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The Journal of Neuroscience, August 1, 2001, 21(15):5461-5472
Developmental Regulation and Specific Brain Distribution of
Phosphorabphilin
Davide L.
Foletti and
Richard H.
Scheller
Howard Hughes Medical Institute, Department of Molecular and
Cellular Physiology, Stanford University School of Medicine, Stanford,
California 94305-5428
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ABSTRACT |
Protein kinases and phosphatases play an important role in
modulating synaptic transmission. The synaptic protein rabphilin associates with synaptic vesicles through the small GTPase
Rab3A, binds Ca2+ and phospholipids, and
interacts with cytoskeletal elements, yet its function remains
controversial. In this study, we have generated phosphospecific
antibodies and studied the developmental, subcellular, and brain
distribution of rabphilin phosphorylated at serine-234 and serine-274.
Our results show that phosphorabphilin is present in
vivo under basal conditions in a specific subset of synapses.
The phosphorylated rabphilin is abundant in the cerebellum, midbrain,
and medulla; phosphorabphilin is specifically enriched in the climbing
fiber synapses of the cerebellar cortex. Its developmental profile
reveals a sharp and transient increase at approximately postnatal day
16, a period critical for the activity-dependent pruning of
supernumerary climbing fibers in the cerebellum. We propose that the
phosphorylation of rabphilin regulates neuronal activity through
development and in a synapse-specific manner.
Key words:
rabphilin; phosphospecific antibodies; protein kinases; development; climbing fibers; immunohistochemistry
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INTRODUCTION |
Synaptic transmission, the main form
of cell to cell communication in the nervous system, is triggered by
Ca2+ and initiated by synaptic vesicle
exocytosis and secretion of neurotransmitters. Many of the proteins
that regulate the targeting, docking, priming, and fusion of synaptic
vesicles with the plasma membrane have been identified. These proteins
belong to families of molecules with homologs that mediate
intracellular vesicle transport and include soluble
N-ethylmaleimide-sensitive factor attachment protein
receptors, Sec1-related proteins, Rabs, and Rab-effectors (for review, see Jahn and Südhof, 1999 ; Lin and Scheller, 2000; Bock et al., 2001). Whereas understanding the function
of these proteins will ultimately elucidate the basic machinery of
synaptic transmission, studying their modulation will yield insights
into some of the mechanisms that control synaptic plasticity and
therefore contribute to learning and memory.
Protein phosphorylation is a major mechanism to control protein
function. It is therefore not surprising that many of the proteins
involved in synaptic transmission have been identified as potential
targets of protein kinases (for review, see Turner et al., 1999).
Despite a large number of in vitro experiments, our
understanding of the in vivo regulation and functional
significance of most of these phosphorylation events remains
fragmentary. To study the in vivo physiological relevance of
phosphosynaptic proteins, we have generated a panel of antibodies that
recognize synaptic proteins only in their phosphorylated states. In
this report, we describe the results obtained with two phosphospecific
antibodies directed against phosphorylated rabphilin. Rabphilin,
originally identified on the basis of its GTP-dependent interaction
with the small GTPase Rab3A (Shirataki et al., 1993), has been
localized to synaptic vesicles (Mizoguchi et al., 1994; Li, 1996), from which it dissociates together with Rab3A during or after exocytosis (Stahl et al., 1996). In addition to Rab3A, several potential interacting molecules have been suggested for rabphilin, including phosphoinositides (Chung et al., 1998), rabaptin 5 (Ohya et al., 1998),
 -actinin (Kato et al., 1996), and  -adducin (Miyazaki et al.,
1994). Whereas these multiple binding partners have implicated rabphilin in exocytosis, endocytosis, and in interactions with the
cytoskeleton, its true function remains controversial. In fact,
overexpression of full-length rabphilin stimulated exocytosis in
pheochromocytoma 12 (PC12) cells, chromaffin cells, and
pancreatic cells (Chung et al., 1995; Komuro et al., 1996; Arribas
et al., 1997), but its microinjection inhibited neurotransmitter
release in squid nerve terminals (Burns et al., 1998). Furthermore, the rabphilin knock-out (KO) displayed no obvious impairments in
synaptic transmission (Schluter et al., 1999). Phosphorylation of
rabphilin occurs within its central domain on serine-234 primarily by
protein kinase A (PKA) and on serine-274 mainly by
Ca2+/calmodulin kinase II (CaMKII)
(Fykse et al., 1995). Studies with hippocampal synaptosomes and
cultured cerebellar granule cells have indicated that rabphilin can be
phosphorylated in vivo in a stimulation-dependent manner
(Fykse, 1998; Lonart and Südhof, 1998). In this report, we
have analyzed the individual contributions of the two phosphorylation
sites on rabphilin. We have identified the regions of the brain that
have high levels of phosphorabphilin, localized it to a specific subset
of synapses, and observed a striking developmental regulation of this modification.
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MATERIALS AND METHODS |
Antibodies and reagents. The mouse monoclonal
antibodies used in this study were: anti-rabphilin from Transduction
Laboratories (Lexington, KY), anti-synaptophysin from Boehringer
Mannheim (Indianapolis, IN), anti-calbindin from Swant (Bellinzona,
Switzerland), and anti-Rab3a from Synaptic Systems (Goettingen,
Germany). The nuclear marker Toto-3 was purchased from Molecular Probes
(Eugene, OR). Secondary antibodies for immunostaining were from Jackson
ImmunoResearch (West Grove, PA) and included fluorescein
isothiocyanate-conjugated AffiniPure goat anti-rabbit IgG and
Texas Red-conjugated AffiniPure goat anti-mouse IgG. Secondary
antibodies for quantitative Western blot analysis were obtained from
Amersham Pharmacia Biotech (Arlington, IL) and included anti-rabbit Ig
from donkey [125I-labeled
F(ab')2 fragment] and anti-mouse Ig from sheep
[125I-labeled
F(ab')2 fragment]. Casein kinase II (CKII;
recombinant from Escherichia coli), PKA (catalytic subunit),
and protein kinase C (PKC) were from Boehringer Mannheim (Indianapolis,
IN). CaMKII and Microcystin-LR were from Calbiochem (La Jolla, CA).
Calf intestinal alkaline phosphatase (CIP) was from New England Biolabs
(Beverly, MA). Paraformaldehyde was from Electron Microscopy Sciences
(Fort Washington, PA). Unless otherwise stated, all other reagents were obtained from Sigma (St. Louis, MO) or Fisher Biotech (Pittsburgh, PA).
Generation and purification of S234-P and
S274-P. Two peptides corresponding to amino
acids 230-239 (TRRASEARMS) and 270-279 (RRANSVQASR) of rabphilin (Li
et al., 1994; Fykse et al., 1995) were synthesized with a
phosphoserine at position 234 or 274, respectively. An additional
cysteine residue was introduced at the C terminus for coupling
purposes. The peptides were coupled to Imject maleimide-activated
keyhole limpet hemocyanin (Pierce, Rockford, IL) and used as
immunogen in rabbit. The polyclonal antisera were affinity purified as
follows. A peptide with unrelated sequence, a peptide with the same
sequence but with unphosphorylated serine (related nonphosphopeptide),
and the peptide used as immunogen (phosphopeptide) were coupled to
Imject maleimide-activated bovine serum albumin (BSA; Pierce). The
conjugates were then linked to cyanogen bromide-activated Sepharose 4B
(Sigma). The polyclonal antisera were first sequentially passed over
columns carrying the peptide with unrelated sequence and the related
nonphosphopeptide to remove nonspecific antibodies. Finally, the
antisera were affinity purified by binding and elution from a column
carrying the phosphopeptide.
Recombinant proteins and in vitro
phosphorylation. A recombinant fragment of rat rabphilin
encompassing amino acids 1-361 [wild-type (WT); a gift from
Dr. Südhof, University of Texas Southwestern Medical Center,
Dallas, TX] as well as single serine to alanine mutants at the
phosphorylation sites (S234A and S274A) were expressed and purified as
described (Li et al., 1994). The mutants were generated by PCR-mediated
site-directed mutagenesis, and the mutations were confirmed by
automated DNA sequencing. The recombinant protein syn1A11 [rat
syntaxin 1A, amino acid (aa) 4-266] was expressed and purified as
described (Yang et al., 1999). Recombinant rabphilin (WT, S234 and
S274) was in vitro phosphorylated by PKA, PKC, and CaMKII
under the following conditions. For PKA: 50 mM
MES-NAOH, pH 6.9, 10 mM
MgCl2, 0.5 mM EDTA, 1.0 mM DTT, 200 µM ATP, and
10 4 U of
PKA per 20 pmol of protein. For PKC: 50 mM
Tris-HCl, pH 7.5, 6 mM
MgCl2, 0.1% -mercaptoethanol, 100 µM CaCl2, 100 nM PMA, 200 µM ATP, and
104 U of
PKC per 20 pmol of protein. For CaMKII: 50 mM
PIPES-NaOH, pH 7.0, 10 mM
MgCl2, 0.1 mg/ml BSA, 5 µg/ml calmodulin, 500 µM CaCl2, 200 µM ATP, and 25 ng of CaMKII. In
vitro phosphorylation of recombinant syn1A11 by CKII was achieved
in 50 mM Tris-HCl, pH 7.4, 130 mM KCl, 10 mM
MgCl2, 1 mM DTT, 30 µM D-Sphingosine, 200 µM ATP, and
104 U of
casein kinase II per 20 pmol of protein. The phosphorylation reactions
were incubated for 30 min at 30°C and stopped by addition of
SDS-PAGE sample buffer. In Western blot experiments, equal amounts of total protein for each sample were resolved by SDS-PAGE and
transferred to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia
Biotech, Piscataway, NJ) according to standard protocols. Western blots
were analyzed by phosphorimaging technology (Molecular Dynamics,
Sunnyvale, CA).
Rat brain fractionation. Total rat brain or dissected rat
brain parts were homogenized with a glass-Teflon homogenizer in the
presence of phosphatase inhibitors. The buffer used was composed of 20 mM HEPES-NaOH, pH 7.4, 200 mM NaCl, 1 mM DTT, 2 mM EDTA, 20 mM
-glycerophosphate, 50 mM NaF, 50 mM Na-pyrophosphate, 2 µM
Microcystin-LR, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 1 mM PMSF. For the preparation of
the homogenate in the absence of phosphatase inhibitors, the following
reagents were omitted: -glycerophosphate, NaF, Na-pyrophosphate,
Microcystin-LR; additionally the homogenate was incubated for 30 min
with 50 U of CIP. For subfractionation into cytosol and membranes, the
homogenate was first centrifuged at 1000 × g for 15 min. The resulting postnuclear supernatant was further centrifuged at
100,000 × g for 1 hr to separate the cytosolic
fraction (supernatant) from the membrane fraction (pellet). Homogenates
were extracted with 1% Triton X-100 for 1 hr at 4°C and then spun at
100,000 × g for 1 hr to pellet the insoluble fraction.
The supernatants containing the solubilized proteins were used for
Western blotting.
Hippocampal cultures and immunostaining. Primary cultures of
hippocampal neurons were prepared from the hippocampi of 18-d-old fetal
rats as described previously (Banker and Cowan 1977; Hazuka et al.,
1999). For immunocytochemistry experiments, cells were fixed in 4%
formaldehyde and 120 mM sucrose in PBS for 20 min at room temperature (RT). After quenching the formaldehyde with 0.1 M glycine in PBS, cells were permeabilized,
and nonspecific sites were blocked in PBS containing 0.4% saponin, 2%
normal goat serum, and 1% bovine serum albumin
(permeabilization/blocking buffer). Primary antibodies, diluted in
permeabilization/blocking buffer, were applied for 1 hr at RT. After
rinsing the cells five times for 5 min with PBS, secondary antibodies
were applied for 1 hr at RT. Finally, cells were washed five times for
5 min and mounted onto slides with Vectashield (Vector Laboratories,
Burlingame, CA) as mounting medium. For immunohistochemistry
experiments on rat brain sections, postnatal day 8 (P8) and adult rats
were anesthetized with an intraperitoneal injection of Nembutal and
then sequentially perfused intracardially with ice-cold 0.1 M phosphate buffer and 4% ice-cold formaldehyde
in 0.1 M phosphate buffer. The brains were then
removed from the skull, post-fixed in the same fixative for 2-4 hr on
ice, and finally cryoprotected by immersion in 20% sucrose for 24 hr.
Frozen brains were cut with a cryostat, generating 16-µm-thick
sagittal sections that were mounted on glass slides. The staining
protocol was performed at room temperature. The sections were first
air-dried for 15 min and then permeabilized for 1-2 hr (this step also
served to block nonspecific binding sites) in permeabilization/blocking
buffer. Primary antibodies diluted in permeabilization/blocking buffer
were applied to the sections for 4 hr in a humidified chamber. After
washing the sections five times for 5 min with
permeabilization/blocking buffer, secondary antibodies were applied for
1-2 hr. Finally, the sections were rinsed as above and mounted with
Vectashield as mounting medium. Microscopy was performed with a
Molecular Dynamics laser confocal imaging system (Stanford University,
Cell Sciences Imaging Facility).
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RESULTS |
Antibodies specific to the serine-234 and serine-274 phosphorylated
forms of rabphilin
To characterize the occurrence and begin to understand the
functional significance of the phosphorylation of rabphilin, we have
generated and purified two polyclonal antibodies that recognize rabphilin only when the serines at position 234 and 274 are
phosphorylated (S234-P and S274-P, respectively). Peptides
encompassing the phosphorylation sites were synthesized in which the
relevant serine residue was included as a phospho-serine (Fig.
1A). The peptides were
coupled to keyhole limpet hemocyanin and used as immunogens in rabbits.
The polyclonal antisera were affinity purified by sequential passage
through columns carrying a peptide with unrelated sequence and a
related non-phosphopeptide (with the same sequence as the peptide used
for immunization) to remove nonspecific antibodies. Finally, the
antisera were affinity purified by specific binding and elution to the
phosphopeptide originally used as the immunogen.
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Figure 1.
The S234-P and S274-P antibodies are
specific for the respective phosphorylated forms of rabphilin.
A, Diagram of the domain structure of rabphilin. The
N-terminal domain is cysteine-rich, binds Zn2+, and
is responsible for the interaction with Rab3. The C-terminal domain
contains two C2 domains (C2A and C2B). The phosphorylation sites at
serine-234 and serine-274 are indicated by the letter P.
The corresponding sequence of the phosphopeptides used as immunogens is
listed below the diagram, S* indicates
the phosphoserine. B-D, A recombinant fragment of
rabphilin containing the two phosphorylation sites at S234 and S274
(WT, aa 1-361) as well as single serine to alanine mutants at the
phosphorylation sites of the same fragment (S234A, S274A), were
in vitro phosphorylated with the indicated kinases. In
lane 1 the recombinant rabphilin was not phosphorylated,
and in lane 11 recombinant syntaxin 1A was
phosphorylated at serine-14 with casein kinase II. One hundred and
fifty nanograms of recombinant rabphilin and 500 ng of
recombinant syntaxin were resolved by SDS-PAGE, transferred to
nitrocellulose, and probed with the S234-P and S274-P antibodies
in B and C, respectively. In
D, the Ponceau staining shows that equal amounts of
protein were loaded in each lane. Neither antibody recognizes the
unphosphorylated rabphilin (B, C, lane 1) or the
phosphoserine present on syntaxin 1A (B, C, lane 11).
S234-P recognizes the WT and the S274A mutant recombinant rabphilin
phosphorylated by PKA (B, lanes 5 and
7) and PKC (B, lanes 8 and
10). A point mutation at S234 completely abolishes this
signal (B, lanes 6 and 9).
Similarly, S274-P recognizes the WT and the S234A mutant recombinant
rabphilin phosphorylated by CaMKII (C, lanes 2 and
3), PKA (C, lanes 5 and
6), and PKC (C, lanes 8 and
9). A point mutation at S274 completely abolishes this
signal (C, lanes 7 and 10).
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Figure 1B-D illustrates the specificity of the
two antibodies. A recombinant fragment of wild-type rabphilin (WT; aa
1-361), as well as serine to alanine mutants at the two
phosphorylation sites of the same fragment (S234A and S274A,
respectively) were used as substrates for in vitro
phosphorylation with CaMKII, cAMP-dependent PKA, and PKC. Recombinant
syntaxin 1A was phosphorylated in vitro with CKII,
resulting in a stoichiometric phosphorylation on serine-14 (Foletti et
al., 2000). Equal amounts of in vitro phosphorylated recombinant syntaxin 1A, wild-type, and mutant rabphilin and
unphosphorylated rabphilin were resolved by SDS-PAGE and transferred to
nitrocellulose. The blots were probed with S234-P (Fig.
1B) and S274-P (Fig. 1C) to confirm the
specificity of the two antibodies. The Ponceau staining in Figure
1D shows that an equivalent amount of proteins was
loaded in each lane. Neither of the antibodies recognized the
unphosphorylated wild-type protein (Fig. 1B,C, lane
1), nor did they cross-react with another phosphoserine-containing
protein (syntaxin 1A phosphorylated on serine-14) (Fig.
1B,C, lane 11). S234-P is specific for rabphilin
phosphorylated at serine-234: the antibody strongly detected WT
rabphilin phosphorylated by PKA and PKC (Fig. 1B,
lanes 5 and 8), and the signal was abolished when
the S234A mutant was used in the phosphorylation reaction (Fig.
1B, lanes 6 and 9). As
expected, the S274A mutant phosphorylated by PKA and PKC was also
recognized by S234-P (Fig. 1B, lanes 7 and
10). Under our in vitro phosphorylation
conditions, PKA and PKC, but not CaMKII, phosphorylated rabphilin at
S234 equally well. Similarly, S274-P is specific for rabphilin
phosphorylated at serine-274: the antibody detected both wild-type and
the S234A mutant phosphorylated by CaMKII, PKA, and PKC (Fig.
1C, lanes 2-3, 5-6, and 8-9,
respectively). The signal was abolished when the S274A mutant was used
in the phosphorylation reaction (Fig. 1C, lanes 4, 7, and
10). Under our in vitro phosphorylation
conditions, rabphilin was efficiently phosphorylated at serine-274 by
PKA and PKC and weakly by CaMKII. Our phosphorylation results with recombinant rabphilin and purified kinases are consistent with previously reported in vitro experiments. Serine-234 was
identified as the major phosphorylation site for PKA, and CaMKII was
suggested to phosphorylate both serine-234 and serine-274, with the
second serine being the preferential site (Fykse et al., 1995). Our
experiments also clearly show, for the first time, that both serine-234
and serine-274 can be efficiently phosphorylated by PKC in
vitro.
Phosphorabphilin is particularly abundant in the cerebellum,
medulla, and midbrain
Having established the specificity of S234-P and S274-P to
in vitro-phosphorylated recombinant rabphilin, we then
investigated the in vivo occurrence of the two
phosphorylations and the specificity of the two antibodies against
native full-length rabphilin in the context of total rat brain
homogenate. Rat brains were homogenized in the presence (PI) or absence
of phosphatase inhibitors, and the homogenate prepared in the absence
of phosphatase inhibitors was additionally treated with CIP. The two
samples were subjected to Western blotting and probed with an antibody
against total rabphilin (total rabphilin; this antibody recognizes
the protein irrespective of its phosphorylation state) and the two
phosphospecific antibodies S234-P and S274-P. The entire gel of
the Western blot is shown in Figure
2A. Total rabphilin was
equally recognized as a protein of ~86 kDa in the homogenates
prepared in the presence and absence of phosphatase inhibitors (PI and
CIP, respectively) (Fig. 2A, lanes 1 and
2). Both S234-P and S274-P detected a protein whose
mobility matched that of rabphilin in the homogenate prepared in the
presence of phosphatase inhibitors (Fig. 2A, lanes 3 and 5). Absence of phosphatase inhibitors and CIP treatment completely abolished the signal (Fig. 2A, lanes 4 and
6), and no other cross-reacting bands were detected
over a molecular weight range of ~15 to ~220 kDa, further
confirming the specificity of the two antibodies.
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Figure 2.
Phosphorylated rabphilin is abundant in the
midbrain, medulla, and cerebellum. A, Rat brain
homogenates were prepared in the presence (PI) or
absence of phosphatase inhibitors. Homogenates prepared in the absence
of PI were additionally treated with calf intestinal phosphatase
(CIP). Two hundred micrograms of total protein was
resolved by SDS-PAGE, transferred to nitrocellulose, and probed with a
commercial antibody against rabphilin (total rabphilin, lanes
1 and 2), as well as the S234-P and S274-P
antibodies (lanes 3-4 and 5-6,
respectively). The total rabphilin antibody recognizes rabphilin as a
single band of ~86 kDa in both PI- and CIP-treated samples
(lanes 1 and 2). S234-P and S274-P
detects a single band with the same molecular weight only from
homogenates prepared in the presence of phosphatase inhibitors
(lanes 3 and 5, respectively). The
absence of phosphatase inhibitors and the additional treatment with CIP
completely abolishes the signal, further confirming the
phosphospecificity of the antibodies (lanes 4 and
6). B, An adult rat brain was
dissected into olfactory bulb (OB), cortex
(CTX), hippocampus (HC), midbrain
(MB), medulla (ME), cerebellum
(CB), and spinal cord (SC). The tissues
were homogenized in the presence of phosphatase inhibitors and
extracted with Triton X-100. Fifty micrograms of total protein from
each sample was resolved by SDS-PAGE, transferred to nitrocellulose,
and probed for total rabphilin (bottom panel), as
well as for its phosphorylated forms using the S234-P and S274-P
antibodies (top and middle panels,
respectively). Although total rabphilin is present in similar amounts
throughout the regions tested, both phosphorylated forms of rabphilin
are particularly enriched in midbrain, medulla, and cerebellum.
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After detecting phosphorabphilin in a total rat brain homogenate, we
sought to determine which regions of the rat brain, if any, were
particularly enriched for the phosphorylated forms of the protein.
Equal amounts of total protein from the olfactory bulb (OB), cortex
(CTX), hippocampus (HC), midbrain (MB), medulla (ME), cerebellum (CB),
and spinal cord (SC) were resolved by SDS-PAGE and transferred to
nitrocellulose. The blots were probed with S234-P, S274-P, and
total rabphilin. Total rabphilin was present in similar amounts in
all areas of the brain tested (Fig. 2B, bottom
panel). In contrast, rabphilin phosphorylated at both
serine-234 (Fig. 2B, top panel) and serine-274
(Fig. 2B, middle panel) was strongly detected
in the cerebellum, medulla, and midbrain, with only fainter signals in
the other regions of the brain. These results clearly show that
rabphilin phosphorylated at both serine-234 and serine-274 is present
in vivo in the rat brain under basal, unstimulated
conditions. The data also indicate that the phosphorylation of
rabphilin is differentially regulated in distinct anatomical regions of
the brain. Finally, the identical distribution of the two
phosphorylated forms of rabphilin suggests that the regulation and
functional significance of the two phosphorylation sites may be linked.
Phosphorabphilin is transiently upregulated during development
Because we have detected phosphorabphilin in the brain of adult
rats under basal, unstimulated conditions, we investigated whether the
phosphorylation of rabphilin is developmentally regulated. We analyzed
the relative developmental profiles of total rabphilin compared with
both forms of phosphorabphilin in rat brains and in the absence of
Rab3a in brains obtained from Rab3a knock-out animals. First, the
brains from rats of various ages, from embryonic day 18 through
development into adulthood, were collected and processed for Western
blotting. The blots were probed for total rabphilin (Fig.
3A, open squares), rabphilin
S234-P (Fig. 3B, open squares), and rabphilin S274-P (Fig.
3C, open squares). The amount of synaptic proteins increases
steadily during development and reaches a plateau around the third week
after birth (Foletti et al., 2000). As shown in Figure 3A
(open squares), rat total rabphilin follows this pattern. In
contrast, the presence of both forms of rat phosphorabphilin during
development is marked by a sharp increase starting at postnatal day 10, peaking at day 16, and quickly declining afterwards (Fig. 3B,C,
open squares). As observed for the distribution in the different
regions of the rat brain, the two phosphorylated forms of rabphilin
behave similarly during development. This further suggests that the
phosphorylation of the two serines may be regulated together and that
both phosphorylation events are implicated in modifying the function of
the protein.
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Figure 3.
Phosphorabphilin is transiently upregulated during
development. The brains of wild-type rats (Rat, open
squares) and Rab3a knock-out mice (KO, closed
circles) of various ages (E, embryonic;
P, postnatal) were homogenized in the presence of
phosphatase inhibitors and extracted with Triton X-100. Twenty-five
micrograms of total protein from each sample was resolved by SDS-PAGE,
transferred to nitrocellulose, and probed for total rabphilin
(A), rabphilin S234-P (B),
and rabphilin S274-P (C). The
graphs illustrate the normalized quantitative
representation of the Western blots shown in the insets
(the Western blots were generated in separate experiments and are not
directly comparable). The developmental regulation of rabphilin and of
both forms of phosphorabphilin in wild-type mice (data not shown) was
equivalent to that observed in rats. We therefore matched the
quantified values of total rabphilin and phosphorabphilin in wild-type mice and rats
(open squares) to normalize the values obtained in Rab3A
knock-out animals (closed circles). A,
The amount of total rabphilin increases steadily during development and
plateaus at approximately P22 in both rats and Rab3a knock-out mice.
B (rabphilin S234-P) and C (rabphilin
S274-P), both phosphorylated forms of rabphilin, exhibit a sharp and
transient increase beginning at P8-P10 and peaking at P16, followed by
a sharp decline in phosphorylation levels. This phenomenon is observed
with different amplitudes in both rats (open squares)
and Rab3a knock-out mice (closed circles).
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Considering the reported interaction between Rab3a and rabphilin, we
next compared the developmental regulation of phosphorabphilin in
wild-type and Rab3a knock-out mice. In the Rab3a knock-out mice, the
amount of rabphilin present in the brain is reduced to ~40-50% of
wild type (Geppert et al., 1994). Moreover, in neurons lacking Rab3a,
rabphilin appears to be trapped in the cell body, unable to reach its
final localization on synaptic vesicles at nerve terminals. Protein
instability and faster degradation were suggested as reasons to explain
the reduced levels of rabphilin in the absence of Rab3A (Li et al.,
1994). Brains from wild-type and Rab3a KO mice of various ages were
collected and processed for Western blotting and probed with total
rabphilin, S234-P, and S274-P. The developmental profiles of
total rabphilin and phosphorabphilin in wild-type mice (data not shown)
were equivalent to those observed in rats (Fig. 3, open
squares). We therefore matched the quantified values of total
rabphilin and phosphorabphilin in wild-type mice and rats to normalize
the values obtained in Rab3A knock-out animals. In Figure
3A-C, the closed circles represent the normalized
developmental profiles of total rabphilin and phosphorabphilin in Rab3a
knock-out animals. As observed in rats, in both wild-type (data not
shown) and Rab3a KO mice (Fig. 3A, closed circles), total
rabphilin increased steadily up to 3 weeks after birth and remained at
a plateau afterwards. As expected, we detected reduced levels of total
rabphilin in the Rab3a KO brains. We observed that the relative level
of rabphilin in the Rab3a KO compared with wild-type animals decreased
during development. In fact, at 1 d after birth, Rab3a KO mice
still had ~85% of the rabphilin amount present in wild-type animals
of the same age, but this proportion decreased to ~45% by postnatal
day 24, the same value detected in adult animals (data not shown).
Rabphilin S234-P and S274-P showed a transient peak at approximately
postnatal day 16 in both wild-type (data not shown) and in Rab3a KO
mice (Fig. 3B,C, closed circles). The peaks of
phosphorabphilin in wild-type and Rab3a knock-out mice appeared broader
than in rat, possibly reflecting differences in the time course of
development in the two species. Interestingly, the amplitude of the
peak of phosphorabphilin in Rab3a KO animals was strongly reduced, to a
level lower than that which could simply be accounted for by the
reduced amount of total rabphilin. It therefore appears that although
the striking transient increase in phosphorylation of rabphilin can
occur in the absence of Rab3a, its magnitude is reduced in the absence of the small GTP-binding protein, suggesting that the interaction between rabphilin and Rab3a is important for its phosphorylation.
Phosphorabphilin staining in cultured
hippocampal neurons
To control for the specificity of the phosphorabphilin antibodies
in immunostaining, we took advantage of the Rab3A knock-out in which
rabphilin is not properly targeted to synapses (Li et al., 1994). We
reasoned that any synaptic staining of phosphorabphilin observed in
brain sections of wild-type animals should be severely reduced or
abolished in sections obtained from Rab3A knock-out animals. Figure
4 shows confocal images of the cerebellar
cortex taken from sagittal sections of adult wild-type (Fig.
4A,B) and Rab3a knock-out animals (Fig.
4C,D). We double stained with S234-P and an antibody
against calbindin to label Purkinje cells (Fig. 4A,C)
and with S234-P and the nuclear marker Toto-3 (Fig.
4B,D). In the cerebellar cortex of wild-type mice,
S234-P detected punctate, synaptic-like structures in the molecular
layer (Fig. 4A, ML) and larger structures
in the granular layer (Fig. 4B). No immunoreactivity was observed in the cerebellar cortex of Rab3a knock-out animals. Equivalent results were obtained with S274-P (data not shown). Additionally, preincubation of both phosphorabphilin-specific antibodies with their respective phosphopeptides (but not with the
non-phosphopeptides) completely abolished the signal (data not shown).
We conclude that the two phosphorabphilin antibodies are specific in
immunostaining.
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Figure 4.
Specificity of the phosphorabphilin antibodies in
immunostaining. In mice lacking Rab3A the overall level of rabphilin is
reduced to ~40% of wild type and the protein is not targeted to the
synapses. We assessed the specificity of S234-P and S274-P by
comparing their immunoreactivity in staining of brain sections obtained
from wild-type and Rab3A knock-out animals. All of the panels are
single confocal images taken from sagittal sections of the cerebellar
cortex of adult wild-type or Rab3A knock-out mice. The staining for
both phosphorylated forms of rabphilin was equivalent.
A, B, Staining of sections obtained from
wild-type animals; C, D, staining in Rab3A knock-out
sections. The panels represent merged images of double labeling for
rabphilin S234-P (green) with an antibody against
calbindin (red in A and C,
stains Purkinje cells) or the nuclear marker Toto-3 (red
in B and D). In the cerebellar cortex of
wild-type animals, phosphorabphilin was detected as a punctate
synaptic-like staining in the molecular layer (A)
and staining of structures in the granular layer
(B). Equivalent double labeling in Rab3A
knock-out sections (C, D) failed to reveal staining for
phosphorabphilin, confirming the specificity of the antibodies.
GL, Granular layer; ML, molecular layer;
Pu, Purkinje cells. Scale bars: A-L, 20 µm.
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To assess the subcellular localization of phosphorabphilin, we cultured
embryonic hippocampal neurons and performed immunocytochemistry with
S234-P, S274-P, total rabphilin, and known markers for synapses and axons. Mature, 14 d in vitro (div)
hippocampal neurons with extensive axonal and dendritic trees and
abundant synapses were used for staining. In double-labeling
experiments in which neurons were stained for total rabphilin and the
synaptic vesicle marker SV2A, we observed rabphilin in a synaptic-like
pattern in the vast majority of the neurons (Fig.
5A-C). In contrast, both
rabphilin S234-P (Fig. 5D-F) and rabphilin S274-P
(Fig. 5G-L), although also clearly present at
synapses, were detected only in a small subset of axons. Rabphilin is a
synaptic protein that has been clearly localized to synaptic vesicles
by electron microscopy studies (Mizoguchi et al., 1994; Li, 1996), so
we expected to find phosphorabphilin colocalized with synaptic vesicle
markers at synapses of cultured hippocampal neurons. Interestingly, and consistent with its nonhomogeneous distribution in the rat brain observed by Western blot analysis (Fig. 2), phosphorabphilin was detected only in a subset of synapses (Fig. 5E,H),
whereas total rabphilin had a widespread synaptic distribution in these
cultures (Fig. 5B). Thus, phosphorabphilin is not only more
abundant in specific regions of the brain but also is present only in a
subset of neurons and likely even only in a subset of synapses within a
given axon. This suggests that the phosphorylation of rabphilin is a
functional modification of the protein required in particular types of
synapses, or synapses in a specific physiological state.
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Figure 5.
Phosphorylated rabphilin is localized to synapses
in a subset of axons in mature cultured hippocampal neurons. Embryonic
cultures of hippocampal neurons at 14 div were fixed and stained with a
panel of antibodies. A-C, Co-staining for the synaptic
vesicle protein SV2A (A) and for total rabphilin
(B, C is the merged image) shows that total rabphilin is
present at synapses in all neurons. D-I, Co-staining
for SV2A (D, G) and rabphilin S234-P
(E) or rabphilin S274-P
(H) reveals that phosphorylated
rabphilin is also present at synapses (arrows in
D-I), but only in a small subset of axons.
Large arrowheads in D-F point to axons
devoid of rabphilin S234-P staining, and small
arrowheads in G-I point to SV2A-labeled
synapses that do not have detectable rabphilin S274-P. F
and I are the respective merged images.
J-L, Co-staining for the plasma membrane protein
syntaxin1A (J) and rabphilin S274-P (K,
L is the merged image) shows the synaptic localization of
phosphorylated rabphilin in an axon that runs along and makes contacts
with a dendrite. Scale bars: A-I, 10 µm;
J-L, 2 µm.
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Phosphorabphilin is enriched in the climbing fiber synapses of the
cerebellar cortex
We next focused our attention on the cerebellum, because abundant
phosphorabphilin was detected by Western blot in this region of the
brain. Confocal images were obtained at medium and high magnification
from double-labeled sagittal sections of adult and postnatal day 8 rat brains.
Figure 6A-C shows a
double staining with Toto-3, a nuclear marker (Fig.
6A), and the antibody against total rabphilin (Fig. 6B,C is the merged image). The characteristic
structure of the cerebellar cortex is illustrated by the nuclear
staining. Between the granular layer (GL) and the molecular layer (ML)
lies the single-cell layer of Purkinje cells. Total rabphilin was
strongly and homogeneously detected in the molecular layer and in
structures in the granular layer; some of these structures likely
represent glomeruli or rosettes, the synapses of mossy fibers onto the
dendrites of granule cells. The other structures, with a characteristic staining just underneath the somas of the Purkinje cells, may be basket
cell nerve terminal plexuses, or pinceaux, which are wrapped around the
base and initial axon segments of the Purkinje neurons. In Figure
6D-F a similar double-labeling experiment was conducted with Toto-3 (Fig. 6D) and S234-P (Fig.
6E,F is the merged image). As for total rabphilin,
phosphorabphilin was detected in structures that are likely to be
glomeruli and pinceaux in the granular layer, but the staining in the
molecular layer appeared much more restricted. Figure 6G-I
shows a co-staining for calbindin, a marker for Purkinje cells (Fig.
6G), and S234-P (Fig. 6H,I is the
merged image). The phosphorabphilin staining in the molecular layer
appeared punctate-like and concentrated in the portion closest to the
Purkinje cell layer. The same staining pattern was obtained with
S274-P (Fig. 7).
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Figure 6.
Phosphorabphilin is present in the rat cerebellar
cortex. All of the panels are single confocal images taken from
sagittal sections of the cerebellar cortex of adult rats. The staining
for both phosphorylated forms of rabphilin was equivalent.
A-C, Double staining with Toto-3 (A,
stains the nuclei) and the total rabphilin antibody (B,
C is the merged image) shows that total rabphilin is
homogeneously expressed in the molecular layer and in some structures,
likely glomeruli and pinceaux, in the granular layer.
D-F, Double labeling with Toto-3
(D) and S234-P (E, F is the
merged image) results in a similar staining of structures in the
granular layer but a more restricted staining in the molecular layer
for phosphorabphilin compared with total rabphilin.
G-I, The nonhomogeneous presence of phosphorabphilin in
the molecular layer is further confirmed in a co-staining for calbindin
(G, stains the Purkinje cells) and rabphilin S234-P
(H, I is the merged image). The staining appears
punctate and concentrated in the portion of the molecular layer closest
to the Purkinje cell layer. Scale bars: A-F, 200 µm; G-I, 100 µm.
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Figure 7.
Phosphorabphilin is present in a subset of
synapses in the molecular layer and in the granular layer. All of the
panels are single (A-F) or collapsed
(G-L) serial confocal images taken from sagittal
sections of the cerebellar cortex of adult rats. The staining for both
phosphorylated forms of rabphilin was equivalent. A-C,
High magnification images of double staining for nuclei with
Toto-3 (A) and phosphorabphilin with
Ser274-P (B, C is the merged image). In the molecular
layer intense staining for phosphorabphilin is observed in small
synaptic-like structures (some of which are indicated by the
arrows; Pu marks the large nucleus of a
Purkinje cell at the boundary between the molecular and granular
layers). D-F, The synaptic identity of the
phosphorabphilin staining in the molecular layer was confirmed by a
double staining for the synaptic vesicle protein synaptophysin
(D) and for rabphilin S234-P (E, F
is the merged image). The molecular layer is packed with synapses, for
the most part contributed by the parallel fibers, the axons of
the granule cells. The double staining shows clear colocalization of
the phosphorabphilin-containing structures with a small subset of
particularly brightly stained synapses (some of which are indicated by
arrows), whereas no phosphorabphilin is detected in the
rest of the synapses in the molecular layer. G-L, The
phosphorabphilin staining in the granular layer was further
characterized at high magnification with double staining with the
S274-P (H) or S234-P
(K) antibodies together with staining for nuclei
with Toto-3 (G) or synaptic vesicles with
-synaptophysin antibody (J). I
is the merged image of G and H, and
L is the merged image of J and
K. In the granular layer, phosphorabphilin is detected
in areas free of cell bodies of the densely packed granule cells
(arrowheads in G-I). This is
where the glomeruli are located, as confirmed by the
colocalization with synaptophysin (arrowheads in
J-L). Note that only a subset of glomeruli stains
positive for phosphorabphilin. Scale bars: A-L, 10 µm.
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We further analyzed the phosphorabphilin staining in both the granular
and molecular layers at higher magnification (Fig. 7). In Figure 7,
A-F, phosphorabphilin identified with S274-P (Fig. 7B,E) is shown in a double staining with Toto-3 (Fig.
7C is the merged image of A and B) and
the synaptic marker synaptophysin (Fig. 7F is the merged
image of D and E). In the molecular layer, phosphorabphilin was detected in strongly labeled small synaptic-like structures (Fig. 7B,C, arrows), whose identity as synapses
was confirmed by their overlap with the staining for synaptophysin (Fig. 7E,F). The molecular layer contains few cells
and a large number of synapses, primarily contributed by the contacts
between parallel fibers and Purkinje cell dendrites. In contrast to
total rabphilin, which was homogeneously present at synapses throughout the entire molecular layer (Fig. 6A-C),
phosphorabphilin was clearly restricted to a subset of synapses that
were strongly labeled with the anti-synaptophysin antibody. In the
granular layer, phosphorabphilin was found in glomeruli or rosettes, as
indicated by the double labeling with S274-P (Fig.
7H) and Toto-3 (Fig. 7G,I is the merged image). This was confirmed by the colocalization of phosphorabphilin and a synaptic marker in sections stained with anti-synaptophysin (Fig.
7J) and S234-P (Fig. 7K,L is the merged
image). Interestingly, as observed for total rabphilin (Fig.
6A-C) (Li et al., 1994), some but not all the
glomeruli contain phosphorabphilin (Fig. 7J-L,
arrowheads indicate glomeruli positive for both
synaptophysin and phosphorabphilin).
The presence of phosphorabphilin in a subset of synapses in the
molecular layer, as opposed to the widespread distribution of total
rabphilin, prompted us to further investigate the nature of these
synapses. Figure 8A-C
is an example of a double-labeling experiment with antibodies against
calbindin (Fig. 8A) and rabphilin S234-P (Fig.
8B,C is the merged image). Shown is part of the
molecular layer in the cerebellar cortex, with calbindin detected in
the dendrites of the Purkinje cells (Fig. 8A, the
arrowheads indicate the proximal trunk of a Purkinje cell
dendrite). Phosphorabphilin is found in synapses (Fig. 8B,
arrows) that decorate the proximal aspect of the dendrite (Fig.
8C). This staining is characteristic of climbing fiber
synapses, one of the two major input pathways of the cerebellum. To
confirm the identity of these synapses, we took advantage of their
particular developmental regulation. Early in development, climbing
fibers establish synapses only on the soma of Purkinje cells. Later, as
the Purkinje cells mature and extend their dendrites into the molecular
layer, these synapses disappear and new synapses are formed, mostly on
the proximal part of the dendritic tree. We stained rat brain sagittal
sections from postnatal day 8 animals. In the experiment shown in
Figure 8D-F, we used Toto-3 to stain nuclei (Fig.
8D) and detected phosphorabphilin with S274-P
(Fig. 8E,F is the merged image). Purkinje cells, identified by their large nucleus in Figure 8D (Pu),
lie at the boundary between the granular and molecular layers. Their
cell body was decorated with synapses positive for phosphorabphilin (Fig. 8E,F). In Figure
8G-I, rabphilin S274-P (Fig.
8H) was detected together with calbindin (Fig.
8G,I is the merged image). Intense synaptic-like
phosphorabphilin staining was again observed on the soma of the
Purkinje cells, but no staining was observed on their short immature
dendrites. The synaptic nature of the phosphorabphilin staining was
confirmed in the double-staining experiment of Figure 8J-L. The staining generated by S274-P (Fig.
8K) overlapped with some of the synapses labeled with
anti-synaptophysin (Fig. 8J,L is the merged image).
As observed above, only synapses on the Purkinje cell bodies (Pu)
contained phosphorabphilin (Fig. 8J-L, arrows),
whereas other synapses in both the granular and molecular layers were
phosphorabphilin-negative (Fig. 8J,L, arrowheads). Therefore, the presence of phosphorylated rabphilin is selective for
the climbing fiber synapses, whereas total rabphilin is also found in
the numerous parallel fiber synapses, the other excitatory input onto
the Purkinje cell dendrites in the molecular layer. This is consistent
with what we observed in cultured neurons and demonstrates that the
phosphorylation of rabphilin is physiologically regulated in a specific
subset of synapses.
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Figure 8.
Phosphorabphilin is enriched in climbing fiber
synapses. All of the panels are single confocal images taken from
sagittal sections of the cerebellar cortex of adult rats
(A-C) or postnatal day 8 animals
(D-L). The staining for both phosphorylated
forms of rabphilin was equivalent. A-C, Double staining
for calbindin (A, stains the Purkinje cell dendrites)
and rabphilin S234-P (B) in the molecular layer.
The merged image (C) reveals that the
phosphorabphilin-containing synapses (arrows) decorate
the proximal aspect of the Purkinje cell dendrites
(arrowheads). This staining is characteristic for
climbing fiber synapses. To confirm the identity of these synapses, we
double-stained brain sections of postnatal day 8 animals. At this age
the climbing fibers make synapses exclusively on the cell body of the
Purkinje cells, later in development these synapses disappear and new
synapses are formed climbing up the Purkinje cell dendrites.
D-F, Double staining with the nuclear marker Toto-3
(D, note the very large nuclei of the Purkinje cells)
and with the S274-P antibody (E). As shown in
the merged image (F), phosphorabphilin staining
is restricted to the cell body of the Purkinje cells.
G-I, The double staining for calbindin to stain
Purkinje cells (G) and for rabphilin S274-P
(H) confirms the presence of
phosphorabphilin-containing synapses on the cell bodies but not on
the growing dendrites of the Purkinje cells
(I). J-L, The synaptic
nature of the staining for phosphorylated rabphilin is further
corroborated by a double labeling with antibodies for the synaptic
vesicle protein synaptophysin (J) and for
rabphilin S274-P (K, L is the merged image). The
phosphorabphilin immunoreactivity on the cell bodies of Purkinje cells
colocalizes with the synaptic staining of synaptophysin
(arrows), but other synapses in the molecular and
granular layer (some of which are indicated by the
arrowheads) are not stained for phosphorabphilin. Scale
bars: A-L, 10 µm.
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DISCUSSION |
Although the basic core machinery for the release of
neurotransmitters is likely shared by most neurons, its regulation by modulatory mechanisms determines in part the strength of individual synapses and therefore contributes to the process of synaptic plasticity. One such modulatory mechanism is protein phosphorylation. Protein kinases and phosphatases have been implicated in regulating synaptic plasticity (for review, see Schulman, 1995; Tokuda and Hatase,
1998), and numerous synaptic proteins have been reported as potential
targets for phosphorylation (for review, see Turner et al., 1999). Many
studies on phosphoproteins have been performed in vitro with
recombinant proteins and purified kinases. In vivo studies
have generally relied on phosphoamino acid analysis, phosphopeptide mapping, and metabolic labeling with inorganic
32P followed by immunoprecipitation of the
protein under study. These approaches do not allow accurate
localization of the protein of interest at a subcellular level or in
specific synapses within the brain and are not easily amenable to
developmental studies. To address these issues, we have generated
phosphospecific antibodies, reagents that detect a protein only when it
is phosphorylated at a specific residue. In this report, we describe
our work with two phosphospecific antibodies directed against
serine-234 and serine-274 of rabphilin, a synaptic protein that
interacts with the small GTPase Rab3a and whose function is still controversial.
We show that rabphilin is phosphorylated in vivo under basal
conditions at both serine-234 and serine-274. Western blot analysis revealed that although rabphilin is quite homogeneously expressed, both
forms of phosphorabphilin are particularly abundant in the cerebellum,
midbrain, and medulla. We further investigated the specific
distribution of phosphorabphilin by immunocytochemistry in cultured
hippocampal neurons and immunohistochemistry on rat brain sections. In
neurons, although there was a widespread synaptic presence of
rabphilin, both rabphilin S234-P and rabphilin S274-P were detected
only in a small subset of synapses. In the cerebellum, rabphilin
appeared localized to all synapses of the molecular layer, but both
forms of phosphorabphilin were found exclusively in the climbing fiber
synapses. Although it has been shown previously that not all synapses
contain rabphilin (Li et al., 1994; Schluter et al., 1999; Von
Kriegstein et al., 1999), we show here that even within the synapses
that express the protein, only in a subset is rabphilin modified by
phosphorylation. In the cerebellum, Purkinje cells constitute the only
output of information. The dendrite of a single Purkinje cell, with
150,000-175,000 excitatory synaptic connections, receives more
synaptic inputs than any other neuron in the brain (Ito, 1984). The
vast majority of these synapses are contributed by parallel fibers, the
axons of granule cells. In contrast, the other source of excitatory
input to each Purkinje cell is provided by some 300 synapses coming
from a single climbing fiber, an axonal projection from the inferior
olive (Ito, 1984). Parallel and climbing fibers contact separate,
nonoverlapping regions of the Purkinje cell dendrite. Climbing fiber
synapses are established on the thick, smooth part of the dendrite,
whereas the parallel fiber synapses are made exclusively on the spiny branchelets (the small spine-covered tertiary dendrites of the Purkinje
cell) (Palay and Chan-Palay, 1974). We have shown that under basal
conditions, rabphilin is present at both types of synapses in the
cerebellar cortex, but that phosphorabphilin is detected only in the
climbing fibers. Phosphorabphilin is therefore present at synapses that
exert a critical effect on in-flow of cerebellar information,
suggesting that it may regulate aspects of the physiological state of
these particular synapses.
Another potential role for phosphorabphilin was revealed by a survey of
its developmental profile. Rabphilin levels increased steadily after
birth and reached a plateau after ~3 weeks, a pattern followed by
several synaptic proteins (Foletti et al., 2000). In contrast, both
rabphilin S234-P and rabphilin S274-P levels showed a sharp increase
that started at postnatal day 10-12, peaked at day 16, and declined
afterwards. The amplitude of this phenomenon was decreased but not
abolished in mice lacking Rab3a. Intriguingly, the peak of
phosphorabphilin level at postnatal day 16 exactly falls into the
critical period for activity-dependent synapse elimination in the
cerebellum. Refinement of redundant connections formed at earlier
developmental stages progresses through synapse elimination, one of the
final steps in neuronal circuit formation (Lohof et al., 1996). The
synapses between climbing fibers and Purkinje cells undergo such a
pruning mechanism. In early postnatal days, multiple climbing fibers
contact individual Purkinje cells. Elimination of supernumerary
climbing fibers reduces their number to a strict one-to-one
relationship with each Purkinje cell (Changeaux et al., 1973; Crepel,
1982; Ito, 1984; Lohof et al., 1996). This process progresses over
three distinct phases, the last of which depends on NMDA
receptor-mediated activity and occurs within a narrow time-window
between postnatal day 15 and 16 (Rabacchi et al., 1992; Kakizawa et
al., 2000). This is the time during development when we observe peak
levels of phosphorabphilin in the brain, and immunohistochemistry shows
abundant phosphorabphilin in climbing fiber synapses. It is therefore
tempting to speculate that phosphorylation of rabphilin within climbing
fiber synapses may be involved in the events responsible for synapse
elimination. In the analysis of rabphilin knock-out mice, the synaptic
architecture of the brain, including the cerebellum, appeared normal
(Schluter et al., 1999). Because climbing fiber synapses are massively
outnumbered by parallel fiber synapses, it is not surprising that the
analysis of the synaptic staining in the cerebellar cortex did not
reveal any aberration in the organization of the climbing fibers. It would be interesting to test, electrophysiologically, if there is
evidence for supernumerary climbing fibers contacting each Purkinje
cell in the cerebellum of rabphilin knock-out animals.
Taken together, these results suggest that the phosphorylation of
rabphilin is a modulatory mechanism to regulate the function of the
protein to respond to particular physiological needs during synaptogenesis and synaptic activity in a cell- and synapse-specific manner.
Rabphilin has a tripartite domain structure. The N-terminal portion of
the protein contains a Zn2+-finger and is
responsible for the binding to Rab3a (Yamaguchi et al., 1993; Li et
al., 1994; Ostermeier and Brunger, 1999). The C-terminal region of
rabphilin is comprised of two C2 domains that have been shown to bind
Ca2+ and phospholipids (Yamaguchi et al.,
1993; Oishi et al., 1996; Chung et al., 1998; Ubach et al., 1999). The
two phosphorylation sites are in the middle of the molecule between the
N-terminal domain that binds Rab3a and connects rabphilin to the
synaptic vesicle, and the C-terminal region with the two C2 domains
that bind phospholipids in Ca2+-dependent
manner. It therefore seems that they are optimally located to modulate
the activity of the protein.
The function of rabphilin remains controversial. A rabphilin knock-out
study showed that rabphilin is not essential, in fact the knock-out
animals are viable and fertile without obvious impairments in synaptic
transmission (Schluter et al., 1999). Yet, evidence from overexpression
and microinjection experiments strongly implicates rabphilin in the
exocytotic process. Presynaptic microinjection of full-length rabphilin
or its N- and C-terminal domains inhibited neurotransmitter release in
squid nerve terminals (Burns et al., 1998). Similarly, microinjection
of the N- and C-terminal fragments of rabphilin into mouse metaphase II
eggs inhibited cortical granule exocytosis at fertilization (Masumoto
et al., 1996). Furthermore, overexpression of full-length rabphilin or
various fragments and mutants of the protein in PC12 cells, chromaffin
cells, and pancreatic cells have been shown to stimulate or inhibit
exocytosis, respectively (Chung et al., 1995; Komuro et al., 1996;
Arribas et al., 1997; Joberty et al., 1999).
As mentioned above, rabphilin is not present at all synapses. Of
particular interest is its absence from ribbon synapses in the outer
plexiform layer of the retina in rodents (Von Kriegstein et al., 1999).
The presynaptic aspect of these terminals contains electron-dense
projections (ribbons) at the plasma membrane. Ribbons are thought to
bind synaptic vesicles and guide them to the active zone for fusion, a
process that accelerates the delivery of vesicles for continuous
exocytosis. It is possible that the absence of rabphilin (and synapsin,
but not the other synaptic proteins tested) from these synapses
provides insight into its function. Synapsin is one of the
best-characterized synaptic phosphoproteins and its role in the release
of synaptic vesicles from the reserve pool is regulated by
phosphorylation (Greengard et al., 1993; Hosaka et al., 1999). At rest,
synaptic vesicles in the reserve pool are thought to be anchored to the
cytoskeleton through dephosphorylated synapsin. After an action
potential and stimulation of protein kinases (CaMKII and PKA), synapsin
becomes phosphorylated, changes its conformation and disengages
synaptic vesicles from the cytoskeleton. By analogy, rabphilin bound to
synaptic vesicles via Rab3a may be involved in synaptic vesicle
mobilization and targeting from the reserve pool to the active zone.
This role could be bypassed in ribbon synapses, where the ribbon itself
constitutes the physical link between the pool of synaptic vesicles and
the site of exocytosis. Although synapsin may exert its role in most if
not all synapses, we propose that phosphorylation of rabphilin
modulates its activity, possibly in the mobilization and targeting of
synaptic vesicles, in a specific subset of synapses or under particular
physiological conditions.
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FOOTNOTES |
Received March 6, 2001; revised May 11, 2001; accepted May 17, 2001.
We thank Dr. Susan Palmieri for assistance with confocal microscopy and
Dr. Susan McConnell and Jeremy Blitzer for critical reading of this manuscript.
Correspondence should be addressed to Richard H. Scheller, Genentech,
Inc., 1 DNA Way, South San Francisco, CA 94080-4990. E-mail:
scheller{at}gene.com.
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ABSTRACT
INTRODUCTION
