(Received for publication, November 30, 1995; and in revised form, January 15, 1996)
From the
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.
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 ()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
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-SNAP
SNARE 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 (
,
,
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.
Figure 1:
Phosphorylation of
Munc-18 by PKC. A, effect of PKC, PKA, and CaMKII on
phosphorylation of Munc-18. His-Munc-18 was incubated for 3
min with PKC in the presence of Ca
and PS.
His
-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
-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
-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).
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.
Figure 3:
Inhibition by syntaxin of the
PKC-catalyzed phosphorylation of Munc-18. GST-syntaxin-1a,
His-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).
Figure 4:
Inhibition of the interaction of Munc-18
with syntaxin by its phosphorylation. His-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
-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.
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 NSF
SNAP
SNARE 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 NSFSNAP
SNARE 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.