From the Department of Physiology, University of Michigan, Ann
Arbor, Michigan 48109-0622 and the Kennedy Krieger
Research Institute, Baltimore, Maryland 21205
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ABSTRACT |
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The Munc-18-syntaxin 1A complex has been postulated to act as a negative control on the regulated exocytotic process because its formation blocks the interaction of syntaxin with vesicle SNARE proteins. However, the formation of this complex is simultaneously essential for the final stages of secretion as evidenced by the necessity of Munc-18's homologues in Saccharomyces cerevisiae (Sec1p), Drosophila (ROP), and Caenorhabditis elegans (Unc-18) for proper secretion in these organisms. As such, any event that regulates the interaction of these two proteins is important for the control of secretion. One candidate for such regulation is cyclin-dependent kinase 5 (Cdk5), a member of the Cdc2 family of cell division cycle kinases that has recently been copurified with Munc-18 from rat brain. The present study shows that Cdk5 bound to its neural specific activator p35 not only binds to Munc-18 but utilizes it as a substrate for phosphorylation. Furthermore, it is demonstrated that Munc-18 that has been phosphorylated by Cdk5 has a significantly reduced affinity for syntaxin 1A. Finally, it is shown that Cdk5 can also bind to syntaxin 1A and that a complex of Cdk5, p35, Munc-18, and syntaxin 1A can be fashioned in the absence of ATP and promptly disassembled upon the addition of ATP. These results suggest a model in which p35-activated Cdk5 becomes localized to the Munc-18-syntaxin 1A complex by its affinity for both proteins so that it may phosphorylate Munc-18 and thus permit the positive interaction of syntaxin 1A with upstream protein effectors of the secretory mechanism.
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INTRODUCTION |
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Many of the key proteins involved in membrane targeting and
synaptic vesicle neurotransmitter release have been identified, and a
fundamental set of interactions has been defined and placed into a
model termed the SNARE hypothesis (1-4). Essential to the SNARE
hypothesis is the idea that a complex of proteins is formed from
soluble cytosolic proteins and from proteins integral to the synaptic
vesicle (termed v-SNAREs) and target membranes (termed t-SNAREs). The
soluble proteins include the ATPase
N-ethylmaleimide-sensitive fusion protein
(NSF)1 and a family of
proteins necessary for membrane attachment and activation of NSF termed
SNAPs (soluble NSF attachment
proteins). The SNAP receptors
(i.e. the SNAREs) were identified as synaptic vesicle-associated membrane protein (VAMP, also termed synaptobrevin), and the plasma membrane proteins syntaxin and
synaptosome-associated protein of 25 kDa (SNAP-25) (2, 5). In the SNARE
hypothesis, the core of the vesicle docking interaction results from
the interaction of v-SNAREs with cognate t-SNAREs via coiled-coil
domains to create a protein complex that further recruits cytosolic
factors /
- and
-SNAP and then NSF to form a 20 S complex. This
assemblage of proteins is required for the donor and target membranes
to come into close apposition and, upon ATP hydrolysis by NSF, become fusion-competent (4, 5). A specialization imposed on the SNARE
hypothesis with regard to neurotransmitter and neurohormone release is
the strict Ca2+-dependence of release, which necessitates
incorporation of a Ca2+ sensor into the fusion machine (1,
3).
Consistent with the SNARE hypothesis, proteins that regulate SNARE complex assembly in either a positive or negative fashion may substantially alter a cell's secretory response. The presence of regulatory proteins is suggested functionally both by the highly controlled nature of neurotransmitter release and by the description of multiple forms of synaptic plasticity that are of presynaptic cause (6). Proteins from two gene families have been identified as key regulators of SNARE complex assembly. These include members of the small GTP-binding family (e.g. Rabs) and of the SEC1 family (7, 8). The SEC1 gene is one of 10 genes identified as essential for the final stages of protein secretion in the yeast, Saccharomyces cerevisiae (9). Sec1p protein has sequence similarity to three other yeast proteins, Sly1p, Vps33p (Slp1p), and Vps45p, which are also important for vesicle targeting and fusion (10). The mammalian homologue of yeast Sec1p was initially identified as a syntaxin-binding protein (Munc-18) (11) and independently isolated by two groups and designated n-Sec1 and rbSec1 (12, 13), although they represent identical genes. rbSec1 is expressed in two alternatively spliced isoforms termed rbSec1A and -B, although, no functional differences have been found (14). Recent reports have demonstrated the existence of at least five additional mammalian Munc-18 homologues, with several forms being expressed ubiquitously (15-19).
Sec1p interacts with yeast syntaxins (Sso1p, Sso2p) based on genetic interactions (20), and Munc-18 has been shown to interact with mammalian syntaxins (11-13, 21) particularly with syntaxin isoforms 1a, 2, and 3 but not with isoforms 4 or 5 (15, 21). Munc-18 has not, however, been found to be part of the 20 S SNARE/SNAP/NSF protein assemblage and was thus postulated to be a negative regulator of v- and t-SNARE protein interactions (7, 15, 21). Sec1p protein homologues have also been reported to be important for vesicle trafficking. For example, Unc-18 (for uncoordinated) (22) and ROP (for Ras opposite) (23) represent neural enriched SEC1 homologues in Caenorhabditis elegans and Drosophila, respectively. Mutation in the unc-18 gene in C. elegans caused abnormal accumulation of acetylcholine and resistance to acetylcholinesterase inhibitors (22, 24), while ROP overexpression in Drosophila reduced the number of spontaneous vesicle fusions in half and significantly decreased evoked responses in response to repetitive stimulation (25). A separate study has shown a Drosophila ROP mutant to result in reduced light-evoked synaptic responses (26).
Recent investigations have found that Munc-18, like SNAP-25 and syntaxin, is not restricted to the synaptic region but distributed throughout the axon and soma, suggesting the possibility of additional actions of Munc-18 protein (14). Consistent with this possibility, Munc-18 has recently been reported to interact with members of a family of double C2 domain proteins termed DOC2 (27) and also with cyclin-dependent kinase 5 (Cdk5; also termed PSSALRE and Nclk for neuronal Cdc2-like kinase), to which it can be tightly bound (28). Cdk5, found in adult neural tissue, phosphorylates neurofilament protein and microtubule-associated tau protein (29-33). Cdk5 is a proline-directed kinase that can bind cyclin D but in brain is often associated with and activated by a 35-kDa protein (34). Munc-18 possesses predicted consensus sequences for phosphorylation by protein kinase A, tyrosine kinase, protein kinase C, casein kinase II, and, interestingly, two sites for Cdk5 (12, 35). A recent in vitro study demonstrated that phosphorylation by protein kinase C of Munc-18 shifted its binding affinity for recombinant GST-syntaxin fusion protein (36).
The purpose of the present investigation was to define the interactions between Cdk5, Munc-18, and syntaxin that occur at nerve endings and to determine their regulation by Cdk5 kinase activity. Furthermore, we investigated whether Munc-18 is subject to phosphorylation within isolated mammalian secretory nerve endings.
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EXPERIMENTAL PROCEDURES |
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Materials and Chemicals--
Recombinant pGEX plasmid constructs
encoding glutathione S-transferase fusion proteins,
pGEX-kg-Munc-18 (rat), pGEX-syntaxin 1A11 (rat), and
pGEX-2T-CDK5 (human) and pGEX-2T-P25 (bovine) were gifts of R. Scheller
and J. Wang, respectively. The plasmids were individually transformed
into Escherichia coli strain TG-1. The syntaxin construct
used throughout was restricted to the cytoplasmic domain of syntaxin
1A. Antibodies used included a mouse monoclonal and rabbit polyclonal
anti-Munc18 (Transduction Laboratories), a mouse monoclonal
anti-syntaxin (HPC-1, Sigma), a rabbit polyclonal anti-Cdk5 directed
against the carboxyl terminus (Santa Cruz Biotechnology Inc.), and a
rabbit polyclonal COOH-terminally directed anti-p35 (Santa Cruz
Biotechnology, Inc.). Protein kinase C purified from rat brain (,
,
mixture) was obtained from Upstate Biotechnology. 32PO4 and [
-32P]ATP
radioactive tracers were obtained from NEN Life Science Products.
Olomoucine and iso-olomoucine were from L. C. Laboratories.
Expression and Purification of Recombinant Proteins-- Recombinant GST fusion proteins were expressed in E. coli (TG-1) and subsequently purified by glutathione linked to Sepharose 4B (Pharmacia Biotech Inc.) as described (37, 38). Host bacteria were lysed using a French press at 1200 p.s.i. Removal of the GST moiety from the purified fusion protein, when necessary, was accomplished by thrombin (human, Sigma) treatment (0.2 NIH unit/µl, 4 h, 22 °C) in phosphate-buffered saline followed by the addition of phenylmethylsulfonyl fluoride (175 µg/ml) to inhibit thrombin action, centrifugation, and collection of the supernatant. Expressed fusion proteins were generally used immediately following purification for in vitro protein/protein interactions or protein phosphorylation studies. Induction of appropriate recombinant protein expression and its subsequent purification was verified by SDS-PAGE and Coomassie Blue staining or by Western blotting and probing with specific antibodies.
Determination of Protein Interactions in Vitro Using GST Fusion Protein-- Protein interactions were determined by incubation of syntaxin 1A, Cdk5, p25, and Munc-18 fusion proteins (individually or combinations) with an individual GST fusion protein, which was immobilized on glutathione-Sepharose 4B beads. Proteins were incubated in protein binding buffer with end-to-end rotation for 1 h or overnight at 4 °C. Protein binding buffer contained 4 mM Hepes/NaOH (pH 7.4), 0.1 M NaCl, 1 mM EDTA, 3.5 mM CaCl2, 3.5 mM MgCl2, and 0.5% Nonidet P-40. Following incubation, protein bound to the GST fusion protein was collected by centrifugation of the samples and washed three more times in binding buffer. Controls for each experiment consisted of analysis of fusion protein binding to GST protein alone immobilized on glutathione beads and elimination of binding through inclusion within the reaction of a 2-5-fold molar excess of a GST-free form of the fusion protein. In each case, protein bound on the beads and the final wash supernatant were dissolved in SDS-sample buffer, boiled and subjected to SDS-PAGE, and Western blotted onto nitrocellulose (0.2-µm Protran, Schleicher & Schuell), which was probed with different antibodies. ECL (Amersham Corp.) was performed according to the manufacturer's instructions, and chemiluminescence was quantitated with a GS-250 Molecular Imager (Bio-Rad) or visualized by x-ray film. The EC50 in saturation binding experiments was defined as half-maximal binding between two proteins with concentrations of the reactants kept below the point where significant nonspecific binding to GST immobilized on beads occurred. Quantitation of protein binding was based on pixel intensity obtained by phosphor imaging as above. To facilitate comparison between experimental determinations, data for each protein concentration were generally expressed as a percentage of the value observed upon binding saturation.
Analysis of Cdk5 Protein Kinase Activity-- Determination and monitoring of Cdk5 kinase activity utilized either purified Cdk5 and p25 fusion proteins or native Cdk5 immunoprecipitated with polyclonal anti-Cdk5 and protein A-agarose beads (Pierce) from rat brain homogenate. The rat brain sample was prepared by homogenization of whole brain in 5 ml of lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 8.0), 1% Nonidet P-40, 40 mM sodium pyrophosphate, 100 mM sodium fluoride, 175 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. The homogenate was then left on ice for 30 min, after which it was centrifuged at 100,000 × g for 30 min. A 1-ml aliquot of the resulting supernatant was used for Cdk5 immunoprecipitation after preclearing with 60 µl of a 50% slurry of protein A-agarose beads. Protein kinase activity of aliquots of immunoprecipitated protein and Cdk5/p25 fusion protein (1 µM) were determined using a Cdc2 kinase assay kit (Upstate Biotechnology, Inc.) by following incorporation of 32P radiotracer (0.025 µCi/µl, 3000 Ci/mmol) into a histone H1 peptide (22 µM) from bovine calf thymus containing predicted Cdc2 phosphorylation sites. Incubation of reactants was carried out for 15 min at 30 °C. Labeling was quantitated following reaction termination via binding of the histone substrate to phosphocellulose P81 paper, which was then extensively rinsed in 0.75% phosphoric acid and liquid scintillation-counted. Background controls included no added kinase. Specificity of tracer incorporation to Cdk5 activity was tested by inclusion of protein kinase C inhibitor peptide (RFARKGALRQKNV; 5 µM), protein kinase A inhibitor peptide (TYADFIASGRTGRRNAI; 0.5 µM), and a calmodulin-dependent protein kinase inhibitor (R24571; 5 µM) in the reaction mixture. In addition, kinase activity was tested for sensitivity to the highly specific Cdk5 inhibitor olomoucine (25 µM) or the much less active analog iso-olomoucine (25 µM). Labeled phosphate incorporation into Munc-18 fusion protein by both immunoprecipitated Cdk5 and purified Cdk5/p25 kinase activity was also determined by subjecting reaction samples to SDS-PAGE followed by autoradiography.
Modulation of Protein Interactions by Protein Kinase-mediated
Phosphorylation--
GST-Munc-18 purified from bacterial lysates by
immobilization onto glutathione-Sepharose 4B beads was eluted off the
beads in buffer containing 10 mM reduced glutathione and 50 mM Tris-HCl (pH 8.0) at room temperature for 10 min. The
eluate was then dialyzed (Slide-a-lyzer, Pierce, 10-kDa cut-off)
overnight at 4 °C against buffer containing 50 mM
Tris-HCl (pH 7.5), 1 mM EGTA, 6.25 mM -glycerol phosphate, 10 mM MgCl2, 100 µM CaCl2. The sample was recovered, and
protein concentration was determined (Bio-Rad protein assay).
GST-Munc-18 was then phosphorylated with either protein kinase C (0.42 µg/ml) or Cdk5 immunoprecipitated from rat brain according to the
manufacturer's instructions (Upstate Biotechnology). Protein kinase C
reactions included Ca2+ (1 mM),
phosphatidylserine (83.3 µg/ml), and diglyceride (8.3 µg/ml)
activators along with a protein kinase A inhibitor peptide (R24571, 3.3 µM). Phosphate incorporation was determined by inclusion of [
-32P]ATP radiotracer (0.017-0.025 µCi/µl,
3000 Ci/mmol) during the kinase reaction, which was followed by
SDS-PAGE, extraction of the Munc-18 protein band from the gel, and
liquid scintillation counting of each sample. Analysis of effects of
Munc-18 phosphate incorporation on binding to syntaxin 1A were
performed using nonradioactive ATP and by rebinding phosphorylated
Munc-18 to glutathione-Sepharose 4B beads (1 h, room temperature)
following the kinase reaction. The glutathione-Sepharose-linked
GST-Munc-18 was then centrifuged and washed extensively with protein
binding buffer. The phosphorylated Munc-18 fusion protein at a
concentration of 200 nM was next incubated with increasing
concentration of syntaxin 1A11 fusion protein with
rotational agitation for 30 min at 4 °C in protein binding buffer.
Following incubation, samples were centrifuged, pellets were
extensively washed, and protein was bound to beads solubilized in
SDS-sample buffer and subjected to SDS-PAGE and Western blotted. Blots
were probed with monoclonal anti-syntaxin followed with a horseradish
peroxidase-linked secondary antibody. Immunoreactive syntaxin bound to
GST-Munc-18 was visualized with ECL and quantitated by phosphor imaging
analysis. Nonphosphorylated Munc-18 incubated with syntaxin 1A served
as control. GST alone was not phosphorylated by the above kinases, and
no Cdk5 phosphorylation consensus sequences occur in syntaxin
1A11. Over the concentration range examined, no binding of
GST to syntaxin 1A11 was observed.
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RESULTS |
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Cyclin-dependent Kinase 5 Interaction with Munc-18-- Cyclin-dependent kinase 5- and Munc-18 protein-specific antibodies were used on immunoblots to demonstrate localization of the respective proteins within lysates of the mammalian neurohypophysis (i.e. neural lobe). Immunoreactivity within the lysates was found to comigrate with immunoreactivity to the respective recombinant bacterially expressed GST fusion proteins for Cdk5 or Munc-18 that had been thrombin-digested to release the GST moiety (Fig. 1A). Further localization of Cdk5 and Munc-18 to nerve endings was demonstrated by their continued presence in a preparation of purified isolated nerve endings (>90% peptidergic nerve endings) termed neurosecretosomes. Both Munc-18 and Cdk5 antibody were also capable of immunoprecipitating the respective proteins from neural lobe homogenates.
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Cyclin-dependent Kinase 5 Phosphorylation of Munc-18-- Munc-18 possess consensus phosphorylation sequences for a variety of protein kinases including two (Ser158, Thr574) for the proline-directed kinase Cdk5. Cdk5 immunoprecipitated from nerve ending lysates demonstrated enzymatic activity as measured by labeled phosphate incorporation into a histone peptide (Fig. 3A). Phosphorylation activity was inhibited by 73% (mean, n = 3) by the specific Cdk5 inhibitor olomoucine (25 µM; reported IC50 = 3 µM (39)). A less active olomoucine analog, iso-olomoucine (IC50 > 1 mM) was found to have limited inhibitory effects (~15%) on the immunoprecipitated kinase activity. Protein A-agarose beads alone following incubation with the neural lobe lysate served as control samples and demonstrated low kinase activity of nonspecifically adhered proteins.
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In Situ Munc-18 Phosphorylation-- While kinase-mediated phosphorylation of Munc-18 decreases its interaction with syntaxin, it has not yet been shown that Munc-18 exists as a phosphoprotein in situ. To determine if Munc-18 exists as a phosphoprotein in situ, we compared the pattern of Munc-18 immunoreactivity for control and alkaline phosphatase-treated brain Munc-18 immunoprecipitates on blots following two-dimensional gel electrophoresis (isoelectric and SDS-PAGE). As shown in Fig. 5, multiple Munc-18 immunoreactive spots migrating at 67 kDa, but which differed in isoelectric point, were observed in control immunoprecipitates. Importantly, dephosphorylation of the immunoprecipitated protein by alkaline phosphatase prior to two-dimensional gel electrophoresis resulted in a loss of reactivity of two spots at the acidic end concomitant with an increase of Munc-18 immunoreactivity toward the basic end of the isoelectric gel, thereby demonstrating that a proportion of Munc-18 in situ exists as phosphoprotein. Specificity of the immunoreactive signal on the blots for Munc-18 was demonstrated by a complete loss of immunoreactive signal upon incubation of the Munc-18 primary antibody with excess Munc-18 fusion protein prior to probe of the blots.
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Syntaxin Interaction with Cyclin-dependent Kinase 5 and Munc-18-- We next examined whether syntaxin, which has been reported to interact with a number of the proteins of the exocytotic mechanism including Munc-18 (21), was capable of interacting with Cdk5. Initial experiments evaluated binding using GST-syntaxin 1A attached to glutathione-Sepharose 4B beads and incubated with cytosol from neural lobe. Analysis of Cdk5 immunoreactivity within the pelleted GST-syntaxin and its comparison with GST protein run as control demonstrated that syntaxin exhibited a specific interaction with cellular Cdk5 (Fig. 6A). Further analysis aimed at defining the properties of the binding interaction were performed in vitro using a constant concentration of GST-Cdk5 fusion protein (200 nM) that was incubated with increasing concentration of syntaxin 1A fusion protein. Binding of syntaxin to GST-Cdk5 was found to be saturable with an EC50 value of 0.53 µM (n = 5, Fig. 6B). The addition of p25 fusion protein (200 nM) to the binding reaction was found to be without significant (p > 0.05, n = 3) effect on the GST-Cdk5 interaction with syntaxin (open symbol in Fig. 6B), although the presence of ATP (2 mM) resulted in a 2-fold increase of syntaxin bound (118,183 ± 15,777 counts for control versus 208,826 ± 22,002 counts with ATP, n = 3).
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Formation and Disassembly of a Cdk5-p25, Munc-18, Syntaxin Protein Complex-- Based on our results demonstrating that Munc-18 can interact with Cdk5 and with syntaxin and that Cdk5 can itself interact with syntaxin, we attempted to determine if these proteins might associate in vitro into a stable heteroligomeric protein complex. An initial series of experiments was performed using GST-p25 fusion protein linked to glutathione-Sepharose 4B beads that was incubated in a sequential manner with each of the proteins forming the putative protein complex. Following incubation of GST-p25 with each individual fusion protein, the mixture was centrifuged and extensively washed prior to incubation with the next fusion protein. Immunoblot analysis of the pellets and final washes from each reaction demonstrated that each additional protein component was present in the pellet but not in the final wash following its incubation reaction (Fig. 7A). The data also demonstrate that GST-p25 fails to directly interact with Munc-18 and that the addition of Munc-18 in this experiment results via its interaction with Cdk5, which interacts with the GST-p25 fusion protein. Overall, these data are supportive of the formation of a heteroligomeric complex containing Cdk5, Munc-18, syntaxin 1A, and GST-p25.
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DISCUSSION |
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The family of proteins related to yeast Sec1p is essential for maintenance of normal secretory activity as evidenced by the missorting of secretory products and the accumulation of secretory vesicles with mutations of Sec1p in yeast or the homologous proteins in C. elegans (Unc-18) and Drosophila (ROP) (9, 22, 24-26). In part, the necessity of the Sec1-related proteins is believed to result from their direct and high affinity interaction with members of the t-SNARE family of syntaxin proteins and from the control by this complex of a v- and t-SNARE protein interaction required for vesicle fusion (7, 15, 21). The present study focused on elucidating the regulatory control of protein interactions of the mammalian Sec1 homologue Munc-18 (also termed n-Sec1, rbSec1) at nerve endings with specific examination of the role of the proline-directed cyclin-dependent kinase 5 to which Munc-18 has been reported to bind (28). We have shown here that bacterially expressed Munc-18 fusion protein interacts with Cdk5 found in nerve ending lysates. This Cdk5 has bound its neural specific p35 activator protein and has therefore been rendered catalytically active. Our data further demonstrate that Munc-18 is subject to phosphorylation by p35-activated Cdk5 and that this phosphorylation significantly reduces Munc-18 binding to syntaxin in vitro. We also report here that in addition to their interaction with Munc-18, Cdk5 and the p35-Cdk5 complex are capable of interacting with syntaxin. Last, we demonstrate that a stable protein complex composed of p35, Cdk5, Munc-18, and syntaxin fusion proteins can be formed in vitro and that dissociation of the complex can be initiated by the addition of ATP. We interpret these data as consistent with a secretory model in which the interaction between Munc-18 and syntaxin 1A at mammalian nerve endings is under regulatory control by the association and phosphorylation of Munc-18 by p35-activated Cdk5. This phosphorylation of Munc-18 would then promote its dissociation from syntaxin and allow the formation of the heterotrimeric 7 S core complex, which is composed of syntaxin, SNAP-25, and VAMP proteins.
The dissociation of the hydrophilic Munc-18 from the integral membrane syntaxin protein is probably under strict regulatory control based on a reported 1000-fold higher binding affinity of Munc-18 than VAMP for syntaxin and of the absence of Munc-18 within the 7 S SNARE particle (21, 37). At present, regulation of binding interactions among the several proteins involved in the vesicle recruitment, docking and fusion process are poorly understood, although some have been demonstrated to be modified by phosphorylation. That the phosphorylation state of Munc-18 plays a critical role in regulating the affinity of the Munc-18/syntaxin interaction has been recently demonstrated by an in vitro assay with fusion proteins, which evaluated the effects of protein kinase C phosphorylation (36). Our results extend this analysis to demonstrate that native p35-Cdk5 immunoprecipitated from nerve ending lysates, as well as p25-activated recombinant Cdk5, was capable of phosphorylating Munc-18 and thereby reducing a Munc-18/syntaxin 1A interaction. Surprisingly, despite a substantially greater equilibrium level of phosphate labeling of Munc-18 with protein kinase C than with Cdk5, the functional result on syntaxin 1A binding was much greater for Cdk5-phosphorylated Munc-18. Furthermore, our findings present the first evidence that phosphorylation state may be physiologically relevant by demonstrating that a proportion of Munc-18 is sensitive to alkaline phosphatase treatment, and Munc-18 thus exists as a phosphoprotein in intact nerve endings. Munc-18 has been reported to bind to syntaxin isoforms 2 and 3 as well as 1A, although the effects of Munc-18 phosphorylation state on these additional interactions have not yet been evaluated. Regulatory control of Munc-18 binding interactions may not, however, be completely mediated by phosphorylation state, since nitric oxide (NO) has also recently been reported to directly inhibit interaction of Munc-18 with syntaxin in vitro (41). In addition, interaction of the yeast Sec1p homologue Sly1p with the t-SNARE syntaxin homologue Sed5p has recently been demonstrated to be regulated by a transient interaction of the Rab protein Ypt1p with Sed5p (8). An additional interaction between Munc-18 and members of a new family of C2 domain proteins termed DOC2 has also recently been described (27). It is thus possible that Munc-18 protein interactions are under multiple regulatory control. Further, as mammalian Munc-18 homologues and splice variants have been reported, there may also occur differential regulation of specific isoforms. For example, rbSec1 bound to bovine adrenal medullary secretory granules interacts with immobilized recombinant syntaxin, while free rbSec1 does not (42), suggesting that additional interactions such as with DOC 2 (27) may regulate conformational state and binding affinity.
Although Cdk5 shows a ubiquitous distribution, its phosphorylation activity is limited to neural tissue, where it is most highly expressed. A prior study had suggested that Munc-18 protein, like p35, was a specific activator of Cdk5 enzymatic activity (28). However, our data argue against a kinase activating function for Munc-18. For example, immunoprecipitation of Cdk5 from neural lobe lysate was found to co-precipitate p35 but not Munc-18, yet that immunoprecipitate did express kinase activity against a histone peptide containing proline-directed phosphorylation consensus sequences. Furthermore, using purified bacterially expressed fusion proteins together with an in vitro kinase assay, we have found that Cdk5 enzymatic activity is strongly activated by recombinant p25 but not by recombinant Munc-18. Demonstration that the enzymatic activity being monitored for both the immunoprecipitated and recombinant proteins was specific to Cdk5 kinase activity was provided by the sensitivity of phosphorylation to the specific Cdk inhibitor olomoucine and by the insensitivity to inhibitors of protein kinase C, protein kinase A, and calcium-calmodulin kinase II. Cdk5 has also been reported to associate with cyclin D1 and D3 in fibroblasts and in vitro, although binding results in only slight activation of phosphorylation activity (29, 43). Despite its role to activate Cdk5, the p35 protein has no sequence similarity to the cyclins that normally regulate Cdk activity. Furthermore, Cdk5 is unique among the Cdks in that it has not been found to be involved in cell proliferation but rather occurs in postmitotic neurons, where it is believed to control cytoskeletal functions and neurite outgrowth (29, 44). A marked increase in Cdk5 activity in vivo occurs during the neurogenic process and coincides with increased p35 levels (34, 45). Our findings that Munc-18 can be phosphorylated by p35- (or p25-) activated Cdk5 in vitro and that GST-Munc-18 is capable of a specific interaction with p35-Cdk5 from a neural lobe lysate suggest a functional relationship. The exact sites on Munc-18 phosphorylated by Cdk5 remain undetermined, although two Cdk5 phosphorylation consensus sequences in Munc-18 (Ser158, Thr574) present likely PO4 sites.
Our findings demonstrating an interaction between Munc-18 with Cdk5 at nerve endings are consistent with a recent report showing the co-purification of Munc-18 (p67) with Cdk5 activity from rat brain and binding of Cdk5 in a rat brain homogenate to p67 immobilized on an affinity matrix (28). However, we report here that only approximately 50% of the immunoreactive Cdk5 present in the nerve ending lysate could be bound to GST-Munc-18, although saturable binding relations between GST-Munc-18 and Cdk5 were found. This upper limit to Cdk5 binding may relate to a limiting amount of p35 protein in the nerve endings and to GST-Munc-18's preferential binding of the catalytically active p35-Cdk5 complex. Our p35 and Cdk5 immunoprecipitation data are largely in support of such a model. For example, immunodepletion of p35 from neural lobe lysate led to only a partial precipitation of the Cdk5 present in the lysate. In comparison, immunoprecipitation of Cdk5 was found to deplete p35 immunoreactivity from the lysate even under conditions where Cdk5 was not completely precipitated. Furthermore, analysis of binding interactions among GST-Munc-18, Cdk5, and p25 fusion proteins in vitro demonstrated that the addition of ATP to the incubation to produce a catalytically active p35-Cdk5 protein complex greatly enhanced the level of Cdk5-p35 binding to GST-Munc-18. The data suggest that ATP present in nerve ending lysate likely facilitated activation and thus transient interaction of Cdk5-p35 protein kinase complex to the GST-Munc-18 substrate. Regulation of Cdk5 activity levels may, therefore, be under strict control by the level of expression of p35, similar to that reported for cyclin control of other Cdks.
We have found, in addition to an interaction between Cdk5 and Munc-18,
that Cdk5 is capable of establishing a specific and saturable binding
interaction with syntaxin 1A of moderate affinity (EC50 = 0.52 µM). This adds Cdk5 to an increasing list of
cytosolic proteins and membrane proteins that have now been
demonstrated to posses an affinity for interaction with syntaxin. These
proteins include -SNAP, VAMP (synaptobrevin), SNAP-25,
synaptotagmin, and voltage-gated N type Ca2+ channels (37,
46, 40). The interactions have been shown to be largely mediated by
assembly of coiled-coil structures with the carboxyl-terminal domain of
syntaxin. Whether this specific region is important in Cdk5 binding
remains undetermined. Our data also indicate that the ability of Cdk5
to interact with syntaxin was unchanged in the presence of p25 fusion
protein, suggesting that Cdk5 does not have to be in an enzymatically
active conformational state to bind. One may envision that the
interaction of Cdk5 with syntaxin may act to further promote, localize,
or stabilize the interaction of Cdk5 or the Cdk5-p35 heterodimer with
the Munc-18-syntaxin complex. Indeed, we demonstrate here, using
sequential binding reactions of bacterially expressed fusion proteins,
that in the absence of ATP, one can assemble in vitro a
stable protein complex containing the p25, Cdk5, Munc-18, and syntaxin
proteins. In vitro, Munc-18 has been reported to bind
syntaxin in a 1:1 molar ratio (12). These data do not, however, exclude
yet larger multimeric complexes, and the stoichiometry of the
interactions within the larger protein complex remains to be fully
investigated. In addition, it will be of importance to determine the
sequence of protein interactions that occur in situ. Of
particular importance, however, are our findings demonstrating that the
addition of ATP to the preformed complex is able to support kinase
activity and lead to rapid disassembly of the complex.
This report has focused on regulation of binding interactions by phosphorylation state of the mammalian neural specific Sec1 family member, Munc-18. However, a number of mammalian Sec1/Munc-18 homologues have been identified that are more ubiquitously distributed and exhibit different binding specificity for syntaxin isoforms (e.g. Munc 18c) or lack binding to known syntaxins (e.g. r-vps33a, r-vps33b, h-vps45) (15-19). Although selectivity in pairing of Munc-18 homologues with syntaxins or syntaxin homologues may be an important determinant of secretory characteristics or specificity to different vesicle transport pathways, it remains unknown whether their binding interactions are similarly under strict regulatory control by Cdk5 phosphorylation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS31888 and NS36227 (to E. L. S.).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: Dept. of Physiology, 7804 Medical Sciences II Bldg., University of Michigan, Ann Arbor, MI 48109-0622. Tel.: 313-763-4477; Fax: 313-963-8813; E-mail: esterm{at}umich.edu.
1 The abbreviations used are: NSF, N-ethylmaleimide-sensitive fusion protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; Cdk, cyclin-dependent kinase.
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REFERENCES |
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