From the ¶ Neurology Service, Department of Veterans Affairs
Medical Center, Lexington, Kentucky 40511 and the Departments
of Biochemistry and § Neurology and
Pharmacology, University of Kentucky Medical Center,
Lexington, Kentucky 40536
Received for publication, August 14, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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Critical to SNARE protein function in
neurotransmission are the accessory proteins, soluble
N-ethylmaleimide-sensitive factor (NSF) attachment protein
(SNAP), and NSF, that play a role in activation of the SNAREs for
membrane fusion. In this report, we demonstrate the
depolarization-induced, calcium-dependent phosphorylation of NSF in rat synaptosomes. Phosphorylation of NSF is coincident with
neurotransmitter release and requires an influx of external calcium.
Phosphoamino acid analysis of the radiolabeled NSF indicates a role for
a serine/threonine-specific kinase. Synaptosomal phosphorylation of NSF
is stimulated by phorbol esters and is inhibited by staurosporine, chelerythrine, bisindolylmaleimide I, calphostin C, and Ro31-8220 but
not the calmodulin kinase II inhibitor, Kn-93, suggesting a role for
protein kinase C (PKC). Indeed, NSF is phosphorylated by PKC in
vitro at Ser-237 of the catalytic D1 domain. Mutation of this
residue to glutamic acid or to alanine eliminates in vitro phosphorylation. Molecular modeling studies suggest that Ser-237 is
adjacent to an inter-subunit interface at a position where its
phosphorylation could affect NSF activity. Consistently, mutation of
Ser-237 to Glu, to mimic phosphorylation, results in a hexameric form
of NSF that does not bind to SNAP-SNARE complexes, whereas the
S237A mutant does form complex. These data suggest a negative regulatory role for PKC phosphorylation of NSF.
The molecular mechanisms of neurotransmitter
(NT)1 release have been the
subject of much attention in recent years resulting in an evolving
model known as the SNARE hypothesis (1-3). The SNARE hypothesis holds
that membrane proteins in the vesicle (v-SNAREs, e.g.
synaptobrevins) bind to a heterodimer in the target membrane (t-SNAREs,
heterodimers of syntaxins and SNAP-25-like proteins). v- and t-SNAREs
bind to form a 7 S complex (1, 4), composed of a bundle of four
parallel, coiled-coil domains (5-8) that, through reconstitution
studies, has been demonstrated to be minimally required for bilayer
fusion (9). The N-ethylmaleimide-sensitive factor (NSF) and
the soluble NSF attachment proteins (SNAPs, not to be confused with
SNAP-25) affect the composition and structure of the SNARE complex.
SNAPs act as adapters and are required for binding of NSF to the 7 S
complex. ATP hydrolysis by NSF causes the resulting 20 S complex to
disassemble into monomeric SNAREs (4, 10), a step required for vesicle
trafficking (11-13). Detailed kinetic experiments have placed one role
of NSF and SNAPs at steps prior to v-/t-SNARE binding (14-16),
suggesting a model in which NSF disassembles cis 7 S
complexes that exist in the same bilayer (17-19). The resulting
monomeric SNAREs can then form trans 7 S complexes that
span the opposing bilayers of the vesicle and target membrane. In this
manner, NSF acts as a chaperone to activate or "prime" the SNARE
proteins for subsequent trans complex formation and membrane fusion.
Studies by Schweizer et al. (20), using NSF-derived peptides
microinjected pre-synaptically, showed that NSF affects the efficiency
and kinetics of neurotransmission in the squid giant axon. Studies of
the comatose mutant in Drosophila indicate that active NSF is not required for initial neurotransmitter release but is
instead required for sustained release upon subsequent stimulations
(21-23). These data are consistent with a role for NSF in maintaining
a "ready-release" pool of synaptic vesicles, which is competent for
fast response to calcium influx. Consistently, SNAPs, when injected
into crayfish axons, increase the probability of neurotransmitter
release but not the rate (24). On a molecular level, these data support
the model that NSF functions prior to neurotransmitter release to
activate SNAREs for membrane fusion and perhaps after fusion to recycle
spent SNAREs (25).
NSF is a homo-hexameric ATPase whose subunits can be divided into three
domains (N, D1, and D2) (11, 26, 27). The amino-terminal domain is
required for binding to the SNAP-SNARE complex. ATP binding and
hydrolysis by D1 is essential for NSF activity. Mutants in the D1
domain that fail to bind ATP also fail to interact with SNAP-SNARE
complex (27). D1 mutants that fail to hydrolyze ATP are dominant
negative inhibitors because they fail to disassemble 7 S SNARE
complexes (11, 27, 28). The D2 domain is required for hexamerization
since its deletion yields a monomer (11). Although this domain has high
affinity for ATP and low ATPase activity, neither function is essential
for NSF activity (11, 29). In a current model of NSF structure, the
hexameric D2 domain anchors the catalytically active D1 domains and
SNAP/SNARE-binding N domains.
Much work has focused on the role of phosphorylation by protein kinase
C (PKC) in the control of neurotransmission (reviewed in Ref. 30). In
particular, it has clearly been shown that activation of PKC by phorbol
esters can lead to an enhancement of L-glutamate release
(31-34). Several studies indicate that PKC translocation from cytosol
to membrane is associated with both PKC activation and enhanced
L-glutamate release (32, 35, 36). Moreover, phosphorylation
of PKC substrates that are selectively localized in nerve terminals,
such as B-50/GAP-43 and myristoylated alanine-rich PKC substrate, has
been related to neurotransmitter release (37-39). Among candidate
protein targets for PKC that have received scrutiny are those involved
in the docking/fusion process. In one such study, activation of PKC by
phorbol esters induced phosphorylation of SNAP-25 and increased
depolarization-dependent norepinephrine release from PC12
cells (40). That study and others (30) have demonstrated a potential
role for PKC in neuroendocrine secretion but have yielded little
molecular insight into the precise effects of phosphorylation. Several
studies have attempted to fill this gap by focusing on the components
of the membrane fusion machinery (e.g. SNARE, SNAPs, and
NSF). Those studies have demonstrated that various machinery elements
can be phosphorylated in vitro with purified kinases
(e.g. PKA, PKC, and CaMKII) and that the phosphorylation
does affect their protein-protein interactions (30).
In the present study, we provide the first demonstration of
depolarization-induced, calcium-dependent phosphorylation
of NSF in rat synaptosomes. Staurosporine, chelerythrine,
bisindolylmaleimide I, calphostin C, and Ro31-8220, but not Kn-93,
inhibit phosphorylation of NSF. Conversely, phorbol 12-myristate
13-acetate (PMA) treatment enhances phosphorylation, thus pointing to a
role for PKC. In vitro, NSF is specifically phosphorylated
on Ser-237 by PKC. Mutation of Ser-237 to glutamic acid or alanine
yields a form of NSF that cannot be phosphorylated by PKC; however,
only the S237E mutant fails to bind SNAP-SNARE complexes. From a
regulatory standpoint, control of NSF activity via phosphorylation
offers a unique mode to modulate trafficking fluxes.
Materials--
ATP and AMP-PNP were obtained from Roche
Molecular Biochemicals. [
Wild-type NSF and NSF mutants were produced as recombinant proteins in
Escherichia coli and purified as described (27). Production
of His6-free NSF was accomplished using the pPROExHt expression system and the TEV protease (Life Technologies, Inc.). Site-directed mutagenesis was accomplished using the QuickChange kit
according to manufacturer's instructions (Stratagene, La Jolla, CA),
and mutations were confirmed by dideoxy nucleotide sequencing. His6- Bath Experiments with Synaptosomes--
An in situ
preparation of Percoll gradient purified rat cerebral cortical
synaptosomes (42) was used to measure both K+-evoked,
Ca2+-dependent NSF phosphorylation and NT
release. The composition of all buffers has been described (43).
Synaptosomes were resuspended in Krebs/sucrose buffer without phosphate
and incubated with 32Pi (7,000 Ci/mmol) for 30 min at 37 °C under 95% O2, 5% CO2.
32Pi-loaded synaptosomes were then placed in
Krebs-Ringer bicarbonate buffer with either 1.5 mM
Ca2+ or 10 mM Mg2+. Samples were
depolarized by addition of K+ (25 mM final
concentration), and the process was stopped at 30 s by the
addition of SDS/orthovanadate (8). L-Glutamate release was
measured in parallel samples using an enzyme-coupled fluorometric assay
(44, 45). Synaptosomes were depolarized on addition of 25 mM KCl to an incubation mixture containing NADP (1 mM), glutamate dehydrogenase (50 units/ml), and either
CaCl2 (1.5 mM) or MgCl2 (10 mM). NADPH fluorescence was monitored using excitation and
emission wavelengths of 340 and 460 nm, respectively. Data were
accumulated at 1-s intervals using a Perkin-Elmer Life Sciences LS5B
spectrofluorometer fitted with stirred, thermostated cuvettes at
37 °C.
Immunoprecipitation of NSF--
Prior to immunoprecipitation
(IP), Triton X-100 was added to the solubilized samples to neutralize
the SDS. Samples were then diluted in IP buffer (10 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.2 mM sodium o-vanadate,
0.2 mM phenylmethylsulfonyl fluoride), and protein
G-Sepharose beads with covalently coupled anti-NSF antibody were added
(1). NSF was immunoprecipitated by incubation for 1 h at 4 °C.
The beads were harvested and washed 5 times in 0.5 ml of IP buffer; the
protein-antibody complexes were eluted with SDS-PAGE sample buffer and
analyzed by SDS-PAGE and immunoblotting.
NSF Phosphorylation by PKC or CaM Kinase II in
Vitro--
Recombinant NSF or NSF mutants (6 µg) were treated for
1 h at 25 °C with PKC (0.5 milliunits) or CaM kinase II (0.05 milliunits) and 1 mM ATP plus 1 µl of
[ Characterization of Phosphorylated NSF--
IP samples or
in vitro phosphorylated NSF were resolved by SDS-PAGE and
transferred to PVDF (47). The position of NSF was detected by enhanced
chemifluorescence with the anti-NSF antibodies noted above using the
Attophos detection system (Amersham Pharmacia Biotech). 32P
labeling and chemifluorescence immunodetection were quantified using
PhosphorImager analysis as noted above.
Radiolabeled NSF samples from in situ and in
vitro labeling experiments were recovered from the PVDF membranes
after Western blotting. Phosphoamino acid analysis was performed by
two-dimensional electrophoresis after acid hydrolysis of the
PVDF-blotted material as described by Duclos et al. (48).
Phosphopeptide analysis was done on NSF that had been phosphorylated
with PKC in vitro (see above). After incubation, the
radiolabeled NSF was recovered by precipitation with trichloroacetic
acid, and the pellets were washed extensively to remove unincorporated
label. The protein was denatured in 4 M urea, 4 M guanidine HCl, 7 mM DTT, and 100 mM Tris-HCl, pH 8.5, then cysteine thiols derivatized by
addition of 2 M acrylamide for 2 h at 37 °C. The
protein was desalted by acetone precipitation and digested overnight at
37 °C in 80 mM Tris-HCl, pH 8.5, 1% (v/v) hydrogenated
Triton X-100 (Calbiochem), and 10% (v/v) acetonitrile with either 1 µg of trypsin (Promega, Madison, WI) or 0.1 µg of endoproteinase
Lys C (Roche Molecular Biochemicals). The digestion reactions were
acidified with trifluoroacetic acid, and the peptides were fractionated
by C18 reverse phase HPLC and monitored by both UV absorbance and
liquid scintillation counting. Radiolabeled peptides, thus purified,
were covalently attached to acrylamide-derivatized discs (Sequelon-AA
discs, Millipore Corp., Bedford, MA) and subjected to chemical
sequencing in an Applied Biosystems 494 Peptide/Protein sequencer
(Foster City, CA). A portion (40%) from each cycle was analyzed
directly by online HPLC to identify and quantify released
phenylthiohydantoin-derivatized amino acids. The remainder was
collected and monitored for 32P by Cherenkov counting using
the preset 3H windows.
SNAP-SNARE Complex Binding Assay--
The complex formation
procedure was modified from our method described previously (29).
GST-syntaxin 1 (cytosolic domain) was incubated with pre-swollen,
glutathione-agarose beads (100 µg of protein per 100 µl of beads)
at 4 °C in phosphate-buffered saline with 0.01% (v/v) Tween 20, 0.1% (v/v) Depolarization-dependent Phosphorylation of NSF in
Isolated Synaptosomes--
The use of purified synaptosomes,
metabolically labeled with high specific activity
32P-inorganic phosphate, permits direct correlation of
Ca2+- and depolarization-dependent
neurotransmitter release with specific protein phosphorylation events.
Fig. 1A shows PhosphorImager
and enhanced chemifluorescence immunoblot analyses of SDS-PAGE resolved and blotted NSF isolated by immunoprecipitation with immobilized antibody from pre-labeled synaptosomes after various treatments. Incubation for 30 s following depolarization with 25 mM KCl in the presence of 1.5 mM
Ca2+ (Fig. 1B, Ca Rel) led to
substantial 32P labeling of NSF. Little 32P
incorporation was observed either on depolarization in the presence of
10 mM Mg2+ (Fig. 1B, Mg
Rel) to block calcium entry through synaptic plasma membrane
calcium channels or without KCl addition to induce depolarization (Fig.
1B, Ca Pre and Mg Pre). Indeed, 32P
incorporation for all controls was less than 10% that obtained with
Ca2+ + K+ depolarization as judged by
quantifying the relative extents of labeling under these conditions as
shown in Fig. 1B (hatched bars). It should be
noted that equal amounts of NSF were recovered from the lysed rat brain
synaptosomes by immunoprecipitation with either the rabbit polyclonal
anti-NSF antibody used for the samples shown in Fig. 1A or
with a mouse monoclonal antibody (2E5, data not shown). In addition, no
NSF protein or radiolabel was recovered with protein G-Sepharose alone.
In a related experiment, no radiolabeled
The relative extents of NSF 32P labeling correlated exactly
with the extents of glutamate release from synaptosomes measured under
the same conditions at 30 s of incubation in the release solution
(Fig. 1B, solid bars). Depolarization in the presence of
Ca2+ gave 6-fold greater glutamate release than
depolarization in the presence of Mg2+, whereas release
without depolarization was negligible. These data are derived from
continuous release assays using the coupled glutamate dehydrogenase
spectrophotometric assay described under "Experimental Procedures."
Release reached a plateau value after ~2 min in every case. The
results for both phosphorylation index and glutamate release assays
were highly reproducible as shown in the figure.
Characterization of NSF Phosphorylation in Synaptosomes--
To
characterize further the nature of the Ca2+- and
depolarization-dependent phosphorylation of NSF and the
possible identity of the responsible protein kinase(s),
32P-labeled NSF was prepared from synaptosomes treated
exactly as shown in Fig. 1A (Ca Rel) and
subjected to two-dimensional thin layer electrophoresis phosphoamino
acid analysis following acid hydrolysis (48). Both phosphoserine and
phosphothreonine, but not phosphotyrosine, were detected from this
in situ labeled NSF (Fig.
2A). This suggests the
involvement of one of the many neuronal Ser/Thr protein kinases
(i.e. PKC, PKA, and CaMKII). NSF contains potential kinase
recognition sites for PKC, CaMKII, and casein kinase II but does not
have consensus PKA sites. When recombinant NSF was labeled in
vitro with purified PKC, phosphoamino acid analyses showed the
presence of only phosphoserine (Fig. 2B). Experiments have
shown that CaMKII but not PKA or casein kinase II can modify NSF
in vitro ((49) see Fig. 3).
Protein kinase inhibitors were used to further examine the kinase(s)
responsible for NSF phosphorylation in synaptosomes. Fig. 2C
shows that the depolarization-dependent phosphorylation of NSF
was inhibited by the inclusion of 5 µM staurosporine but
not by the addition of 4 µM Kn-93, which is 10 times the
reported Ki for CaM kinase II (50). Other inhibitors, more specific for protein kinase C, also affected phosphorylation. Bisindolemaleimide I (100 nM), calphostin
C (500 nM), chelerythrine (6.6 µM), and
Ro31-8220 (100 nM) inhibited NSF phosphorylation by 55%
for chelerythrine and 80% for calphostin C (Fig. 2D). The
appearance of phospho-NSF was not enhanced significantly by 1 µM okadaic acid suggesting that protein phosphatases 1 and 2A may not be involved in NSF dephosphorylation. Treatment of synaptosomes with the PKC activator PMA (0.16 µM)
increased NSF phosphorylation by 77% in the presence of EGTA and by
61% in the presence of calcium (Fig. 2C). Under these same
conditions, PMA did not stimulate glutamate release; however, it did
enhance both the extent of release (199.7 ± 32.9 versus 126.3 ± 12.0 pmol/mg synaptosome protein
(p = 0.05)) and the initial rate of release (253.8 ± 43.6 versus 126.8 ± 18.6 pmol/mg synaptosome
protein/min (p = 0.03)).
Characterization of NSF and SNAP Phosphorylation in
Vitro--
Previous studies (49) reported that CaMKII can
phosphorylate NSF in vitro; however, PKC was not tested. To
reconcile those studies with present data, the ability of purified rat
brain-derived CaMKII and PKC to phosphorylate recombinant NSF was
compared (Fig. 3). Both kinases phosphorylated NSF in vitro;
however, the level of NSF phosphorylation with PKC (0.1-1 mol of
PO4/mol of NSF subunit; n = 3) was 100-fold
greater than that obtained with CaMKII under similar conditions.
Consistent with Hirling and Scheller (49), both CaMKII and PKC
phosphorylated
There are 12 canonical PKC recognition sites on the NSF subunit and
several potential noncanonical recognition sites. To determine the
exact sites where the recombinant NSF was phosphorylated in vitro, labeled NSF was subjected to peptide mapping coupled with radiochemical sequencing. Initial two-dimensional tryptic peptide maps
gave a single major radioactive spot, which yielded phosphoserine after
hydrolysis (data not shown). Subsequently, PKC-phosphorylated NSF was
digested with either trypsin or Lys C, and the released 32P-labeled phosphopeptides were fractionated by
reverse-phase HPLC as shown in Fig.
4A. Only one major
radiolabeled peak (other than the unabsorbed fraction containing
inorganic phosphate) was observed in each case. However, the tryptic
fragment eluted early in the acetonitrile gradient, commensurate with a
small peptide, whereas the Lys C product eluted much later, as expected
for a longer peptide. Radiochemical sequencing of the trypsin fragment
isolated from the separation shown in Fig. 4A gave release
of the bulk of the 32P at cycle 4 (data not shown), whereas
release of radiolabel on degradation of the Lys C phosphopeptide was
observed after cycle 4 and cycle 12 as shown in Fig. 4B.
These data suggest that the phosphorylated serine in the recombinant
NSF was 4 residues from an arginine or lysine (trypsin digest) and 12 residues from a lysine (Lys C digest). (It should be noted that the
primary sequence detected on phenylthiohydantoin-amino acid analysis of
these fractions was that for the amino-terminal portion of the
recombinant molecule containing the His6 tag preceded by
the sequence MRGS. Removal of this tag by TEV protease digestion,
however, did not lead to a significant reduction in in vitro
phosphorylation (data not shown)). The only residue that completely
fulfills these criteria, Ser-237, is part of the sequence
RRAFASRVF that is a noncanonical PKC recognition site but is
conserved in 17 of the 18 NSF sequences in the data base. To
demonstrate that this serine was phosphorylated in vitro,
site-directed mutagenesis was used to change the residue to either an
alanine or a glutamate. The resulting S237A and S237E mutant forms of
NSF retain native oligomeric structure (see below) but are not
phosphorylated significantly by PKC in vitro as shown in
Fig. 4C. Thus, Ser-237 is the only modification site
consistent with the radiochemical sequencing data, and its mutation
results in a nonphosphorylatable form of NSF.
Potential Role of Ser-237 Phosphorylation--
Initial experiments
did not detect any significant difference between in vitro
phosphorylated and unmodified NSF in SNAP/SNARE binding assays (data
not shown). The stoichiometry of phosphorylation was variable in those
experiments. Therefore, we were not certain if all of the subunits of
the NSF hexamer were modified. Previous reports have shown that NSF can
bind to SNAP-SNARE complexes when monomeric or when the hexamer lacks a
full complement of intact subunits (27) suggesting that NSF binding
might not be affected unless all of the subunits are modified. The
Ser-237 to Glu point mutation described above should mimic the negative
charge that would be present in wild-type NSF after phosphorylation.
Therefore, all six subunits would have the identical negative charge at
the Ser-237 position mimicking complete phosphorylation of an NSF hexamer. The resulting mutant protein, S237E, was hexameric and had the
same chromatographic properties on Superose 6 as wild-type NSF (data
not shown). However, when tested for SNAP-SNARE complex binding, the
S237E mutant showed reduced binding under standard conditions (Fig.
4D). The lack of binding is most likely due to the
introduction of a negative charge at Ser-237 since another mutant,
S237A, showed wild-type levels of SNAP/SNARE binding. This defect in
SNAP/SNARE binding is consistent with an inability of the NSF molecule
to attain a binding competent conformation. Such binding incompetence
occurs when NSF is in the ADP-bound form (29) and has been seen in
mutants that lack or have mutations in the N-domain
(27)2 or that are unable to
bind nucleotide in the D1 domain.
In this study, we demonstrate a depolarization-induced
phosphorylation of NSF in rat synaptosomes that is dependent on influx of Ca2+ into the synaptosome. Phosphoamino acid analysis of
radiolabeled NSF shows the presence of phosphoserine and
phosphothreonine but no phosphotyrosine suggesting a role for a
neuronal Ser/Thr kinase. Previous work has shown that NSF is not a
substrate, in vitro, for casein kinase II or PKA (49). Here,
we show that the depolarization-induced phosphorylation of NSF in
situ is not sensitive to the CaM kinase II inhibitor Kn-93 but is
sensitive to staurosporine as well as bisindolemaleimide I, calphostin
C, chelerythrine, and Ro31-8220, suggesting a role for PKC. We further
demonstrate that PKC phosphorylates NSF in vitro,
specifically at Ser-237, which lies in the catalytically important D1
domain. Mutation of Ser-237 to either Ala or Glu eliminates in
vitro phosphorylation by PKC confirming the phosphopeptide analysis. Both mutants are hexameric, based on sizing chromatography. However, only the S237E mutant loses its ability bind to SNAP-SNARE complexes. This inactivation of NSF can be partially understood using
the known structure for the homologous D2 domain. A negative charge at
Ser-237 (such as a phosphate or glutamate) is in a position to affect
greatly the flexibility of the catalytic D1 domain (see below).
We believe our observations are the first that connect control
of a physiologic process to a phosphorylation event involving a protein
of the docking/fusion apparatus. They are significant because they
indicate that in response to depolarization, synaptosomal PKC may
down-regulate NSF activity by affecting its ability to interact with
its substrates, SNAPs and SNAREs. We were able to document this
phosphorylation because the synaptosomal preparation offers an isolated
region of the cell where the docking/fusion components involved in
neurotransmitter release are concentrated; 0.5-1% of the total
synaptosomal protein is NSF.
Role of PKC in Neurotransmission--
Numerous studies have shown
a presynaptic role for PKC in neurotransmission (for a recent review
see Ref. 30). It is generally accepted that activation of PKC, by
either phorbol esters or agonist, leads to an increase in the
stimulus-induced release of neurotransmitters. Several studies, all
in vitro, have attempted to identify the relevant PKC
substrates responsible for this enhancement of secretion. Others have
attempted to associate changes in the phosphorylation profile of PKC
substrates or the compartmentalization of PKC isozymes with physiologic
processes (e.g. LTP) and pathologic processes (e.g. kindled epilepsy). Thus LTP of synaptic transmission
in the hippocampus, a model of learning and memory (for review see Ref.
51), has been associated with PKC translocation to the membrane (35,
52) and to phosphorylation by PKC of F1/GAP-43 (53). Similarly, kindled
epilepsy evoked by stimulation at multiple brain sites has been
associated with both transient (54, 55) and long term (56, 57)
increases in membrane-associated PKC enzyme activity but not protein
levels. However, no direct association has been made between enhanced
glutamate release associated with LTP or kindling and phosphorylation
of the secretory machinery (43, 58). Our studies provide the first
evidence for the linkage between phosphorylation of secretory
machinery, namely NSF, and neurotransmitter release and also provide a
potential site for alteration in LTP and kindling.
To date, SNAP-25 (40, 59, 60), syntaxins (60-62), Molecular Analysis of the Effects of NSF Phosphorylation--
Two
models of NSF function in neurons suggest two distinct interpretations
of the phosphorylation data presented here. Initially studies of NSF
function in Drosophila and chromaffin cells suggested that
NSF is important for the maintenance of the "ready release" pool of
vesicles by disassembling cis-SNARE complexes to promote the
formation of functional trans-SNARE complexes (22, 23, 67).
However, in a more recent study, inactivation of NSF through the
addition of N-ethylmaleimide was shown to increase the pool of hyperosmotically sensitive synaptic vesicles (68). This pool is
thought to be equivalent to the kinetically defined ready
release pool. In this second model, inhibition of NSF is thought
to block disassembly of trans-SNARE complexes thereby
maintaining the functional complexes for membrane fusion. In these two
models, NSF inactivation by phosphorylation could lead to distinct
outcomes. Based on the first model, phosphorylation of NSF would
down-regulate a nerve terminal by lessening the activation of SNAREs
and therefore lowering the pool of ready release vesicles. For the
second model, inactivation of NSF would lead to an increase in fusion
competent vesicles because it would lengthen the lifetime of the active
trans-SNARE complexes. At this stage, it is difficult to be
more specific since both up- and down-regulation of fusion-competent
synaptic vesicle pools has been observed.
Structurally, phosphorylation of NSF at Ser-237 could have rather
drastic effects. Since no crystal structure is available for the D1
domain of NSF, we must use the structure of the homologous, but not
identical, D2 domain as the basis for discussion. By structure-based sequence alignments (69), Ser-237 would be in the middle of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (4,500 Ci/mmol) and
[32P]PO
,
,
and
isoforms) was from Promega (Madison, WI) and CaM kinase II,
also from rat brain, was from Calbiochem.
L-
-Phosphatidyl-L-serine, diolein, and
chelerythrine were obtained from Sigma; staurosporine,
bisindolylmaleimide I, calphostin C, Ro31-8220, Kn-93, and the inactive
isomer Kn-92 were from Calbiochem. All inhibitors and activators were
suspended in Me2SO. Nitrocellulose (0.2 µm) and
PVDF membranes for immunoblotting were from Schleicher & Schuell and
Waters (Milford, MA), respectively. Horseradish peroxidase-conjugated,
anti-immunoglobulin secondary antibodies were from Sigma.
Polyethyleneimine cellulose plates for thin layer chromatography were
from Selecto Scientific (Norcross, GA). Glutathione immobilized on
cross-linked 4% beaded agarose was from Sigma. Calmodulin was purified
from bovine testis as described previously (41). All chemicals were
reagent grade.
-SNAP, His6-
-SNAP, and GST-syntaxin
1 (cytosolic domain 1-265 amino acids) were produced as recombinant
proteins in E. coli and purified as described (7, 27).
Protein concentrations were measured with the Bio-Rad protein assay
reagent using ovalbumin as a standard. The anti-NSF monoclonal (2E5)
and polyclonal antibodies were described previously (11, 27). The
anti-
-SNAP antibody was from Gamma One Laboratories (Lexington, KY).
-32P]ATP (4, 500 Ci/mmol) in PKC buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 10 mM
MgCl2 1 mM CaCl2, 1 mM
DTT, 200 µg/ml L-
-phosphatidyl-L-serine, and 10 µg/ml diolein) or CaM kinase II buffer (50 mM
Tris-HCl, pH 7.6, 1 mM DTT, 10 mM
MgCl2, 1 mM CaCl2, 100 mM KCl, 1 µM calmodulin) in a total reaction
volume of 40 µl. Phosphorylation reactions were terminated by
addition of 1% SDS, 1% 2-mercaptoethanol and boiling for 5 min. The
radiolabeled proteins were resolved by SDS-PAGE, and the gels were
stained with 0.02% Coomassie Brilliant Blue R-250, 40% methanol, and
10% acetic acid, washed overnight, and dried. The levels of
phosphorylation were assessed by analysis with a Storm PhosphorImager
(Molecular Dynamics, Sunnyvale, CA) with ImagQuant software.
-mercaptoethanol, and 2 mM EDTA. After
1 h of incubation, the beads were washed four times in the same
buffer, and then equal volumes of the beads were aliquoted into the
reaction tubes. Particle formation reactions were performed in a final
volume of 500 µl containing 15 µl of beads with GST-syntaxin 1 in
binding buffer (20 mM HEPES/KOH, pH 7.4, 250 mM
imidazole, 150 mM potassium acetate, 5 mM EGTA, 1 mM AMP-PNP, 5 mM MgCl2, 1% (w/v)
glycerol, 1% (w/v) Triton X-100, and 10% (w/v) ovalbumin) and
saturating amounts of
-SNAP and wild-type or mutant NSF. After
3 h of incubation at 4 °C with rotation, the beads were washed
five times in binding buffer without ovalbumin. The bound proteins were
eluted with SDS-PAGE sample buffer, subjected to SDS-PAGE, and analyzed
by Western blotting using the 2E5 monoclonal antibody, which equally
detects both mutant and wild-type NSF. In all cases NSF binding was
SNAP-dependent.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-SNAP from either resting
or stimulated synaptosomes was detected in immunoprecipitates using
anti-
-SNAP antibodies (data not shown).
View larger version (37K):
[in a new window]
Fig. 1.
Depolarization-induced,
calcium-dependent phosphorylation of synaptosomal NSF.
A shows PhosphorImager
(32P) and immunoblot (Ab)
analysis of NSF immunoprecipitated from solubilized synaptosomes
pre-loaded with 32P and then treated for 30 s under
conditions that trigger NT release (Ca Rel, 1.5 mM Ca2+, 25 mM K+) or
various controls (Ca Pre, 1.5 mM
Ca2+, 1 mM K+; Mg Rel,
10 mM Mg2+, 25 mM K+;
Mg Pre, 10 mM Mg2+, 1 mM
K+). The position of NSF was detected by enhanced
chemifluorescence and the 32P label was detected by
PhosphorImager analysis following a 5-day exposure. Quantification of
relative labeling (B) was obtained by dividing
32P PhosphorImager densities by enhanced chemifluorescence
intensities for these samples. The values shown (hatched
bars) are averaged values for two experiments normalized to
Ca Rel as 100%. Error bars represent deviation
from the average. Glutamate release (solid bars) was
measured at 30 s from a parallel set of samples using the
continuous spectrophotometric assay outlined under "Experimental
Procedures." The data shown are averages of six separate experiments
with error bars indicating the range of values.
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Fig. 2.
In situ and in vitro
phosphorylation of NSF by protein kinase C. A
shows the results of two-dimensional thin layer electrophoresis
analysis of phosphoamino acids recovered from the
32P-labeled NSF from one of the Ca Rel samples
shown in Fig. 1. B is a similar analysis of in
vitro, PKC-phosphorylated, recombinant NSF shown in the
right panel of Fig. 3. Regions outlined by dotted
lines denote the positions of the indicated phosphoamino acid
standards (1 µg each), which were added to the sample prior to
analysis and detected by ninhydrin. C, the indicated
inhibitors (staurosporine and Kn-93) or activator (PMA) were
added, from Me2SO (DMSO)-dissolved stocks, to
synaptosomes prior to depolarization with potassium in the presence of
calcium or EGTA where noted. The incubations were stopped by the
addition of SDS and boiling. NSF was recovered by immunoprecipitation
and detected by PhosphorImager analysis. Kn-93 (and its inactive isomer
Kn-92), were added at 10 times the Ki for CaMKII.
Okadaic acid (OA) was added at levels that would block
protein phosphatases 1 and 2A. The numbers above
each lane of the right-hand panel of C represent
the ratio of PhosphorImager signal to Coomassie-stained NSF protein.
D, synaptosomes were incubated with calphostin C
(Calph, 500 nM), chelerythrine (Chel,
6.6 µM) and Ro31-8220 (Ro31, 100 nM) or bisindolemaleimide I (Bis, 100 nM) 15 min prior to depolarization. Reactions were stopped,
and NSF was recovered by immunoprecipitation. Phosphorylation was
evaluated as the ratio of PhosphorImager signal to Coomassie-stained
NSF protein. The data represent two separate experiments, which were
compared by setting the depolarization-induced phosphorylation
(Cal Rel) as 100%. Error bars represent
deviation from the average. Calphostin C was activated, after addition
to synaptosomes, by exposure to fluorescence lighting for 30 min at
room temperature (46).
View larger version (53K):
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Fig. 3.
Comparison of NSF and SNAP phosphorylation by
Ca2+-dependent protein kinases in
vitro. Samples of recombinant NSF (6 µg), -SNAP
(10 µg), or
-SNAP (10 µg) were treated for 1 h at 25 °C
in a total volume of 40 µl with CaM kinase II (0.05 milliunits) or
PKC (0.5 milliunits) + 1 mM [
-32P]ATP
(specific activity = 4,500 Ci/mmol). Phosphorylated samples were
immediately disrupted by boiling for 5 min in 1% SDS, 1%
2-mercaptoethanol, resolved by SDS-PAGE, and then electroblotted onto
PVDF yielding the PhosphorImager analyses shown following 48 h of
exposure. Samples contained various combinations of Ca2+ (1 mM), calmodulin (1 µM), phosphatidylserine
(200 µg/ml), or diolein (10 µg/ml) as indicated.
Autophosphorylation of the CaMKII
-chain is the major labeled band
in the left-hand panel.
- and
-SNAP under these conditions (Fig. 3)
indicating that the low extent of phosphorylation of NSF observed with
CaMKII is an inherent property of NSF as a substrate. Further
characterization of in vitro phosphorylation of NSF by PKC
showed that the reaction was time-dependent, saturable
(data not shown), and required the PKC activators phosphatidylserine, diolein, and calcium (Fig. 3). As noted above, phosphoamino acid analysis of the in vitro PKC-phosphorylated NSF showed the
presence of only phosphoserine (Fig. 2B). These data suggest
that PKC may be one of the enzymes responsible for in situ
phosphorylation of NSF. (It should be noted that the presence of
phosphothreonine in the in situ but not in vitro
phosphorylated NSF could be explained by contaminating phosphoproteins
in the material isolated from synaptosomes. Alternatively, the
commercial mixture of PKC isoforms (predominantly
,
, and
)
used in vitro may not contain all of the kinases
involved.)
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Fig. 4.
Identification and functional significance of
Ser-237 as the protein kinase C phosphorylation site in NSF.
A shows the HPLC profile of the phosphopeptides recovered
from trypsin (dashed line) and Lys C (solid line)
digestion of in vitro phosphorylated NSF. B shows
the 32P radiolabel recovered from the Lys C generated
phosphopeptides after each cycle of the chemical sequencing reactions
corrected for repetitive yield (92.5%). C shows the results
of in vitro phosphorylation of NSF by purified PKC.
His6-tagged NSF mutants S237A and S237E were phosphorylated
by PKC, in vitro, and separated by SDS-PAGE. The left
side shows the Coomassie-stained gel used to generate the
PhosphorImage at the right. D shows the results
from a SNAP/SNARE binding experiment. Mutants S237E and S237A mutants
or wild-type NSF were incubated with GST-syntaxin and -SNAP in the
presence of AMP-PNP. Complexes were recovered on GSH-agarose beads and
the amount of wild type (wt) or mutant NSF bound was
determined by Western blotting (Bound). The band at the
left side is pure NSF standard.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-SNAPs
(49), Munc18 (63, 64), synaptotagmins (65, 66), and now NSF are
substrates for PKC at least in vitro. In most of these
reports the PKC-mediated phosphorylation prevents the association of
proteins (i.e. phospho-Munc18a and syntaxin-1 (63) or
phospho-
-SNAP and syntaxin-1 (49)). In this report, this also
appears to be true based on the behavior of the S237E mutant NSF, which
cannot bind to the SNAP-SNARE complex. At this point, it is unclear how
these PKC-mediated phosphorylation events can regulate the secretory
cycle in the synaptosome. Here, we report depolarization-dependent phosphorylation of NSF that
requires calcium influx, suggesting that NSF phosphorylation occurs
after exocytosis. The physiological role of this modification in
controlling NSF function has yet to be determined.
1
helix, which is solvent-accessible and is adjacent to the inter-subunit
interface that is important for hexamerization. This residue (Ser-237)
is well within 15 Å of the adjacent subunit particularly the loop
between the
8 and
9 helices. Such a position could be important
since the
8 helix has several key residues that make contacts with
the ATP nucleotide (red, Fig.
5B) and with the adjacent
subunit (yellow, Fig. 5B). Phosphorylation at Ser-237 could affect the positioning or flexibility of the
8 and
therefore the conformational changes associated with ATP hydrolysis or
with nucleotide exchange. When one models in the residues that are present in the predicted
8-
9 loop of D1, the potential for an effect becomes more striking (Fig. 5B). This region of D1
has a net higher positive charge than the corresponding region of D2
(Fig. 5A) with the Arg-463 and Lys-466 being within 9 Å of the phosphoamino acid (or the modeled Glu in Fig. 5B). This
is well within the reach of charge influences from a negatively charged phosphate on the adjacent subunit. Such charge-charge interactions could restrict the movement of the
8 helix and thereby could affect
the catalytically important conformational change(s) in the D1 domain.
At this stage, the proposed effects of the negative charge at the
Ser-237 position remain speculative until a structure is available for
the D1 domain.
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Fig. 5.
Proposed molecular mechanism for the
regulation of NSF activity by Ser-237 phosphorylation.
A shows the structure-based sequence alignment of the
8-loop-
9 region of the D2 domain compared with D1. B,
the position of Ser-237 in the D1 domain was modeled onto the crystal
structure of the D2 domain (69). This representation shows a S237E
mutation made in one subunit (red) and the potentially
interacting residues on the adjacent subunit (blue). The
residues in the inter-helix loop have been modeled based on the D1
sequence. The
8 helix is green, and the
9 is
purple. The yellow residues in
8 make up part
of the inter-subunit interface that is critical to hexamer formation,
and those in red make up part of the nucleotide-binding
pocket of the blue subunit. B was constructed
with Swiss PDB Viewer and rendered with Pov-Ray.
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ACKNOWLEDGEMENTS |
---|
We thank Ramona Alcala, Charlotte Randle, and Carol Beach for their expert technical assistance. We also thank Todd Schraw for assistance in creating Fig. 5.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Veterans Affairs Research Service, Department of Veterans Affairs, and by the University of Kentucky Medical Center grants (to J. T. S.) and National Institutes of Health Grants NS21868 (to T. C. V.) and HL56652 (to S. W. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 859-323-6702 (ext. 245); Fax: 859-323-1037; E-mail: jslevin@pop.uky.edu.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M007394200
2 E. A. Matveeva et al. manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NT, neurotransmitter;
NSF, N-ethylmaleimide-sensitive factor;
SNAP, soluble NSF attachment protein;
SNAP-25, synaptosomal associated
protein of 25 kDa;
PKA, protein kinase A;
PKC, protein kinase C;
CaMKII, Ca2+/calmodulin dependent protein kinase II;
PMA, phorbol 12-myristate 13-acetate;
PAGE, polyacrylamide gel
electrophoresis;
HPLC, high pressure liquid chromatography;
LTP, long
term potentiation;
GST, glutathione S-transferase;
DTT, dithiothreitol;
AMP-PNP, adenosine 5'-(,
-imido)-triphosphate;
PVDF, polyvinylidene difluoride;
IP, immunoprecipitation;
v-SNARE, vesicle SNAREs;
t-SNARE, target membrane SNARE.
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