Phosphorylation of the N-Ethylmaleimide-sensitive Factor Is Associated with Depolarization-dependent Neurotransmitter Release from Synaptosomes*

Elena A. MatveevaDagger , Sidney W. WhiteheartDagger , Thomas C. VanamanDagger , and John T. Slevin§||

From the  Neurology Service, Department of Veterans Affairs Medical Center, Lexington, Kentucky 40511 and the Departments of Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- ATP and AMP-PNP were obtained from Roche Molecular Biochemicals. [gamma -32P]ATP (4,500 Ci/mmol) and [32P]PO<UP><SUB>4</SUB><SUP>2+</SUP></UP> (7,000 Ci/mmol) were from ICN (Costa Mesa, CA). PKC from rat brain (mix of alpha , beta , and gamma  isoforms) was from Promega (Madison, WI) and CaM kinase II, also from rat brain, was from Calbiochem. L-alpha -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.

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-alpha -SNAP, His6-gamma -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-alpha -SNAP antibody was from Gamma One Laboratories (Lexington, KY).

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 [gamma -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-alpha -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.

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) beta -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 alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -SNAP from either resting or stimulated synaptosomes was detected in immunoprecipitates using anti-alpha -SNAP antibodies (data not shown).



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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.

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)).



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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).



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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), alpha -SNAP (10 µg), or gamma -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 [gamma -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 alpha -chain is the major labeled band in the left-hand panel.

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 alpha - and gamma -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 alpha , beta , and gamma ) used in vitro may not contain all of the kinases involved.)

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.



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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 alpha -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.

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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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), alpha - and gamma -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-alpha -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.

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 alpha 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 alpha 8 and alpha 9 helices. Such a position could be important since the alpha 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 alpha 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 alpha 8-alpha 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 alpha 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 alpha 8-loop-alpha 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 alpha 8 helix is green, and the alpha 9 is purple. The yellow residues in alpha 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.



    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'-(beta ,gamma -imido)-triphosphate; PVDF, polyvinylidene difluoride; IP, immunoprecipitation; v-SNARE, vesicle SNAREs; t-SNARE, target membrane SNARE.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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