates that the CA1 synaptosomes are responsive to
stimulation and that the lack of an increase in rabphilin
phosphorylation under our treatment conditions is not attributable to a
failure of these synaptosomes to respond.
View larger version (28K):
[in this window]
[in a new window]
|
Figure 6.
Modulation of synapsin phosphorylation in CA1 and
CA3 synaptosomes by forskolin and K+-induced
Ca2+ influx. Synapsins were immunoprecipitated from
32P-labeled CA1 or CA3 synaptosomes treated singly or in
combination with forskolin and elevated K+, as
described in the legend to Figure 5. Immunoprecipitates were analyzed
accordingly. Data shown are mean ± SEM from 10 independent experiments performed in duplicate; statistically significant changes
are marked by asterisks.
|
|
| |
DISCUSSION |
In presynaptic mossy fiber terminals of the CA3 region of the
hippocampus, PKA activation induces a presynaptic form of LTP, which we
refer to as CA3 LTP (Huang et al., 1994; Weisskopf et al., 1994).
Recent studies have shown that, in mice lacking the synaptic vesicle
protein rab3A, CA3 LTP cannot be induced normally (Castillo et al.,
1997). This result directly proves a participation of the molecular
machinery for vesicular exocytosis in CA3 LTP. It suggests that rab3A
is, in an unknown manner, required for PKA-dependent long-term
potentiation of neurotransmitter release. Rab3A itself is not a PKA
substrate, suggesting that PKA does not act directly on rab3A. However,
rab3A is thought to act by binding to effector proteins in a
GTP-dependent manner, and one of its two known effectors, rabphilin,
was shown previously to be a substrate for PKA in vitro
(Fykse et al., 1995). In the present study we set out to address two
questions: (1) Is rabphilin also phosphorylated by PKA in a system
resembling nerve terminals of living neurons? (2) Is there a difference
in the phosphorylation of rabphilin between CA1 and CA3 nerve terminals
that correlates with the ability of PKA to induce LTP in mossy fiber
synapses of the CA3 region, but not in Schaffer collateral/commissural fiber synapses of the CA1 region?
To resolve these questions, we have isolated synaptosomes, pinched-off
nerve endings, from the CA1 and CA3 regions of the hippocampus and have
analyzed the phosphorylation of rabphilin in these synaptosomes.
Synapsin phosphorylation was studied as a control. We quantitatively
compared the phosphorylation of rabphilin and synapsins to baseline
phosphorylation states under four treatment conditions: (1) okadaic
acid as a relatively nonspecific phosphatase inhibitor to induce a
general increase in phosphorylation states, (2) forskolin as an
activator of adenylyl cyclase to increase cAMP levels and PKA activity,
(3) elevated K+ to trigger Ca2+
influx into the synaptosomes to activate
Ca2+-dependent kinases, and (4) a combination of
forskolin and K+ treatment.
Our data result in two basic conclusions. (1) Rabphilin is
phosphorylated in synaptosomes, and its phosphorylation is increased several-fold by activations of PKA and by Ca2+
influx. This suggests that it constitutes a physiological substrate for
PKA and for Ca2+-activated kinases, probably CaM
KII. (2) Although rabphilin is phosphorylated and its phosphorylation
is increased by a generic phosphatase inhibitor in both CA1 and CA3
synaptosomes, forskolin and Ca2+ influx enhance
rabphilin phosphorylation only in CA3, but not in CA1, synaptosomes.
The region-specific regulation of rabphilin phosphorylation parallels
that of the action of PKA in inducing CA3 LTP. It is unlikely to be
artifactual, because synapsin phosphorylation was increased similarly
in both types of synaptosomes with forskolin and K+
stimulation.
The region-specific phosphorylation of rabphilin is surprising because
phosphorylated rabphilin and active PKA are present in both types of
synaptosomes. These results reveal an unexpected regional
specialization of protein phosphorylation in nerve terminals, the first
demonstration of region-specific phosphorylation of a synaptic protein.
The region-specific phosphorylation of rabphilin is particularly
striking in view of the behavior of synapsins, which are colocalized
with rabphilin on the same vesicles and are substrates for the same
kinases but are still not subject to the same region-specific
phosphorylation.
Two questions arise: (1) What is the mechanism that allows the
region-specific phosphorylation of rabphilin? (2) What does this
phosphorylation mean for CA3 LTP? Although we have no definitive answer
to either question at present, the fact that forskolin and
K+ depolarization have similar effects on synapsin
phosphorylation in CA1 and CA3 synaptosomes suggests that PKA and CaM
kinases are similarly present in both types of nerve terminals. Thus
the selective phosphorylation of rabphilin in CA3 synaptosomes could be
attributable to a compartmentalization of the kinases, a possibility raised by observations with other phosphorylation events (for review,
see Faux and Scott, 1996). Alternatively, it could be attributable to
differential activities of phosphatases in the two types of nerve
terminals. Independent of which mechanism is operative, the net result
is a differential phosphorylation state of rabphilin in the two types
of nerve terminals, which thereby adds a layer of regulation on top of
activation of protein phosphorylation and protein expression.
As regarding the question of the functional significance of the
differential phosphorylation of rabphilin in CA3 versus CA1 synaptosomes, the correlation of this phenomenon with the selective induction of PKA-dependent LTP in mossy fiber nerve terminals (CA3 LTP)
and the requirement for rab3A in the induction is intriguing. This
suggests the possibility that rab3A may be required for CA3 LTP because
it interacts with rabphilin and reversibly recruits rabphilin to nerve
terminals (Li et al., 1994; Stahl et al., 1996). At present this
possibility cannot be readily tested because forskolin has additional
actions besides activating PKA and inducing LTP in mossy fiber nerve
terminals (Hosi et al., 1988; Huang et al., 1995; Castillo et al.,
1997). These parallel actions are unimpaired in the rab3A knock-out
mice so that, although CA3 LTP can no longer be induced, forskolin
still enhances neurotransmitter release. Therefore so that rabphilin
phosphorylation can be correlated with LTP, studies on rabphilin
knock-outs will be required. Nevertheless, the specificity for the CA3
region of the PKA-dependent phosphorylation of rabphilin and of the
PKA-dependent induction of LTP is suggestive of a role of rabphilin
phosphorylation in CA3 LTP. In addition, the region-specific
phosphorylation of rabphilin implies that the phosphorylation states of
substrate proteins can be regulated locally if the kinases and
substrates are present ubiquitously, even if the substrates are
colocalized on the same organelle.
| |
FOOTNOTES |
Received Aug. 25, 1997; revised Oct. 27, 1997; accepted Oct. 31, 1997.
This study was supported by a Human Frontier Science Program grant,
National Institutes of Health Grant RO1-MH52804, and the Perot Family
Foundation. We thank Drs. E. Kandel, R. Nicoll, R. Malenka, M. S. Brown, and J. L. Goldstein for invaluable discussions.
Correspondence should be addressed to Dr. Tom Südhof, Room
Y5.322, University of Texas Southwestern Medical Center, 5323 Harry
Hines Boulevard, Dallas, TX 75235.
| |
REFERENCES |
-
Castillo PE,
Janz R,
Südhof TC,
Malenka RC,
Nicoll RA
(1997)
The synaptic vesicle protein rab3A is essential for mossy fiber long-term potentiation in the hippocampus.
Nature
388:593-598[Medline].
-
Faux MC,
Scott JD
(1996)
Molecular glue: kinase anchoring and scaffold proteins.
Cell
85:9-12[Web of Science][Medline].
-
Ferro-Novick S,
Jahn R
(1994)
Vesicle fusion from yeast to man.
Nature
370:191-193[Medline].
-
Fykse EM,
Li C,
Südhof TC
(1995)
Phosphorylation of rabphilin-3a by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro.
J Neurosci
15:2385-2395[Abstract].
-
Garrels JI
(1979)
Two-dimensional gel electrophoresis and computer analysis of proteins synthesized by clonal cell lines.
J Biol Chem
254:7961-7977[Abstract/Free Full Text].
-
Geppert M,
Bolshakov VY,
Siegelbaum SA,
Takei K,
De Camilli P,
Hammer RE,
Südhof TC
(1994)
The role of Rab3A in neurotransmitter release.
Nature
369:493-497[Medline].
-
Geppert M,
Goda Y,
Stevens CF,
Südhof TC
(1997)
Rab3A regulates a late step in synaptic vesicle fusion.
Nature
387:810-814[Medline].
-
Greengard P
(1987)
Neuronal phosphoproteins. Mediators of signal transduction.
Mol Neurobiol
1:81-119[Web of Science][Medline].
-
Hajos F,
Wilkin G,
Wilson J,
Balazs R
(1975)
A rapid procedure for obtaining a preparation of large fragments of the cerebellar gromelruli in high purity.
J Neurochem
24:1277-1278[Medline].
-
Hosi T,
Gaber SS,
Aldrich RW
(1988)
Effect of forskolin on voltage-gated K+-channels is independent of adenylate cyclase activation.
Science
240:1652-1655[Abstract/Free Full Text].
-
Huang Y-Y,
Li XC,
Kandel ER
(1994)
cAMP contributes to mossy fiber LTP by initiating both a covalently modified early phase and macromolecular synthesis-dependent late phase.
Cell
79:69-79[Web of Science][Medline].
-
Huang Y-Y,
Kandel ER,
Varshavsky L,
Brandon EP,
Qi M,
Idzerda RL,
McKnight GS,
Bourtchouladze R
(1995)
A genetic test of the effects of mutation in PKA on mossy fiber LTP and its relation to spatial and contextual learning.
Cell
83:1211-1222[Web of Science][Medline].
-
Kato M,
Sasaki T,
Imazumi K,
Takahashi K,
Araki K,
Shirataki H,
Matsuura Y,
Ishida A,
Fujisawa H,
Takai Y
(1994)
Phosphorylation of rabphilin-3A by calmodulin-dependent protein kinase II.
Biochem Biophys Res Commun
205:1776-1784[Web of Science][Medline].
-
Li C,
Takei K,
Geppert M,
Daniell L,
Stenius K,
Chapman ER,
Jahn R,
De Camilli P,
Südhof TC
(1994)
Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C.
Neuron
13:885-898[Web of Science][Medline].
-
Li L,
Chin LS,
Shupliakov O,
Brodin L,
Sihra TS,
Hvalby O,
Jensen V,
Zheng D,
McNamara JO,
Greengard P,
Anderson P
(1995)
Impairment of synaptic vesicle clustering and of synaptic transmission and increased seizure propensity in synapsin I-deficient mice.
Proc Natl Acad Sci USA
92:9235-9239[Abstract/Free Full Text].
-
Lisman J,
Malenka RC,
Nicoll RA,
Malinow R
(1997)
Learning mechanisms: the case for CaM-KII.
Science
276:2001-2002[Abstract/Free Full Text].
-
Martin TFJ
(1997)
Stages of regulated exocytosis.
Trends Cell Biol
7:271-276. [Web of Science][Medline]
-
Nagy A,
Delgado-Escueta AV
(1984)
Rapid preparation of synaptosomes from mammalian brain using nontoxic iso-osmotic gradient material (Percoll).
J Neurochem
43:1114-1123[Web of Science][Medline].
-
Nicoll RA,
Malenka RC
(1995)
Contrasting properties of two forms of long-term potentiation in the hippocampus.
Nature
377:115-118[Medline].
-
Rosahl TW,
Geppert M,
Spillane D,
Herz J,
Hammer RE,
Malenka RC,
Südhof TC
(1993)
Short term synaptic plasticity is altered in mice lacking synapsin I.
Cell
75:661-670[Web of Science][Medline].
-
Rosahl TW,
Spillane D,
Missler M,
Herz J,
Seling DK,
Wolff JR,
Hammer RE,
Malenka RC,
Südhof TC
(1995)
Essential functions of synapsins I and II in synaptic vesicle regulation.
Nature
375:488-496[Medline].
-
Salin PA,
Malenka RC,
Nicoll RA
(1996)
Cyclic AMP mediates a presynaptic form of LTP at cerebellar parallel fiber synapses.
Neuron
16:797-803[Web of Science][Medline].
-
Sheperd GM
(1990)
In: The synaptic function of the brain, 3rd Ed. New York: Oxford UP.
-
Shirataki H,
Kaibuchi K,
Sakoda T,
Kishida S,
Yamaguchi T,
Wada K,
Miyazaki M,
Takai Y
(1993)
Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.
Mol Cell Biol
13:2061-2068[Abstract/Free Full Text].
-
Spillane DM,
Rosahl T,
Südhof TC,
Malenka RC
(1995)
Long-term potentiation in mice lacking synapsins.
Neuropharmacology
34:1573-1579[Web of Science][Medline].
-
Stahl B,
Chou JH,
Li C,
Südhof TC,
Jahn R
(1996)
Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras.
EMBO J
15:1799-1809[Web of Science][Medline].
-
Stevens CF
(1993)
Quantal release of neurotransmitter and long-term potentiation.
Cell
10:55-63.
-
Südhof TC
(1995)
The synaptic vesicle cycle: a cascade of protein-protein interactions.
Nature
375:645-653[Medline].
-
Terrian DM,
Johnston D,
Claiborne BJ,
Ansah-Yiadom R,
Strittmatter WJ,
Rea MA
(1988)
Glutamate and dynorphin release from a subcellular fraction enriched in hippocampal mossy fiber synaptosomes.
Brain Res Bull
21:343-351[Web of Science][Medline].
-
Wang JKT,
Walaas SI,
Greengard P
(1988)
Protein phosphorylation in nerve terminals: comparison of calcium/calmodulin-dependent and calcium/diacylglycerol-dependent systems.
J Neurosci
8:281-288[Abstract].
-
Wang Y,
Hofman K,
Südhof TC
(1997)
RIM: a putative Rab3 effector in regulating synaptic vesicle fusion.
Nature
388:590-593[Medline].
-
Weisskopf MG,
Castillo PE,
Zalutsky RA,
Nicoll RA
(1994)
Mediation of hippocampal mossy fiber long-term potentiation by cyclic AMP.
Science
23:1878-1882.
Copyright © 1998 Society for Neuroscience 0270-6474/98/182634-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
C. L. Brett, R. L. Plemel, B. T. Lobinger, M. Vignali, S. Fields, and A. J. Merz
Efficient termination of vacuolar Rab GTPase signaling requires coordinated action by a GAP and a protein kinase
J. Cell Biol.,
September 22, 2008;
182(6):
1141 - 1151.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. A. Gaffield and W. J. Betz
Synaptic Vesicle Mobility in Mouse Motor Nerve Terminals with and without Synapsin
J. Neurosci.,
December 12, 2007;
27(50):
13691 - 13700.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. P. Kwan, L. Xie, L. Sheu, T. Ohtsuka, and H. Y. Gaisano
Interaction Between Munc13-1 and RIM Is Critical for Glucagon-Like Peptide-1 Mediated Rescue of Exocytotic Defects in Munc13-1 Deficient Pancreatic {beta}-Cells
Diabetes,
October 1, 2007;
56(10):
2579 - 2588.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H.-C. Lu, D. A. Butts, P. S. Kaeser, W.-C. She, R. Janz, and M. C. Crair
Role of efficient neurotransmitter release in barrel map development.
J. Neurosci.,
March 8, 2006;
26(10):
2692 - 2703.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Baba, T. Sakisaka, S. Mochida, and Y. Takai
PKA-catalyzed phosphorylation of tomosyn and its implication in Ca2+-dependent exocytosis of neurotransmitter
J. Cell Biol.,
September 26, 2005;
170(7):
1113 - 1125.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Boczan, A. G. M. Leenders, and Z.-H. Sheng
Phosphorylation of Syntaphilin by cAMP-dependent Protein Kinase Modulates Its Interaction with Syntaxin-1 and Annuls Its Inhibitory Effect on Vesicle Exocytosis
J. Biol. Chem.,
April 30, 2004;
279(18):
18911 - 18919.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Calixto, E. Thiels, E. Klann, and G. Barrionuevo
Early Maintenance of Hippocampal Mossy Fiber--Long-Term Potentiation Depends on Protein and RNA Synthesis and Presynaptic Granule Cell Integrity
J. Neurosci.,
June 15, 2003;
23(12):
4842 - 4849.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. D. Burgoyne and A. Morgan
Secretory Granule Exocytosis
Physiol Rev,
April 1, 2003;
83(2):
581 - 632.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. J. O. Evans, M. C. Wilkinson, M. E. Graham, K. M. Turner, L. H. Chamberlain, R. D. Burgoyne, and A. Morgan
Phosphorylation of Cysteine String Protein by Protein Kinase A. IMPLICATIONS FOR THE MODULATION OF EXOCYTOSIS
J. Biol. Chem.,
December 14, 2001;
276(51):
47877 - 47885.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Staunton, B. Ganetzky, and M. L. Nonet
Rabphilin Potentiates Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Function Independently of rab3
J. Neurosci.,
December 1, 2001;
21(23):
9255 - 9264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. L. Foletti and R. H. Scheller
Developmental Regulation and Specific Brain Distribution of Phosphorabphilin
J. Neurosci.,
August 1, 2001;
21(15):
5461 - 5472.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. L. Foletti, J. T. Blitzer, and R. H. Scheller
Physiological Modulation of Rabphilin Phosphorylation
J. Neurosci.,
August 1, 2001;
21(15):
5473 - 5483.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Hilfiker, A. J Czernik, P. Greengard, and G. J Augustine
Tonically active protein kinase A regulates neurotransmitter release at the squid giant synapse
J. Physiol.,
February 15, 2001;
531(1):
141 - 146.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Shew, S. Yip, and B. R. Sastry
Mechanisms Involved in Tetanus-Induced Potentiation of Fast IPSCs in Rat Hippocampal CA1 Neurons
J Neurophysiol,
June 1, 2000;
83(6):
3388 - 3401.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. V. Goussakov, K. Fink, C. E. Elger, and H. Beck
Metaplasticity of Mossy Fiber Synaptic Transmission Involves Altered Release Probability
J. Neurosci.,
May 1, 2000;
20(9):
3434 - 3441.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. Prange and T. H. Murphy
Correlation of Miniature Synaptic Activity and Evoked Release Probability in Cultures of Cortical Neurons
J. Neurosci.,
August 1, 1999;
19(15):
6427 - 6438.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
O. M. Schluter, E. Schnell, M. Verhage, T. Tzonopoulos, R. A. Nicoll, R. Janz, R. C. Malenka, M. Geppert, and T. C. Sudhof
Rabphilin Knock-Out Mice Reveal That Rabphilin Is Not Required for Rab3 Function in Regulating Neurotransmitter Release
J. Neurosci.,
July 15, 1999;
19(14):
5834 - 5846.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. A. Blanpied and G. J. Augustine
Protein kinase A takes center stage in ATP-dependent insulin secretion
PNAS,
January 19, 1999;
96(2):
329 - 331.
[Full Text]
[PDF]
|
|
|
|
|
|
|
G Joberty, P. Stabila, T Coppola, I. Macara, and R Regazzi
High affinity Rab3 binding is dispensable for Rabphilin-dependent potentiation of stimulated secretion
J. Cell Sci.,
January 10, 1999;
112(20):
3579 - 3587.
[Abstract]
[PDF]
|
|
|
|
|
|
|
G. Lonart and T. C. Sudhof
Assembly of SNARE Core Complexes Prior to Neurotransmitter Release Sets the Readily Releasable Pool of Synaptic Vesicles
J. Biol. Chem.,
September 1, 2000;
275(36):
27703 - 27707.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
