©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Munc-18/n-Sec1/rbSec1 by Protein Kinase C
ITS IMPLICATION IN REGULATING THE INTERACTION OF Munc-18/n-Sec1/rbSec1 WITH SYNTAXIN (*)

(Received for publication, November 30, 1995; and in revised form, January 15, 1996)

Yasuyuki Fujita (1) Takuya Sasaki (1) Koji Fukui (1) Hirokazu Kotani (1) Toshihiro Kimura (1) Yutaka Hata (3)(§) Thomas C. Südhof (3) Richard H. Scheller (4) Yoshimi Takai (1) (2)(¶)

From the  (1)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan, the (2)Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan, the (3)Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, Texas 75235, and the (4)Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser and Ser. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.


INTRODUCTION

Recent results from biochemical and genetic studies allow formation of a model for how synaptic vesicles are docked and fused with the presynaptic plasma membrane (for reviews, see (1, 2, 3) ). According to this model, named the SNARE (^1)hypothesis, syntaxin and SNAP-25 on the presynaptic plasma membrane serve as t-SNAREs, whereas VAMP/synaptobrevin on synaptic vesicles serves as a v-SNARE. Syntaxin-1a and -1b were isolated as proteins interacting with synaptotagmin/p65, a synaptic vesicle membrane protein (4, 5) . Syntaxin-1a was also identified as a surface protein of various neurons recognized by a monoclonal antibody, HPC-1(6, 7) . The t-SNAREs and v-SNARE first form a ternary complex followed by assembly of the NSF-SNAP system, eventually causing the docking and/or the fusion of the vesicles with the membrane.

SEC1 was isolated as a gene essential for vesicle transport in yeast(8) . Unc-18 and rop were identified as SEC1 homologs in Caenorhabditis elegans and Drosophila, respectively(9, 10) . An unc-18 mutant inhibits neurotransmitter release from the presynaptic nerve terminals in Caenorhabditis elegans(11) , and a rop mutant shows a loss of normal synaptic response to a light stimulus in Drosophila(12) . Munc-18 was isolated as a syntaxin-binding protein and turned out to be a mammalian homolog of yeast Sec1p(13) . A mammalian homolog of yeast SEC1 was also isolated by the polymerase chain reaction method and named n-Sec1 and rbSec1(14, 15) . These three genes, Munc-18, n-Sec1, and rbSec1, were identical. Munc-18 interacts with syntaxin with the highest affinity (K approx 80 nM) among all syntaxin-interacting molecules, and the interaction of Munc-18 with syntaxin inhibits the association of VAMP and SNAP-25 with syntaxin (16) . Overexpression of the syntaxin-related proteins, Sso1p and Sso2p, suppresses a partial loss of SEC1 gene activity in yeast (17) . These results suggest that Munc-18 plays a central role in synaptic vesicle transport by regulating the formation of the NSF-SNAPbulletSNARE complex.

PKC was originally isolated as a Ca- and phospholipid-dependent protein kinase (18, 19, 20) and exerts a wide range of physiological functions (for a review, see a (21) ). The PKC family is divided into three types: conventional PKCs (alpha, beta, and isoforms), novel PKCs (, , , and isoforms), and atypical PKCs ( and isoforms)(21) . Of many functions of PKC, conventional PKCs are involved in Ca-dependent exocytosis in many types of secretory cells (for a review, see a (22) ), including platelets (23) , mast cells(24, 25) , and chromaffin cells(26, 27, 28) . However, the mode of action of PKC in exocytosis has not been understood.

In the present study, we have examined whether Munc-18 is phosphorylated by PKC and whether this phosphorylation affects the interaction of Munc-18 with syntaxin.


EXPERIMENTAL PROCEDURES

Materials and Chemicals

pQE9-Munc-18 was transformed into BL21(DE3) cells. His(6)-Munc-18 was purified by Ni-NTA-agarose column chromatography (Invitrogen). Recombinant GST-syntaxin-1a was purified by glutathione-Sepharose 4B column chromatography as described(4) . A mixture of conventional PKCs (alpha, beta, and isoforms) was purified from rat brain (29) and separated into each isoform by hydroxyapatite column chromatography (30) . The catalytic subunit of PKA was purified from rabbit skeletal muscle(31) . CaMKII and calmodulin were purified from rat brain and testis, respectively(32, 33) .

Phosphorylation of Munc-18 by PKC

Phosphorylation of Munc-18 was performed in a reaction mixture (50 µl) containing 50 mM Tris/HCl at pH 7.5, 6 mM MgCl(2), 1 mM DTT, 50 mM imidazole, 0.06% CHAPS, 100 µM CaCl(2), 50 µM EGTA, 50 µM [-P]ATP (0.5-1.2 times 10^3 cpm/pmol), and the mixture of conventional PKCs at 30 °C for the indicated periods of time. Where specified, each isoform was used. The amounts of His(6)-Munc-18, PKC, PS, and TPA in a reaction mixture were 30 pmol, 0.4 pmol, 0.5 µg, and 100 nM, respectively, when not indicated. The reaction was stopped by the addition of 25 µl of 3 times Laemmli's buffer(34) . Each sample was subjected to SDS-PAGE followed by protein staining with a Coomassie Brilliant Blue and autoradiography. The radioactivity of each band was measured with Fujix BAS2000 Bioimaging Analyzer.

Inhibition by Syntaxin of the PKC-catalyzed Phosphorylation of Munc-18

Forty pmol of GST-syntaxin-1a fusion protein, 30 pmol of His(6)-Munc-18, or both were preincubated at 4 °C for 30 min in the PKC reaction mixture containing PKC, PS, and TPA, but no [-P]ATP. After the incubation, [-P]ATP was added, and incubation was further performed at 30 °C for 10 min. In one set of experiments, each sample was directly subjected to SDS-PAGE. In another set of experiments, after the phosphorylation reaction, the reaction mixture was incubated with 10 µl of glutathione beads in the presence of 0.3% CHAPS at 4 °C for 30 min, followed by centrifugation and extensive washing. The sample was then subjected to SDS-PAGE.

Inhibition of the Interaction of Munc-18 with Syntaxin by Its Phosphorylation

Forty pmol of His(6)-Munc-18 was phosphorylated by PKC maximally for 30 min in the presence of Ca, PS, and TPA. Phosphorylated Munc-18 was incubated with 30 pmol of GST-syntaxin-1a bound to 10 µl of glutathione beads in the presence of 1% CHAPS at 4 °C for 30 min, followed by centrifugation and extensive washing. As a control, 40 pmol of His(6)-Munc-18 was incubated under the same conditions as described above except that [-P]ATP was excluded. The intensity of the bands in SDS-PAGE was determined by densitometry.

Determination of the Phosphorylation Sites of Munc-18 by PKC

Two hundred pmol of Munc-18 was phosphorylated by PKC as described above. Phosphorylated Munc-18 was resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane and digested with Achromobacter protease I(35) . The digested peptides were subjected to a COSMOSIL C(18) column (Nacalai Tesque, Inc., Kyoto, Japan) pre-equilibrated with 0.1% trifluoroacetic acid. Elution was performed with a 80-ml linear gradient of acetonitrile (0-65%) in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The radioactivity of each peptide was counted. The amino acid sequences and phosphorylated residues of the radioactive peaks were determined as described(36) .

Other Procedures

Protein concentrations were determined with bovine serum albumin as a reference protein(37) . Proteins were separated by 10% SDS-PAGE(34) .


RESULTS

Phosphorylation of Munc-18 by PKC

Munc-18 has many consensus amino acid sequences for PKC, PKA, and CaMKII (for a review, see a (38) ). We first examined whether Munc-18 is phosphorylated by these protein kinases. Munc-18 was phosphorylated by the mixture of conventional PKCs in the presence of Ca and PS, but it was rarely phosphorylated by PKA or CaMKII (Fig. 1A). Activities of these three kinases were confirmed using histone IIIss, myelin basic protein, and rabphilin-3A as respective substrates (data not shown). About 1 mol of phosphate was maximally incorporated into 1 mol of Munc-18 by PKC (data not shown). The K(m) value for Munc-18 was about 1 µM (data not shown), which was comparable to that of the other substrates for PKC, such as histone(39) . Munc-18 was equally phosphorylated by each of alpha, beta, and isoforms (data not shown).


Figure 1: Phosphorylation of Munc-18 by PKC. A, effect of PKC, PKA, and CaMKII on phosphorylation of Munc-18. His(6)-Munc-18 was incubated for 3 min with PKC in the presence of Ca and PS. His(6)-Munc-18 was incubated for 3 min with 2.5 pmol of PKA in the same reaction mixture as used for the PKC assay except that EGTA and Ca were excluded. His(6)-Munc-18 was incubated for 3 min with 0.2 pmol of CaMKII in the same reaction mixture as used for the PKC assay except that PS was excluded but that 50 µM Ca and 0.2 nmol of calmodulin were included. Lane 1, PKC; lane 2, PKA; lane 3, CaMKII. Arrowhead, Munc-18. B, effect of various activators on the PKC-catalyzed phosphorylation of Munc-18. His(6)-Munc-18 was phosphorylated by PKC for 3 min in the presence of various combinations of Ca, PS, TPA, and 5 mM EGTA; lane 1, Ca alone; lane 2, PS and EGTA; lane 3, Ca and PS; lane 4, Ca, PS, and TPA. Arrowhead, Munc-18. The results shown are representative of three independent experiments.



Conventional PKCs are activated by Ca and PS, and this activation of PKCs is further enhanced by diacylglycerol or phorbol ester(20, 21) . Consistently, in the presence of both Ca and PS, Munc-18 was phosphorylated by conventional PKCs (Fig. 1B, lane 3). In the presence of Ca or PS alone, Munc-18 was not phosphorylated at all (Fig. 1B, lanes 1 and 2). TPA, a PKC-activating phorbol ester, further enhanced the Ca- and PS-dependent phosphorylation of Munc-18 (Fig. 1B, lane 4).

Determination of the Phosphorylation Sites of Munc-18 by PKC

Munc-18 was fully phosphorylated with [-P]ATP and was completely digested with Achromobacter protease I. The digest was subjected to C(18) column chromatography. More than thirty peptide peaks were observed (Fig. 2A). When the radioactivity of each peak was measured, two major (Peak-1 and -2) and two minor peaks were detected. The amino acid sequences and the phosphorylated residues of the two major peaks were determined. The two minor peaks were too small to be analyzed. Peak-1 and -2 were derived from Peptide-1 and -2, respectively, as shown in Fig. 2B. Peak-1 contained three Ser residues. Ser was phosphorylated, but Ser or Ser was not as shown in Fig. 2C. Peak-2 contained one Thr and two Ser residues, and only Ser was phosphorylated (data not shown). Ser is well conserved in rop and unc-18, but Ser is not.


Figure 2: Phosphorylation sites of Munc-18 by PKC. A, peptide map analysis of fully phosphorylated Munc-18. (-), absorbance; shaded bar, radioactivity. B, amino acid sequences of the phosphopeptides. Asterisk, phosphorylated residue. C, sequencing data of Ser, Ser, and Ser. The amounts of phenylthiohydantoin-serine and DTT-serine of each residue are shown. Lane 1, phenylthiohydantoin-serine; lane 2, DTT-serine.



Inhibition by Syntaxin of the PKC-catalyzed Phosphorylation of Munc-18

We next examined whether the interaction of Munc-18 with syntaxin affects its phosphorylation by PKC. Munc-18 was phosphorylated by conventional PKCs in the absence and presence of GST-syntaxin-1a. The phosphorylation of Munc-18 dramatically decreased in the presence of GST-syntaxin, compared with that in the absence of GST-syntaxin (Fig. 3, A and B, lanes 2 and 3). GST-syntaxin itself was not phosphorylated (Fig. 3, A and B, lane 1). GST alone did not affect the phosphorylation of Munc-18 (data not shown). GST-syntaxin did not affect the PKC-catalyzed phosphorylation of another substrate, histone IIIss (data not shown). To confirm that Munc-18 complexed with syntaxin was not phosphorylated by PKC, the Munc-18 samples phosphorylated by PKC in the absence and presence of GST-syntaxin were separately incubated with glutathione beads, and the proteins bound to the beads were subjected to SDS-PAGE followed by protein staining and autoradiography. When the phosphorylation was performed in the absence of GST-syntaxin, the Munc-18 protein molecule was very faintly detected in the beads, and the phosphorylation in the Munc-18 bound to the beads was slightly detected (Fig. 3, A and B, lane 4). These results indicate that the Munc-18 protein molecule nonspecifically interacts with the syntaxin-free beads to a very small extent. However, when the phosphorylation was performed in the presence of GST-syntaxin, a significant amount of Munc-18 was detected in the beads, but the phosphorylation in the Munc-18 bound to the beads was not detected at all (Fig. 3, A and B, lane 5). These results indicate that the Munc-18 complexed with GST-syntaxin is not phosphorylated by PKC.


Figure 3: Inhibition by syntaxin of the PKC-catalyzed phosphorylation of Munc-18. GST-syntaxin-1a, His(6)-Munc-18, or both were phosphorylated by PKC. In one set of experiments, each sample was directly subjected to SDS-PAGE. Lane 1, syntaxin; lane 2, Munc-18; lane 3, syntaxin and Munc-18. In another set of experiments, after the phosphorylation reaction, the reaction mixture was incubated with glutathione beads. Proteins on the beads were subjected to SDS-PAGE. Lane 4, Munc-18; lane 5, syntaxin and Munc-18. A, protein staining; B, autoradiography. Arrowhead, Munc-18; arrow, syntaxin.



We performed another experiment to confirm that the Munc-18 complexed with GST-syntaxin was not phosphorylated by PKC. Munc-18 was first incubated with GST-syntaxin-1a-bound glutathione beads, followed by centrifugation and extensive washings. The Munc-18 bound to the GST-syntaxin beads was incubated with conventional PKCs in the same way as described above, but no phosphorylation of Munc-18 was detected (data not shown).

Inhibition of the Interaction of Munc-18 with Syntaxin by Its Phosphorylation

Finally, we examined whether the phosphorylation of Munc-18 affects its interaction with syntaxin. Munc-18 was incubated with conventional PKCs in the absence and presence of [-P]ATP, and incubated with GST-syntaxin-1a-bound glutathione beads. Free Munc-18 and the Munc-18 bound to GST-syntaxin beads were separated by centrifugation and quantitated by SDS-PAGE followed by protein staining and autoradiography. The amount of the Munc-18 bound to GST-syntaxin observed after the incubation with [-P]ATP was about 20%, whereas that observed after the incubation without [-P]ATP was about 65% (Fig. 4, A and C). Of the P radioactivity incorporated into Munc-18 after the incubation with [-P]ATP, less than 10% was found in the GST-syntaxin beads, whereas more than 90% was recovered in the supernatant (Fig. 4, B and D).


Figure 4: Inhibition of the interaction of Munc-18 with syntaxin by its phosphorylation. His(6)-Munc-18 was first phosphorylated, and then incubated with GST-syntaxin-1a bound to glutathione beads, followed by centrifugation. A half of the supernatant and the whole precipitated beads were solubilized and subjected to SDS-PAGE. As a control, His(6)-Munc-18 was incubated under the same conditions except that [-P]ATP was excluded. A, protein staining. Lane 1, the supernatant without [-P]ATP; lane 2, the precipitated beads without [-P]ATP; lane 3, the supernatant with [-P]ATP; lane 4, the precipitated beads with [-P]ATP. B, autoradiography. Lane 1, the supernatant with [-P]ATP; lane 2, the precipitated beads with [-P]ATP. Arrowhead, Munc-18; arrow, syntaxin. C, quantification of A. D, quantification of B.




DISCUSSION

We have first shown here that Munc-18 is phosphorylated by PKC, but not by PKA or CaMKII, in a cell-free system. The major phosphorylation sites are Ser and Ser located in the middle portion of the protein. Munc-18 tightly interacts with syntaxin and this interaction inhibits the association of VAMP and SNAP-25 with syntaxin(16) , which may lead to blockage of the formation of the NSFbulletSNAPbulletSNARE complex. We have shown here that the interaction of Munc-18 with syntaxin inhibits the PKC-catalyzed phosphorylation of Munc-18. This result suggests that the Munc-18 complexed with syntaxin does not serve as a substrate for PKC. The phosphorylation site of Munc-18 may be directly or indirectly masked by interaction with syntaxin.

We have moreover shown here that the PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. Both the N- and C-terminal regions of Munc-18 are responsible for the interaction with syntaxin(40) , but the definite syntaxin-binding region of Munc-18 has not been determined. Although Munc-18 is phosphorylated at least at two sites, Ser and Ser, about 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. Therefore, phosphorylated Munc-18 may consist of the Ser-phosphorylated, Ser-phosphorylated, and/or Ser, Ser-phosphorylated forms. We could not separate these three forms by column chromatographies, but our results suggest that either phosphorylation of Ser or Ser, or both, contributes to the inhibition of the interaction of Munc-18 with syntaxin. This region may be involved in the interaction with syntaxin, and Munc-18 may function as a key regulator in the NSF-SNAP-SNARE system through the phosphorylation of this region.

We have not studied here whether the PKC-catalyzed phosphorylation of Munc-18 indeed occurs in intact neuron, because we have not yet succeeded in making a good antibody to precipitate Munc-18. However, our present results have raised several possible physiological functions of the PKC-catalyzed phosphorylation of Munc-18: 1) the phosphorylation of Munc-18 may block its re-interaction with syntaxin, after it dissociates from syntaxin, thus eventually facilitating the formation of the NSFbulletSNAPbulletSNARE complex; 2) Munc-18 has been shown to be widely distributed in the axon and not to be restricted to the nerve terminals(41) . A majority of membrane-bound Munc-18 is not complexed with syntaxin(41) . These results suggest that Munc-18 plays another role and that there is a mechanism to cause this distribution. The PKC-catalyzed phosphorylation of Munc-18 may be involved in this mechanism; and 3) the phosphorylation may confer a novel characteristic on Munc-18 to interact with an unknown protein and to perform an unknown function. Further studies are necessary to establish the physiological function of the PKC-catalyzed phosphorylation of Munc-18.


FOOTNOTES

*
This investigation at Osaka University was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan(1995), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan(1995), and by a grant from the Uehara Memorial Foundation(1995). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Takai Biotimer Project, Exploratory Research for Advanced Technology (ERATO), 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan.

To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. Tel.: 81-6-879-3410; Fax: 81-6-879-3419; ytakai{at}molbio.med.osaka-u.ac.jp.

(^1)
The abbreviations used are: SNARE, SNAP receptor; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide-sensitive fusion protein; SNAP-25, synaptosomal-associated protein of 25 KDa; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; v-SNARE, vesicle SNARE; PKC, protein kinase C; GST, glutathione S-transferase; PKA, cyclic AMP-dependent protein kinase; CaMKII, calmodulin-dependent protein kinase II; PS, phosphatidylserine; DTT, dithiothreitol; TPA, 12-O-tetradecanoylphorbol-13-acetate; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. M. Inagaki for fruitful discussion.


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