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The Journal of Neuroscience, June 15, 2000, 20(12):4535-4544
Phosphorylated Syntaxin 1 Is Localized to Discrete Domains Along
a Subset of Axons
Davide L.
Foletti,
Richard
Lin,
Michael A. F.
Finley, 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 |
Syntaxin 1 is a SNARE protein that plays a central role in
synaptic vesicle (SV) exocytosis. We generated an antibody that specifically recognizes a casein kinase II-mediated phosphorylation on
serine-14 of syntaxin 1. In this report we show that this
phosphorylation occurs in vivo and is developmentally
regulated in the rat brain, rising to a level of 40% of the total
syntaxin in adult animals. Phosphorylated syntaxin is preferentially
associated with SNAP-25 and localizes to discrete domains of the axonal
plasma membrane that do not colocalize with pools of synaptic vesicles.
These phosphosyntaxin domains may define fusion sites for a novel class of vesicles outside classical active zones.
Key words:
syntaxin 1; phosphorylation; casein kinase II; SNAREs; exocytosis; immunohistochemistry
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INTRODUCTION |
Regulation of plasma membrane
dynamics is critical to the function of the nervous system in many
ways. For example, during development, axon and dendrite outgrowth
takes place through membrane addition at the growth cone, whereas
synapse formation includes the insertion and/or localization of
specific proteins at sites of cell contact. In the adult,
neurotransmitter release is accomplished through a cycle of vesicle
exocytosis and endocytosis. Finally, memory formation likely takes
place through the modulation of presynaptic neurotransmitter release
and/or postsynaptic receptor organization and requires the intricate
regulation of membrane trafficking events.
Crucial steps in the dynamics of the plasmalemma are the addition of
new membrane through the process of vesicle fusion and the retrieval of
membrane by endocytosis. Several genes or gene families have been
implicated in the membrane fusion process and are therefore likely
targets for controlling the dynamics of the neuronal plasma membrane.
The products of three of these genes, VAMP (also called
synaptobrevin), SNAP-25, and syntaxin, are collectively referred
to as SNAREs (Südhof, 1995 ). These proteins form a core fusion complex that is composed of a four-helical bundle spanning the
vesicle and target membranes (Poirier et al., 1998a; Sutton et
al., 1998). Formation of this complex is a late step in the membrane
fusion process and perhaps actually drives fusion of the lipid bilayers
(Hanson et al., 1997; Lin and Scheller, 1997).
Given the central role of membrane fusion in the functions of various
membrane compartments, it is critical to understand how the SNAREs may
be regulated. In particular, insight into the regulation of neuronal
SNAREs will most likely be critical in understanding mechanisms of
synaptic development and plasticity. The three neuronal SNAREs, VAMP2,
syntaxin1, and SNAP-25, have been shown to be phosphorylated in
vitro by different kinases: VAMP2 by
Ca2+- and calmodulin-dependent protein
kinase II (Hirling and Scheller, 1996) and casein kinase II (CKII)
(Nielander et al., 1995), syntaxin 1 by casein kinase II (Bennett et
al., 1993b; Hirling and Scheller, 1996; Risinger and
Bennett, 1999), and SNAP-25 by protein kinase A (Risinger and Bennett,
1999) and protein kinase C (Shimazaki et al., 1996). The in
vivo occurrence, functional significance, cell or developmental
specificity, and the stimuli that control the phosphorylation of these
SNAREs remain largely unknown. In this report we investigate the
phosphorylation on serine-14 of syntaxin 1 by casein kinase II using
phosphosyntaxin-specific antibodies. We show that this phosphorylation
occurs in vivo and that phosphosyntaxin levels increase
during development. Specific domains along subsets of axons are marked
by phosphosyntaxin, and immunoprecipitation experiments show that
phosphorylated syntaxin is enriched in complexes with SNAP-25.
Although the phosphosyntaxin occurs in regions of brain that are
actively undergoing synaptogenesis, the labeled axonal domains do not
correspond to synaptic sites. The data suggest a role for casein kinase
II and phosphosyntaxin 1 in defining specific subdomains in the axonal
plasma membrane that are segregated from the synaptic active zones.
These subdomains are likely enriched in the binary SNAP-25/syntaxin
complex and therefore may be primed for the exocytosis of a novel class
of vesicles.
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MATERIALS AND METHODS |
Antibodies and reagents. The mouse monoclonal
antibodies used in this study were anti-MAP2 (Transduction
Laboratories, Lexington, KY), anti-tau and anti-synaptophysin
(Boehringer Mannheim, Indianapolis, IN), anti-HPC-1 (described
in Barnstable et al., 1985), anti-calbindin (Swant, Bellinzona,
Switzerland), and anti-SNAP-25 (Sternberger Monoclonals, Lutherville,
MD). The affinity-purified polyclonal anti-VAMP2 antibody was described
previously (Pevsner et al., 1994). The nuclear marker TOTO-3 was
purchased from Molecular Probes (Eugene, OR). Secondary antibodies for
immunohistochemistry were from Jackson ImmunoResearch Laboratories
(West Grove, PA) and included fluorescein isothiocyanate
(FITC)-conjugated AffiniPure goat anti-rabbit IgG and Texas Red
(TxR)-conjugated AffiniPure goat anti-mouse IgG. Secondary antibodies
for quantitative Western Blot analysis were bought 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. Paraformaldehyde was purchased from Electron Microscopy
Sciences (Fort Washington, PA), and casein kinase II (human,
recombinant from Escherichia coli) was from Boehringer
Mannheim (Indianapolis, IN). Unless stated otherwise, all other
reagents were purchased from Sigma (St. Louis, MO) or Fisher Biotech
(Pittsburgh, PA).
Generation and purification of  Psyn. A peptide
corresponding to amino acids 9-19 of syntaxin 1A (RTAKDSDDDDD)
(Bennett et al., 1992) was synthesized with a phosphoserine at position
14 and an additional cysteine residue at the C terminus (introduced for
coupling purposes). The peptide was coupled to Imject
maleimide-activated keyhole limpet hemocyanin (KLH) (Pierce, Rockford,
IL) and used as immunogen in rabbit. The polyclonal antiserum was
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 conjugated peptides were then
linked to cyanogen bromide-activated Sepharose 4B (Sigma). The
polyclonal antiserum was first sequentially passed over columns
carrying the peptide with unrelated sequence and the related
nonphosphopeptide to remove nonspecific antibodies. Finally, the
antiserum was affinity-purified by binding and elution from a column
carrying the phosphopeptide.
Recombinant proteins and in vitro
phosphorylation. The recombinant proteins syn1A11 [rat syntaxin
1A, amino acid (aa) 4-266], SN25N (mouse SNAP-25 N terminus, aa
1-82), SN25C (mouse SNAP-25 C terminus, aa 142-206), and
VAMP2 1-24 (rat VAMP2, aa 25-96) were expressed and purified as
described (Yang et al., 1999). SDS-PAGE and Western blotting
were performed according to standard protocols. Quantitative Western
blots were analyzed by phosphorimaging (Molecular Dynamics, Sunnyvale,
CA). The preparation of the SDS-resistant core complex was as described
(Yang et al., 1999). In vitro phosphorylation of recombinant
syn1A11 was achieved by incubating the protein for 30 min at 30°C 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
10 4 U
casein kinase II per 20 pmol of protein. The reaction was stopped by
addition of SDS-PAGE sample buffer.
Rat brain fractionation and immunoprecipitation. The
preparation of rat brain homogenates, postnuclear, membrane, and
cytosolic fractions as well as Triton X-100-extracted fractions were as described (Steegmaier et al., 1999). Immunoprecipitation experiments were performed essentially as described (Steegmaier et al., 1999), with
the exception that the antibodies were not directly coupled to the
protein A or protein G-Sepharose beads (Amersham Pharmacia Biotech); instead, a two-step procedure was used.
Rat brain slices. Rat brain slices were prepared as
previously described (McQuinston and Madison, 1999). During
preparation, slices were maintained in a high
Mg2+, low
Ca2+ (3 mM, 1 mM)
Ringer's solution containing the glutamate receptor antagonist
kynurenic acid (1 mM) to prevent excitotoxicity.
Application of activity-modulating drugs was made in normal Ringer's
solution (2.5 mM Ca2+, 1.3 mM Mg2+) lacking kynurenic acid.
Immunohistochemistry. Postnatal day 5 (P5), P9, 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
unspecific sites) in PBS containing 0.4% saponin, 1% BSA, and
2% normal goat serum (permeabilization/blocking buffer). Primary antibodies diluted in permeabilization/blocking buffer were applied to
the sections for 4 hr in a humidified chamber. After the sections were
washed 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
(Vector Laboratories, Burlingame, CA). Microscopy was performed with a
Molecular Dynamics laser confocal imaging system (Stanford University,
Cell Sciences Imaging Facility). Additionally, deconvoluted
reconstructions of serial sections were obtained by acquiring and
processing images with an Olympus IX70 inverted microscope equipped
with a CCD camera and Deltavision software (Molecular Dynamics).
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RESULTS |
Previous reports (Bennett et al., 1993b; Hirling and
Scheller, 1996; Risinger and Bennett, 1999) have shown that syntaxin 1 can be in vitro-phosphorylated by CKII. Immunoprecipitation of synaptotagmin from rat brain lysates resulted in coprecipitation of
CKII, and the kinase was shown to phosphorylate synaptotagmin as well
as syntaxin 1 (Bennett et al., 1993b). Furthermore, in vitro binding studies have shown that the CKII-mediated
phosphorylation of syntaxin 1 enhances its interaction with
synaptotagmin (Risinger and Bennett, 1999). In vitro
phosphorylation of truncated forms of syntaxin 1 and analysis of its
primary structure searching for CKII consensus motifs indicated that
serine-14 was a potential phosphorylation site. To elucidate the
possible in vivo occurrence, distribution and physiological
relevance of a CKII-mediated phosphorylation of syntaxin 1 on
serine-14, we raised an antibody that selectively recognizes the
phosphorylated form of the protein.
The Psyn antibody is specific for syntaxin 1 phosphorylated
on serine-14
A peptide corresponding to aa 9-19 of syntaxin 1A with a
phosphoserine at position 14 was coupled to KLH and used to generate an
immune response in rabbit. The resulting polyclonal antiserum was
passed over columns carrying an unrelated peptide and the related
nonphosphopeptide to remove nonspecific antibodies. Finally, the
antiserum was affinity-purified by binding to the phosphorylated peptide. The affinity-purified polyclonal antibody (Psyn) proved to
be highly specific for the CKII-phosphorylated form of syntaxin 1. In a
dot-blot experiment, Psyn was able to recognize 10 ng of the
immunogenic phosphopeptide and did not cross-react with up to 2 µg of
nonphosphopeptide or with unrelated peptides containing a phosphoserine
or phosphothreonine (Fig.
1A). Purified CKII was used to phosphorylate in vitro recombinant syntaxin 1A that
lacks the membrane anchor (syn1A11). Phosphorylated and
unphosphorylated syn1A11 were then resolved side by side by SDS-PAGE,
transferred to nitrocellulose, and probed with Psyn or HPC-1, a
monoclonal antibody directed against syntaxin 1 (Barnstable et al.,
1985). Although HPC-1 detected equally well the phosphorylated and
unphosphorylated syn1A11, Psyn showed selective recognition of the
phosphorylated protein (Fig. 1B).
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Figure 1.
The antibody Psyn is specific for the
serine-14-phosphorylated form of syntaxin 1. A, Dot-blot
analysis of the specificity of Psyn. The indicated amounts of
peptides coupled to BSA were spotted on a nitrocellulose membrane and
probed with the antibody Psyn. The antibody recognizes only the
phosphopeptide that was used as immunogen (1); no
cross-reactions are detected against the nonphosphorylated peptide with
the same sequence (2), or against unrelated
phosphopeptides containing a phosphoserine (3) or
a phosphothreonine (4). B,
Bacterially expressed syntaxin 1 lacking the membrane anchor
(syn1A11) was phosphorylated in vitro
with purified CKII. Five hundred nanograms of unphosphorylated
(lanes 1 and 3) or phosphorylated
(lanes 2 and 4) syn1A11 were
resolved by SDS-PAGE, transferred to nitrocellulose, and probed with
the antibody Psyn (lanes 1 and 2) or
HPC-1 (lanes 3 and 4). Although
HPC-1 equally recognized the phosphorylated and nonphosphorylated
syn1A11, Psyn detected only the phosphorylated protein.
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Phosphorylated syntaxin 1A and 1B are found throughout the adult
rat brain
Having established the specificity of Psyn in recognizing only
the phosphorylated form of syntaxin, we probed rat brain fractions for
the in vivo presence of phosphosyntaxin. Adult rat brain was homogenized and separated into membrane, cytosolic, and Triton X-100
extracted membrane fractions. The fractions were probed by Western blot
with HPC-1 and Psyn. As shown previously (Ruiz-Montasell et al.,
1996), HPC-1 recognized a doublet of proteins of 35-38 kDa with the
lower band being syntaxin 1A (syn1A) and the upper band corresponding
to syntaxin 1B (syn1B) (Fig.
2A, lane 1).
Similarly, Psyn recognized in the membrane fraction a doublet of
proteins with the same apparent molecular weight as syn1A and syn1B; no proteins were detected in the cytosolic fraction (Fig.
2A, lanes 2 and 3). As expected
from the integral membrane protein characteristic of syntaxin 1, the
two proteins recognized by Psyn are quantitatively extracted from
membranes by incubation with Triton X-100 (Fig. 2A,
lane 4). No additional bands were detected in these
fractions or in the total homogenate fraction (data not shown). Based
on the specificity of the Psyn antibody, and on the molecular weight and the fractionation behavior of the recognized proteins, we conclude
that both phosphosyntaxin 1A and 1B (p-syn1A, p-syn1B, respectively)
are present in the adult rat brain. Figure 2B shows the close sequence similarity of syn1A and syn1B over the length of the
peptide that was used as immunogen. Syn1A and syn1B are closely related
isoforms with a high level of sequence homology (84% identity)
(Bennett et al., 1993a). The two syntaxin isoforms are expressed in a
combinatorial manner in the CNS and PNS, with regions of overlapping
and regions of distinct distribution (Ruiz-Montasell et al., 1996;
Aguado et al., 1999). The physiological significance of the occurrence
of these two close isoforms and their differential distribution remains
to be elucidated. The identity of the sequence centered on the
phosphoserine (Fig. 2B, underlined) likely
explains why the Psyn antibody, which was raised against the
syn1A-derived peptide, also recognizes the phosphorylated syn1B.
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Figure 2.
Psyn recognizes phosphorylated syn1A and syn1B
in rat brain fractions. A, Rat brain was homogenized and
fractionated by differential centrifugation. Total protein (15 µg)
from the membrane fraction (M), cytosolic
fraction (C), and Triton X-100-extracted membrane
fraction (TX) was resolved by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with HPC-1
(lane 1) or Psyn (lanes 2 to
4). HPC-1 detected a doublet in the membrane
fraction; the lower band corresponds to syn1A and the upper band to
syn1B. Similarly, Psyn recognized phosphorylated syn1A and syn1B. As
expected, no bands are detected in the cytosolic fraction, and
phosphorylated syn1A and syn1B are quantitatively extracted from the
membrane fraction by Triton X-100 incubation. B,
Sequence alignment of syn1A and syn1B over the length of the peptide
used as immunogen. Note that although the syn1A peptide was used as
immunogen, the sequence of syn1B centered on serine-14
(underlined) is identical to that of syn1A.
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We next examined the presence of p-syn1A and p-syn1B in several regions
of the rat brain. Homogenates of the different dissected parts of the
brain were analyzed by Western blot. The presence of p-syn1A and
p-syn1B accurately mirrored that of syn1A and syn1B detected by
HPC-1 (Fig. 3, compare top
and bottom panels). Interestingly, some areas of the brain
express the two syntaxin 1 isoforms in similar amounts (cortex,
hippocampus, and olfactory bulb), whereas other regions show a marked
preference for syn1B (cerebellum, medulla, midbrain, and spinal cord).
Despite this differential expression of the two isoforms,
phosphorylated syntaxin 1A and 1B are found throughout the whole rat
brain.
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Figure 3.
Phosphosyntaxin 1A/B are detected throughout the
rat brain. Adult rat brain was dissected in cortex
(CTX), hippocampus (HC), olfactory
bulb (OB), cerebellum (CB), medulla
(ME), midbrain (MB), and spinal cord
(SC). The tissues were homogenized, and the postnuclear
fraction was prepared for Western blotting. Total protein (15 µg) of
each tissue was resolved by SDS-PAGE, transferred to nitrocellulose
membranes, and probed with Psyn (top panel)
and HPC-1 (bottom panel).
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The extent of phosphorylation on syntaxin 1 is upregulated
during development
After detecting significant levels of p-syn1A and p-syn1B in the
brain of adult animals, we investigated the possibility of a
developmental regulation of the extent of CKII-mediated phosphorylation of syntaxin 1. To this end, brains of animals of different ages were
tested for the presence of p-syn1A and p-syn1B by quantitative Western
blot with Psyn and HPC-1. For comparison, we also quantified the
developmental regulation of expression of two synaptic vesicle (SV)
markers, synaptotagmin and VAMP2, and two syntaxin 1-interacting proteins, the soluble factor nsec1 and the plasma membrane protein SNAP-25. A representative Western blot result is shown in Figure 4A. Figure
4B displays its quantitative analysis. For
comparison, the amounts of the different proteins were normalized to
100 arbitrary units at the earliest time point, E18. All proteins
tested showed increased relative levels of expression during
development. The amount of the two SV proteins synaptotagmin and VAMP2
increased steadily, reaching in the adult values of approximately 9- to 12-fold the amounts detected at E18, a trend that likely reflects the
increasing number and maturation of synapses. SNAP-25, nsec1, syn1A,
and syn1B (the last two proteins were quantified together and detected
with HPC-1, which recognizes both the phosphorylated and
nonphosphorylated forms of the protein) showed a more modest increase
in expression, with values in adult that were three- to fourfold above
the amounts at E18. Interestingly, the increase in the extent of
phosphorylation on syn1A and syn1B (quantified together) paralleled the
behavior of the two SV proteins (ninefold increase in adult over E18),
substantially differing from the moderate increase in syn1A and syn1B
expression. Therefore the proportion of phosphorylated syn1A and syn1B
increases with the maturation of the brain.
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Figure 4.
The extent of serine-14-phosphorylation on
syn1A/B increases during the development of the rat brain. Rat brains
of different ages (E, embryonic; P,
postnatal; A, adult) were homogenized, and the
postnuclear fraction was extracted with Triton X-100. Total protein (25 µg) from each lysate was resolved by SDS-PAGE, transferred to
nitrocellulose, and probed with specific antibodies to detect
phosphorylated syntaxin1A/B, syn1A/B, SNAP25, VAMP2, synaptotagmin, and
nsec1. A, Representative Western blot results.
B, Quantitative analysis of the relative amounts of
detected proteins. The data for each protein were normalized to 100 arbitrary units at E18.
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Using known amounts of in vitro phosphorylated or
mock-phosphorylated syn1A11 as standards, we calibrated the signal
generated by Psyn and HPC-1 in Western blot assays. Using this
information we estimated how much of syntaxin 1 is phosphorylated on
serine-14 at different time points in development (p-syn1A and p-syn1B
were again quantified together). We found that although at E18 only 4%
of syntaxin 1 is phosphorylated, the percentage increased to 17% at
P4, to 26% at P12, and finally reached 40% in the adult. This high
level of phosphorylation on syntaxin 1 is particularly striking if one
considers the abundance of this plasma membrane protein in the brain.
Phosphorylated syntaxin 1 is enriched in complexes
with SNAP-25
We next examined the possible regulation of the CKII-mediated
phosphorylation of syn1A and syn1B. We prepared acute rat brain slices
and incubated them under conditions that promote depolarization and SV
exocytosis (56 mM K+ for 1 hr)
or with 50 µM 4-amino-pyridine for 20 min, a treatment that delays action potential repolarization and increases firing rate
by selectively blocking the ID
potassium current (Wu and Barish, 1992). We also tested agents known to
stimulate CKII (30 µM sphingosine for 1 hr),
PKC (10 µM phorbol 12,13-dibutyrate for 1 hr)
(Fig. 5A), or PKA (50 µM Forskolin for 1 hr; data not shown). The
extent of phosphorylation of syn1A and syn1B was quantified by Western
blot with Psyn. Comparison with unstimulated slices showed that
these treatments failed to alter the state of the serine-14
phosphorylation on syn1A and syn1B (Fig. 5A). It thus appears that short-term pharmacological stimulations do not have an
effect on the serine-14 phosphorylation of syntaxin 1, suggesting that
this phosphorylation is unlikely to be involved in any fast change in
syntaxin function as a means of modulation in synaptic activity.
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Figure 5.
Phosphorylated syntaxin 1 is enriched in
complexes with SNAP-25. A, Stimulation of acute rat
brain slices failed to alter the state of serine-14-phosphorylation on
syntaxin1A/B. Acute rat brain slices were prepared from adult animals
and subjected to no stimulation (NS), 1 hr in 56 mM potassium (K+), 20 min in 50 µM 4-amino-pyridine (4AP), 1 hr in 10 µM phorbol 12,13-dibutyrate (PDBu), or 1 hr in 30 µM sphingosine (SPHI). The
slices were homogenized, and the homogenate was extracted with Triton
X-100. Total protein (50 µg) of each lysate was resolved by SDS-PAGE,
transferred to nitrocellulose membranes, and probed with Psyn.
B, In vitro-phosphorylated syn1A11
readily assembles with VAMP21-24, SN25C, and SN25N to form an
SDS-resistant/boiling-sensitive core complex. Syn1A11 was in
vitro-phosphorylated with purified CKII and then mixed with a
twofold molar excess of VAMP21-24, SN25C, and SN25N. After a 6 hr
incubation, unboiled (lane 1) or boiled (lane
2) samples were resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and probed with Psyn. Under unboiled
conditions (lane 1), phosphorylated syn1A11 was found
primarily in a ~50 kDa SDS-resistant complex (arrow)
with little protein left in the monomeric form
(asterisk). After boiling (lane 2), the
SDS-resistant complex disassembled, and the phosphorylated syn1A11 was
detected exclusively in the monomeric form. C,
Phosphorylated syntaxin 1A/B coimmunoprecipitates with SNAP-25. Rat
brain was homogenized, and the postnuclear fraction was extracted with
Triton X-100. The lysate was incubated with a SNAP-25 antibody
followed by protein-G coupled to Sepharose beads. Total protein (25 µg) from the starting material (ST) and
one-fifth of the total immunoprecipitated material (IP)
were loaded on duplicate and resolved by SDS-PAGE, transferred to
nitrocellulose membranes, and finally probed to detect the presence of
phosphorylated syntaxin 1A/B (Psyn), total syntaxin
(HPC-1), SNAP-25, and
VAMP2.
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The phosphorylation on serine-14 is positioned away from the H3 domain
of syntaxin, the C-terminal -helical region situated just before the
membrane anchor. This region has been shown to be essential in the
formation of the four-helix bundle structure with SNAP-25 and VAMP. We
therefore predicted phosphorylated syntaxin 1 to be unaffected in its
ability to interact with the other components of the core complex. To
test this prediction we combined in vitro phosphorylated
syn1A11 with VAMP21-24, SN25C, and SN25N (the portions of VAMP2 and
SNAP-25 that each contribute one helix to the four-helix bundle) under
standard conditions to allow the formation of the minimal core complex
(Poirier et al., 1998b; Fasshauer et al., 1998). As with the
unphosphorylated syn1A11 (data not shown), we observed efficient
formation of the SDS-resistant/boiling-sensitive complex that is a
hallmark of the formation of the four-helix bundle (Fig. 5B)
(Hayashi et al., 1994).
Of the interactions between syntaxin 1 and its partners, that with
nsec1 requires a majority of the cytoplasmic portion of syntaxin (Kee
et al., 1995). We therefore also tested the possibility that the
phosphorylation on serine-14 could alter the affinity of this binding.
A glutathione-S-transferase-nsec1 fusion protein was
immobilized on glutathione beads, and a bead-binding assay was
performed with various concentrations of in
vitro-phosphorylated or mock-phosphorylated syn1A11. The
phosphorylated and unphosphorylated syn1A11 bound to nsec1 with
essentially the same affinity (data not shown).
To further explore the ability of p-syn1A/B to interact with SNAP-25
and VAMP2, we subjected rat brain lysates to immunoprecipitation with
an antibody specific for SNAP-25. The complexes purified with
SNAP-25 contained, together with SNAP-25, phosphorylated syntaxin1A/B and VAMP2 (Fig. 5C). Interestingly, p-syn1A/B
was enriched in the immunoprecipitated complexes. Quantitation of the
recovered proteins showed that the ratio of p-syn1A/B to total syntaxin
increased twofold in the immunoprecipitated material relative to the
starting material. Similarly, the relative ratio of p-syn1A/B to
SNAP-25 was increased 1.8-fold in the SNAP-25 immunoprecipitation
relative to the starting material, whereas the proportion of total
syntaxin 1 to SNAP-25 showed only a 1.1-fold increase.
Phosphorylated syntaxin 1 is present at nonsynaptic sites along a
subset of axons
To further understand the physiological significance of the
phosphorylation on serine-14 of syntaxin 1, we performed
immunohistochemistry on sagittal rat brain sections. We focused our
analysis on the cortex, the hippocampal region, and the cerebellar cortex.
Figure 6A is a
deconvolution reconstruction of serial images taken in the cortex using
Psyn and TOTO-3 as nuclear marker. The staining for p-syn1A/B shows
a punctate distribution that appears to outline a selected number of
processes. Preincubation of Psyn with the phosphopeptide used as
immunogen (but not with the related nonphosphopeptide) or omission of
the primary antibodies in the immunohistochemistry protocol completely
abolished the staining (data not shown). To investigate the nature of
these processes, we costained rat brain sections with Psyn and an
antibody directed against MAP2 (a dendritic marker), or Psyn and an
antibody that recognizes tau (an axonal marker). As shown in Figure
6B, a confocal image taken in the cortex, there is no
colocalization between the dendrites and the processes stained by
Psyn. In fact, the psyn1A/B puncta are typically observed in
processes that appear to run parallel to or in between the
MAP2-containing dendrites (Fig. 6, arrows). With Psyn we
never observed staining of cell bodies, and the total absence of
costaining with dendrites indicates that the labeled processes are
axons. As expected from the high density of axons in the cortex, the
antibody that recognizes tau generated a strong homogeneous labeling,
whereas Psyn stained only a subset of axons (Fig.
6C).
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Figure 6.
Phosphorylated syntaxin is present at nonsynaptic
sites along a subset of axons. Except for the image in
A, which is a deconvoluted reconstruction of serial
sections, all of the images were acquired by a single confocal scan
from the cortex of sagittal rat brain sections. A,
Double staining with Psyn (green) and TOTO-3
(red). p-syn1A/B appears as puncta that outline a subset
of processes. B, Double staining with Psyn
(green) and an antibody against MAP2
(red, stains the dendrites); note the
absence of colocalization. Arrows point to psyn1A/B
processes that run parallel to a dendrite. C, Double
labeling with Psyn (green-yellow) and an
antibody against tau (red) to stain axons. In contrast
to the high density of axons stained for tau (single processes cannot
be resolved), Psyn stains only a selected number of them.
D-F, Double staining with HPC-1
(D, red) and Psyn (E,
green); the merged image in F illustrates
that psyn1A/B is present in a subset of axons.
G-I, Double staining with an antibody
against SV2A (G, red) and Psyn
(H, green); the merged image of
I shows no colocalization. Arrows point
to a representative area in which the SV2A and psyn1A/B puncta appear
mutually exclusive. The pia is up in the direction of the
arrowhead. Scale bars: A, 5 µm;
B, C, 10 µm;
D-F, 50 µm; G,
H, 5 µm.
|
|
The selective staining of a subset of axons is further illustrated by
the costaining with HPC-1 and Psyn. HPC-1 recognizes both the
phosphorylated and unphosphorylated form of syntaxin 1. As shown in
Figure 6D, this results in a homogeneous staining that contrasts with the presence of psyn1A/B in a subset of axons in
the same field (Fig. 6E,F).
The localization of phosphorylated syntaxin 1 to the axonal domain of
neurons is in agreement with previous light and electron microscopy
studies (Galli et al., 1995; Garcia et al., 1995; Sesack and Snyder,
1995). Interestingly, the punctate labeling restricted to a
subdomain of the plasma membrane is in striking contrast with the
homogeneous presence of syntaxin 1 (detected by nonphosphospecific
antibodies) along the entire axonal plasmalemma.
The size and distribution of the psyn1A/B puncta were highly
reminiscent of staining of synapses, but when we double-labeled sections with Psyn and with antibodies to detect SV pools at synapses [antibodies against SV2a (Fig.
6G-I) or antibodies against synaptophysin; data not shown for the cerebral cortex but see Fig.
7, C and F, for
double labeling in the cerebellar cortex], we did not detect any
colocalization between SV markers and psyn1A/B. In fact, the psyn1A/B
and synaptic puncta appeared to be mutually exclusive (Fig.
6G-I, arrows).
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Figure 7.
Psyn strongly labels the
molecular layer in the cerebellar cortex. All of the panels represent
confocal images taken from sagittal sections of the cerebellar cortex.
A, Double staining with Psyn
(green) and TOTO-3 (red, stains
the nuclei) in P5 animals; psyn1A/B is highly enriched in the forming
ML. B, Double staining with Psyn
(green) and an antibody against calbindin
(red, stains the Purkinje cells) in P9 animals. The
maturing Purkinje cell dendrites in the ML appear embedded in Psyn
staining; no psyn1A/B is detected in the EGL, and only low levels are
present in the GL. C, Double staining with Psyn
(green) and an antibody against synaptophysin
(red, stains synaptic vesicles) in P9 animals. No yellow
areas of overlap between psyn1A/B and synaptophysin are detected;
moreover, the cell bodies (decorated with synapses) and dendrites of
Purkinje cells (Pu) are not labeled with Psyn.
D, Double staining with Psyn
(green) and TOTO-3 (red) in adult
animals. The ML, still strongly labeled with Psyn,
extends all the way to the pia. Unlabeled Purkinje cell dendrites are
apparent, and substantial staining is now observed also in the
ML in areas free of cell bodies. E,
Double staining with Psyn (green) and an
antibody against calbindin (red) in adult animals.
Psyn stains intensely the ML and the GL in discrete structures. The
one-cell Purkinje cell layer extends highly branched dendrites in the
ML and axons across the GL; in both regions there is no colocalization
with psyn1A/B. F, Double staining with Psyn
(green) and an antibody against synaptophysin
(red). The psyn1A/B-positive structures in the GL
(inset) are decorated by patches of synapses identifying
them as cerebellar glomeruli. As observed in the cortex, the Psyn
staining does not overlap with that of synapses.
G-I, Double staining with HPC-1
(G) and Psyn (H)
in P5 animals. Although psyn1A/B is present mainly in the forming ML,
HPC-1 detects the presence of unphosphorylated syntaxin 1 in the GL.
I, The merged image with areas of overlap in
yellow. J-L, Double
staining with HPC-1 (J) and Psyn
(K) in adult animals. In the adult,
psyn1A/B is detected in both the ML and the GL with a staining
pattern that matches that obtained with HPC-1 (L,
overlap). EGL, External germinal layer;
GL, granular layer; ML, molecular layer;
Pu, Purkinje cell. Scale bars: A,
B, D-F, 20 µm;
C, 10 µm; G-L, 50 µm.
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|
In summary, these double-labeling experiments show that psyn1A/B
is detected in a selected population of axons with a punctate staining pattern that does not coincide with synapses.
Psyn strongly labels the molecular layer in the
cerebellar cortex
Because our biochemical characterization of the phosphorylation
state of syntaxin 1 revealed a developmental regulation, we stained
sagittal rat brain sections of animals at P5 and P9 and compared them
with adults. Figure 7 shows a series of double-staining experiments
performed on sections of animals of different ages with images acquired
in the cerebellar cortex.
In Figure 7A we costained sections from P5 animals with
Psyn and TOTO-3. In the developing cerebellum we observed strong staining with Psyn in the forming molecular layer (ML),
whereas little psyn1A/B was detected in the granular layer
(GL) and virtually no staining was detected in the external
germinal layer (EGL). During maturation of the cerebellar
cortex, the granule cells in the EGL first become bipolar with their
growing processes oriented parallel to the pial surface and
perpendicular to the Purkinje cell dendrites. These processes, the
axons of the granule cells, form the parallel fibers that elongate over
the surface of already formed fibers in an ordered stacking process
that proceeds from the bottom upward, progressively displacing the EGL.
Then each granule cell extends a third process, perpendicular to the
parallel fibers, that elongates into the ML. Finally, the cell body
migrates along this process to reach its final position in the
molecular layer (Altman, 1972a).
The intense, homogeneous staining of the ML suggests that psyn1A/B is
present in the parallel fibers, consistent with the axonal distribution
observed in the cortex. The parallel fibers are the orderly oriented,
tightly packed axons of the granule cells. The high density of staining
that results from the structure of the ML is a likely explanation of
our inability to detect single puncta as observed in the cortex, where
only a subset of axons was labeled.
The preferential staining of the ML is persistent in P9. In Figure
7B we costained with Psyn and an antibody that
specifically labels Purkinje cells (an antibody against calbindin). At
this time in development the Purkinje cells have already formed their single cell layer just below the molecular layer, where their dendritic
tree is undergoing active growth and branching (Altman, 1972b). As seen
at P5, psyn1A/B is detected primarily in the ML at P9, with staining
that appears to surround the unlabeled Purkinje cell dendrites; no
labeling is observed in the EGL or GL. In Figure 7C we
double-labeled for psyn1A/B and synaptophysin. Confirming the results
obtained in the cortex, the psyn1A/B staining is clearly excluded from
the areas occupied by the unstained dendrites of the Purkinje cells and
does not appear to colocalize with the synaptic puncta scattered
throughout the ML. We next performed equivalent costaining experiments
in sections from adult rat cerebellum: Psyn with the nuclear marker
TOTO-3, with an antibody against calbindin, and with an antibody
against synaptophysin in Figure 7, D, E, and
F, respectively. In the adult the EGL has disappeared and
the ML extends all the way to the pia. As observed in younger animals,
the ML is strongly labeled with Psyn (Fig.
7D-F), the Purkinje cell dendrites are
not stained (unlabeled ghosts in Fig. 7D, no overlap in Fig.
7E), and there is no apparent colocalization with the SV
marker synaptophysin (Fig. 7F). In contrast to what we observed in P5 and P9 animals, we detected significant levels of
psyn1A/B in the granular layer. The Psyn immunoreactivity was
confined to areas free of cell bodies (Fig. 7D) that were intensely labeled, on their profile, with the SV marker synaptophysin (Fig. 7F). The morphology and staining pattern of
these structures identify them as the cerebellar glomeruli, the
cell-free islands where the mossy fiber rosettes form synapses with the
granule cell dendrites, which in turn can be in synaptic contact with the Golgi cell axons (Altman, 1972c). Note again that the synaptic vesicle marker does not overlap with p-syn 1A/B in the glomeruli (Fig.
7F, inset).
To further analyze the developmental changes in the distribution of
psyn1A/B in the molecular and granular layers of the cerebellum, we
costained sections from P5 and adult animals with HPC-1 and Psyn.
In P5 animals, HPC-1 stains both the forming ML and the GL; little
labeling is detected in the EGL (Fig. 7G). In contrast, as
described above, Psyn stains primarily the ML (Fig.
7H), as best shown in the merged image of Figure
7I with the yellow area of overlap restricted to the
molecular layer. So, at an early stage in development, while syntaxin 1 is present in neurons in the ML as well as in the GL, psyn1A/B is
enriched in the parallel fibers of the molecular layer. In the adult,
the distribution of phosphorylated syntaxin 1 becomes more similar to
that of unphosphorylated syntaxin 1: Psyn stains both the ML and the
GL (Fig. 7K) with a pattern and intensity that
matches the staining obtained with HPC-1 (Fig.
7J,L). The distribution and
developmental changes that we observed in the rat cerebellar cortex
stained with HPC-1 to detect total syntaxin 1 are in good agreement
with previous light and electron microscopy studies (Koh et al., 1993;
Veeranna et al., 1996). Interestingly however, Psyn revealed that
only a specific subset of syntaxin 1A/B is phosphorylated in the
developing cerebellar cortex; indeed, the staining was largely
restricted to the parallel fibers in the growing molecular layer. In
the adult, the distribution of p-syn1A/B became indistinguishable from
that of total syntaxin.
The distribution of phosphorylated syntaxin 1 follows the
maturation of the cerebral cortex and hippocampal region during
development
We next examined the distribution of psyn1A/B in the hippocampal
region and in the cortex, again comparing P9 animals with adults. In
the hippocampus of P9 animals we detected intense labeling with Psyn
in the CA3 region (Fig.
8A,B).
In the cortex the stained axons, mostly oriented perpendicular to the
pial surface, are observed only in the inner layers (Fig.
8C). In the adult the psyn1A/B-positive axons are detected
throughout the cortex (Fig. 8D,F), and labeling is
observed up to the pia (Fig. 8E). The hippocampal region of adult animals displays strong staining with a high density of
labeled axons throughout the whole structure (Fig. 8G).
Figure 8H shows a higher magnification of a portion
of the CA1 region; psyn1A/B is detected in axons running in the stratum
oriens and stratum radiatum, as well as in the stratum
lacunosum-moleculare (out of field in Fig. 8H). A
higher magnification of part of the dentate gyrus is shown in Figure
8I; a high density of axons containing psyn1A/B is
found in the hilus, and numerous processes are stained in the stratum
moleculare. Occasionally we observed labeled axons crossing the stratum
granulosum, spanning the region occupied by the granule cell bodies
between the hilus and the stratum moleculare.
View larger version (171K):
[in this window]
[in a new window]
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Figure 8.
The distribution of phosphorylated syntaxin 1 changes in the cortex and hippocampal region during development. All of
the panels represent confocal images of rat brain sagittal sections
double-labeled with Psyn (green) and TOTO-3
(red). A, B, In
P9 animals the CA3 region of the hippocampus shows enriched presence of
psyn1A/B. C, In the cortex of P9 animals, the
psy1A/B-positive axons are found in the inner layers, but the staining
does not extend to the pial surface. D-F, In
adult animals the cortex shows Psyn-stained axons throughout its
layers (D, F), all the way up to the pia
(E). G-I, The hippocampus of
adult animals is intensely stained with Psyn (G).
In the CA1 region (H), numerous axons are observed in
the stratum oriens and stratum radiatum. The dentate gyrus
(I) shows intense staining in the hilus and high
density of labeled axons in the stratum moleculare. CA1, CA1
region of the hippocampus; CA3, CA3 region of the
hippocampus; DG, dentate gyrus; hi, hilus;
str. m., stratum moleculare; str. o., stratum
oriens; str. r., stratum radiatum. The pia is up in the
direction of the arrowhead. Scale bars: A,
G, 100 µm; B, D, H,
I, 50 µm; C, E, 20 µm;
F, 10 µm.
|
|
Taken together, the immunohistochemical studies support and expand our
biochemical data. The staining revealed that of the total pool of
syntaxin 1 molecules, only a subset is phosphorylated by CKII.
Moreover, although conceivably all neurons express syntaxin 1, only a
subset of them possesses p-syn1A/B. The developmental changes in the
distribution of phosphosyntaxin are in agreement with the observed
increased proportion of the phosphorylation and are consistent with a
role in mature axons. The subcellular localization along the axolemma
but outside synapses indicates that this phosphorylation may not have a
direct effect on SV exocytosis but could be involved in the distinction
between active zones and nonsynaptic portions of the axonal plasma membrane.
| |
DISCUSSION |
We have generated an antibody (Psyn) that selectively
recognizes the casein kinase II-mediated phosphorylation of serine-14 on syntaxin 1A/B. Using this antibody we showed that this
phosphorylation occurs in vivo in the rat brain and that the
proportion of phosphorylated syntaxin increases during development:
from ~4% in E18 embryos to ~40% in adult animals. The
developmental regulation and the high proportion of phosphorylated
syntaxin 1 are noteworthy considering that syntaxin 1 is one of the
most abundant membrane proteins in brain. The upregulation in syntaxin
1 phosphorylation paralleled an increase in the levels of SV proteins
and is therefore likely to be functionally linked to the increased
number and maturation of synapses. When we subjected rat brain slices
to treatments that would stimulate synaptic transmission or the
activity of different kinases, we did not observe any effect on the
phosphorylation state of syntaxin 1A/B. The high proportion of
phosphorylated syntaxin 1 and the inability of stimulation to change
this proportion suggest that the casein kinase II-mediated
phosphorylation of syntaxin 1A/B is likely not an activity-induced fast
response that could serve rapid modulation of the vesicle docking and
fusion machinery. A rapid change in the phosphorylation state after
stimulation was observed, for example, in the well characterized case
of phosphorylation of synapsin I (Greengard et al., 1993).
Our biochemical comparison between phosphorylated and unphosphorylated
syntaxin 1A showed that both proteins assembled equally well in the
core complex with VAMP2 and SNAP-25, and they bind with similar
affinity to the interacting protein nsec-1. Immunoprecipitation experiments confirmed that p-syn1A/B can be found in complexes with
SNAP-25 and VAMP2. Interestingly, phosphorylated syntaxin 1A/B was
enriched, compared with total syntaxin, in complexes with SNAP-25.
Is phosphosyntaxin 1A/B and its preferential interaction with SNAP-25
marking plasma membrane domains for specific functions, and if so what
might that function be? The immunohistochemistry experiments revealed
that the Psyn staining was concentrated on a subset of axons with
labeling of puncta that appeared to outline the profile of the
processes, consistent with a plasma membrane localization of the
phosphorylated syntaxin 1A/B. This restricted localization contrasted
the widespread distribution along the entire axonal plasma membrane
observed for total syntaxin 1A/B stained with HPC-1. When we
double-labeled sections for p-syn1A/B and SV markers, we did not see
any colocalization, strongly suggesting that p-syn1A/B is not enriched
at active zones.
The immunohistochemical comparison between young animals and adults
confirmed the developmental increase in phosphorylation of syntaxin
1A/B observed by Western blotting as well as the selective distribution
of p-syn1A/B staining compared with HPC-1 staining.
In the cerebellar cortex of young animals, p-syn1A/B was highly
enriched in the growing molecular layer, with a staining pattern and
progression through development strongly indicative of parallel fiber
staining. Only in the adult did we observe Psyn staining in the
granular layer, whereas total syntaxin was detected in this region of
the cerebellum throughout development. Similar observations were made
in the hippocampus and the cerebral cortex. A highly enriched staining
in the CA3 region of the hippocampus in P9 animals was followed by a
very strong and widespread staining of axons throughout the whole
structure in the adult. In the cortex of young animals, stained axons
oriented perpendicular to the pia are observed only in the inner
layers, but their presence extends all the way to the pial surface in
the mature brain.
In summary, the data are consistent with Psyn detecting p-syn1A/B
preferentially bound to SNAP-25 in binary complexes on the plasma
membrane that are outside of synaptic sites. Could this complex be
marking plasma membrane domains where exocytosis of specifically
synaptic vesicle is inhibited? This interpretation would be consistent
with our findings that the proportion of phosphorylated syntaxin 1A/B
increases during development and by the progression of the stained
axons in both the cerebral and cerebellar cortex. Early in development,
flexibility in the choice of the axonal plasma membrane domain that may
become a mature presynaptic site is dictated by the transient and
developing nature of the synapses. At this time point, a clear and
rigid distinction between synaptic and nonsynaptic plasma membrane
domains seems not only unnecessary but also possibly detrimental. As
axons mature and establish defined synaptic specialization with
dendrites, neurons invoke mechanisms to restrict synaptic vesicle
exocytosis to the active zones. The presence of phosphorylation on
serine-14 of syntaxin could be part of a signaling mechanism to mark
the difference between the two domains. Despite the fact that active
zones occupy only a small portion of the axonal plasma membrane,
several studies have shown that both syntaxin 1 and SNAP-25 are found
all along the axolemma (Galli et al., 1995; Garcia et al., 1995).
Alternatively, because phosphosyntaxin is preferentially associated
with SNAP-25, it may be that the labeled sites along axons are novel
vesicle fusion sites not previously recognized. These axonal domains
are not likely to be fusion sites for synaptic vesicles because these
organelles do not appear clustered at phosphosyntaxin sites. Perhaps
these assembled t-SNARE complexes demarcate fusion sites for a novel
class of vesicles important in nonclassical intercellular communication
between neurons. Finally, the phosphosyntaxin sites may define axonal
domains that mature into new synapses after appropriate stimulation,
perhaps by experience.
The phosphorylation site analyzed in this study is in the very
N-terminal part of the protein, a location that places it away from the
domains that have been shown to be essential in the formation of the
four-helix bundle structure that is believed to be at the base of the
fusion process of SV with the plasma membrane. Interestingly, among the
other plasma membrane syntaxins with broad distribution, syntaxin 3A
and syntaxin 4 are also phosphorylated in vitro by CaMKII
and CKII, respectively (Risinger and Bennett, 1999). In each case the
phosphorylation site was mapped to their N-terminal domain. Although
this location makes it unlikely that these phosphorylations will have a
direct effect on the vesicle fusion process, it indicates that
the phosphorylated N terminus of the protein might have regulatory functions and could interact with other molecules and impart additional functions to syntaxin 1.
| |
FOOTNOTES |
Received Jan. 27, 1999; revised March 20, 2000; accepted March 27, 2000.
We thank Dr. Chris Kaznowski for assistance in obtaining and analyzing
rat brain sections, Dr. Susan Palmieri for assistance with confocal
microscopy, and Dr. Susan McConnell for critical reading of this manuscript.
Correspondence should be addressed to Richard H. Scheller, Howard
Hughes Medical Institute, Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA
94305-5428. E-mail: scheller{at}cmgm.stanford.edu.
| |
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TOP
ABSTRACT
INTRODUCTION
