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INTRODUCTION |
The most generalized form of the
SNARE1 hypothesis describes a
series of biochemical steps, common among diverse cell types and
organisms, that mediate the trafficking of subcellular vesicles (1). In
the case of regulated exocytosis, the formation and dissociation of
SNARE protein complexes is essential and under spatial and temporal
control (1-3). In neurons, the final stages of vesicle priming and
membrane fusion leading to neurotransmitter release are also strictly
Ca2+-dependent (4). In addition to
Ca2+, there are a number of other factors that have been
postulated to regulate the secretory machinery either positively or
negatively. One such factor is the protein Munc18a (also termed nSec1
and rbSec1), a mammalian homologue of the Saccharomyces
cerevisiae Sec1p protein (5-7). Munc18a is at once essential and
inhibitory to secretion (8) because while Sec1p and its homologues are necessary for membrane trafficking and the final stages of protein secretion (9, 10), high affinity binding between Munc18a and syntaxin
1a also inhibits the association of vesicle SNAREs with syntaxin 1a
(5-7, 11). The association of vesicle SNAREs with syntaxin is both
essential (1-3) and sufficient (12) for formation of a protein core
complex that mediates vesicle fusion. Genetic evidence has also
established that the Drosophila Sec1 homologue ROP functions
in vivo to regulate neurotransmitter release via binding to
syntaxin (13). As such, any factor that regulates the interaction of
Munc18a with syntaxin 1a, either by increasing their affinity or by
prompting their dissociation, might be crucial to the ultimate control
of the secretory process.
One candidate for this type of regulation is Cdk5, a member of the Cdc2
family of cell cycle kinases that has recently been found to
co-precipitate with Munc18a from rat brain (14). Unlike the other
members of this family, Cdk5 appears to be neither directly involved in
the cell cycle nor activated by a cyclin (15, 16). Indeed, Cdk5 was
first isolated from brain tissue as part of the Nclk (neuronal
Cdc2-like kinase) complex, where it was found to be associated with a
35-kDa neural specific activator protein now termed p35 (17-19). In
this capacity, Cdk5 has been demonstrated to act as a proline-directed
serine/threonine kinase, phosphorylating neurofilament and tau protein
at (S/T)PX(K/R) sites (20, 21). Moreover, mice that lack p35
or Cdk5 have been shown to suffer from severe cortical lamination
defects, suggesting that the Nclk complex is also essential for proper
neuronal migration and, therefore, for brain development in general
(22, 23). Although Cdk5 can associate with cyclin D, it does not appear
that, despite its demonstrated structural similarities to p35, cyclin D
is able to fully activate Cdk5 (24). This has led to the suggestion that cyclin D exerts an indirect effect on Cdk5 by competing with p35
for binding (25). Cdk5 is further thought to differ from the other cell
cycle kinases in that it does not appear to be directly regulated by
phosphorylation. Thus, it is neither activated by CDK-activating kinase
nor inhibited by Wee1 kinase, although it possesses consensus sites for
both (26). However, recent work has also determined that there may be a
number of regionally specific isoforms of p35 and that levels of p35
are under strict control (27). Thus, regulation of Cdk5 may be as
intricate and highly specialized as that of the other members of the
Cdc2 family (28).
Munc18a is a potential substrate for Cdk5 phosphorylation as it
contains two of the Cdk5 consensus sequences identified from neurofilament and tau protein (9). Furthermore, the phosphorylation state of Munc18a has previously been shown to be a crucial determinant of its interaction with syntaxin 1a. When phosphorylated by protein kinase C, Munc18a has been shown to have a greatly reduced affinity for
syntaxin 1a, although protein kinase C has proven ineffective at
phosphorylating Munc18a already bound to syntaxin 1a (29). Recently it
has been demonstrated that Cdk5 bound to its 35-kDa activator protein
not only binds Munc18a but utilizes it as a substrate for
phosphorylation, and that Munc18a phosphorylated in this manner has a
significantly reduced affinity for syntaxin 1a (30).
The focus of the present investigation was to attempt to more
completely characterize the interaction between Cdk5 and Munc18a and to
establish the likelihood of a regulatory role for Cdk5 in the secretory mechanism.
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EXPERIMENTAL PROCEDURES |
Materials and Chemicals--
Recombinant pGEX plasmid constructs
containing GST-nSec1 (rat), GST-syntaxin 1a (rat), GST-Cdk5 (human),
and GST-p25 (bovine) were gifts of R. Scheller and J. Wang.
Escherichia coli strain TG-1 was used as the host for the
bacterial expression recombinant plasmids. Mouse monoclonal anti-Munc18
was obtained from Transduction Laboratories, and rabbit polyclonal
anti-Cdk5 (C8) was purchased from Santa Cruz Biotechnology, Inc.
Protein kinase C purified from rat brain was purchased from Calbiochem.
Olomoucine and iso-olomoucine were from LC Laboratories.
[
-32P]ATP was purchased from NEN Life Science
Products. Sprague-Dawley rats were asphyxiated with carbon monoxide and
decapitated prior to preparation of neural lobe tissue samples.
Construction of Expression Plasmids and Vectors--
pGEX
plasmid double-stranded DNA with the GST-nSec1 insert was purified from
transformed bacteria strain (TG-1) with QIAprep Spin Miniprep Kit
(Qiagen). For the Munc18a site-directed mutants (S158A, T574A),
cDNA constructs were made by the polymerase chain reaction using
specific oligonucleotide sense (amino acids 152-162, S158A; 566-586,
T574A) and corresponding antisense primers. Oligonucleotide primers
were synthesized by the University of Michigan DNA Core Facility. The
construction of the Munc18a mutant cDNAs was confirmed by chain
termination sequencing using the Sequenase version 2.0 DNA Sequencing
Kit (Amersham Pharmacia Biotech).
The BamHI and EcoRI fragment cut from GST-p25 was
inserted into the BamHI and EcoRI site of the
vector pcDNA in which a Kozak sequence had been inserted to allow
mammalian expression of the p25 cDNA under the control of the
cDNA promoter.
Expression and Purification of Recombinant
Proteins--
Recombinant glutathione S-transferase (GST)
fusion proteins were expressed in E. coli and subsequently
purified by means of their affinity for glutathione-conjugated
Sepharose 4B beads (Amersham Pharmacia Biotech) as described (31).
Expression of recombinant proteins was induced by treatment with 0.2 mM isopropyl-1-thio-b-D-galactopyranoside (Boehringer Mannheim) for 4 h at 37 °C. The bacteria were lysed by treatment with a French cell press (1000 pounds/square inch pressure
differential) and subsequently with 1% Triton X-100 for 1 h at
4 °C. When necessary, cleavage of the GST moiety from the fusion
protein was accomplished by treatment with human thrombin (Sigma) at
0.2 NIH units/µl for 16 h at 20 °C. Alternatively, the entire
GST fusion protein was eluted from the Sepharose 4B beads by treatment
with 10 mM glutathione for 15 min at 20 °C. Protein
production and purification was confirmed by Coomassie Blue staining
and Western blotting.
Translocation and Cdk5 Activity Measurements--
The cytosolic
versus particulate distribution of Cdk5 was examined in cell
or tissue samples under control conditions or following exposure to
membrane-depolarizing stimuli. Control physiological saline contained
40 mM NaCl, 100 mM
N-methyl-D-glucamine-Cl, 5 mM
KHCO3, 2.2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES (pH 7.2). Elevated potassium saline solutions were
prepared by appropriate addition of KCl (50 or 100 mM),
concomitant with an equivalent reduction in concentration of
N-methyl-D-glucamine-Cl. Calcium-free solutions
omitted CaCl2 and included 1 mM EGTA. Following treatment, cells were lysed in buffer containing 2 mM EDTA,
2.25 mM
-glycerol phosphate, 20 mM Tris (pH
7.5), 175 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 50 µM olomoucine. Lysates were
then centrifuged at 100,000 × g for 25 min at 4 °C. The
supernatant constituted the cytosolic fraction. The resulting pellet
was suspended in lysis buffer containing 0.2% Triton X-100, sonicated
3 × 5 s with 1-min intervals between each sonication and
then re-centrifuged at 20,000 × g for 25 min at
4 °C. The resulting supernatant was the particulate fraction. Cdk5
content in each fraction was measured by SDS-PAGE, Western blotting,
and probing for Cdk5 immunoreactivity by ECL detection. Visualization and quantitation of the signal was performed off both x-ray film and a
GS-250 Molecular Imager. Cdk5 kinase activity was determined as
described previously using a Cdc2 kinase assay kit (Upstate Biotechnology Inc.) by following incorporation of 32P
radiotracer into a histone H1 peptide from bovine calf thymus containing a predicted Cdc2 phosphorylation site. Specificity of the
reaction to Cdk5 kinase activity was tested by inclusion of supplied
peptide inhibitors of protein kinase C and protein kinase A within the
reaction mixtures, together with an inhibitor of
calmodulin-dependent protein kinase (R24571; 5 µM). In addition, kinase activity was tested for
sensitivity to the Cdk5 inhibitor olomoucine or the much less active
analogue iso-olomoucine.
Munc18a and mutant Munc18a phosphorylation reactions were performed by
using Cdk5 immunoprecipitated from rat brain. Cdk5 immunoprecipitate
was prepared by homogenizing rat brain in lysis buffer and spinning the
resulting lysate at 30,000 × g for 30 min at 4 °C.
The supernatant was collected and precleared with protein A-linked
agarose beads for 1 h at 4 °C. Aliquots (1 ml) of the
precleared supernatant were then treated for 1 h at 4 °C with 2 µl of anti-Cdk5 and subsequently with 100 µl of protein A-linked
agarose beads for 1 h at 4 °C. The agarose beads were gathered
by centrifugation and washed 3 times with phosphorylation buffer (50 mM Tris-HCl (pH 8.0), 1 mM EGTA, 10 mM MgCl2, 0.1 mM CaCl2,
1 mM dithiothreitol). 30 µl of the beads (approximately 20 ng of Cdk5 immunoprecipitate) were then added to 300 µl of phosphorylation buffer containing ATP at 0.5 mM and
substrate (i.e. wild type and mutant Munc18s) at 1 µM. The reaction mixtures were prepared and kept at
4 °C to prevent the start of the phosphorylation reactions.
Olomoucine and iso-olomoucine were also added in 50 and 200 µM concentrations, respectively, to certain reactions. The reactions mixtures were kept at 4 °C and then spiked with 3 µl
(30 µCi) of [
-32P]ATP and incubated for 30 min at
30 °C. For kinase assay quantification, an aliquot of the reaction
mixture was blotted onto phosphocellulose paper, washed with 0.75%
phosphoric acid, and the incorporated radioactivity determined by
liquid scintillation counting. Specificity of 32P
incorporation into wild type or mutant Munc18a was determined by
subjecting aliquots of each reaction mixture to SDS-PAGE followed by
visualization and quantitation with a GS-250 Molecular Imager (Bio-Rad).
Determination of Fusion Protein Interactions and Analysis of
Regulation by Cdk5--
Binding relations between Munc18a or mutant
Munc18s and syntaxin 1a were performed by incubation of GST-Munc18a
proteins at 300 nM (600 nM for Munc18a T574A)
bound to glutathione-Sepharose 4B beads with given concentrations of
syntaxin 1a in binding buffer. 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. After overnight incubation with
rotation at 4 °C, samples were centrifuged, washed extensively, and
pellets resuspended in SDS-sample buffer. Analysis of syntaxin 1a
binding was determined by SDS-PAGE of each sample, followed by Western
blotting and probing for syntaxin 1a immunoreactivity by ECL.
Quantitation of the signal was performed by phosphorimaging.
For kinase-induced protein dissociation studies, the
Munc18a· syntaxin 1a heterodimer complex was formed by incubating
12 µg of Munc18a (either wild type or mutant) with 50 µg of
GST-syntaxin bound to glutathione-Sepharose 4B in 300 µl of protein
binding buffer for 1 h at 4 °C. The Sepharose beads were then
pelleted and washed extensively with protein binding buffer to remove
all the unbound Munc18a. Next, the complex was eluted off the purified Sepharose 4B beads by treatment with 100 µl of 10 mM
glutathione for 15 min at 20 °C. The supernatant containing the
eluted complex was then injected into a 10K Dialysis Cassette (Pierce)
and incubated in 750 ml of phosphorylation buffer with constant
stirring for 2 h at 4 °C in order to remove the glutathione.
The Munc18a·GST·syntaxin 1a complex was then recovered from the
cassette, and aliquots of approximately 15 µg of total protein were
added to 300 µl of phosphorylation buffer containing 0.5 mM ATP and either Cdk5 immunoprecipitated from rat brain
lysate or 0.42 µg/ml protein kinase C (the protein kinase C reaction
was conducted in the presence of 100 µM
CaCl2, 83.3 µg/ml phosphatidylserine, and 8.3 µg/ml
diglyceride). Immunoprecipitated Cdk5 rather than bacterially expressed
recombinant p25/Cdk5 protein was utilized for these experiments as it
demonstrated higher specific catalytic activity, represented mammalian
expressed Cdk5 protein, and could be more rapidly prepared. The
reactions were incubated for 30 min at 30 °C with constant
agitation, following which the Cdk5 immunoprecipitate on agarose beads
was centrifuged. The supernatant was then mixed with 100 µl of
glutathione-Sepharose 4B beads for 1 h at 4 °C to bind and
subsequently pellet all the syntaxin 1a by centrifugation. The obtained
pellet was washed 3 times with phosphorylation buffer. Aliquots of the
pellet and supernatant from each sample were subject to SDS-PAGE,
Western blotted, and probed for Munc18a immunoreactivity by ECL.
Visualization of the signal was by both x-ray film and a GS-250
Molecular Imager.
Cell Preparation, Transfection, and Secretion
Experiments--
Chromaffin cell preparation, transient transfection,
and secretion experiments were performed as described previously (32). For intact chromaffin cells, secretion experiments were performed in a
physiological salt solution containing 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 15 mM HEPES (pH 7.4), and 0.5 mM ascorbate.
Secretion from digitonin-permeabilized cells was conducted in potassium
glutamate solution containing 139 mM potassium glutamate,
20 mM PIPES (pH 6.6), 2 mM MgATP, and 5 mM EGTA with no added Ca2+
(Ca2+-free) or 5 mM EGTA buffered with calcium
to set a free calcium concentration of 30 µM. In
non-transfected chromaffin cells, secretion was investigated by
preincubating the cells for 3 h in Dulbecco's modified Eagle's
medium/Ham's F-12 containing 10% heat-inactivated fetal calf serum,
[3H]norepinephrine, and 0.5 mM ascorbate.
Cultures were rinsed for at least 30 min in medium without added
[3H]norepinephrine before inducing release with the
nicotinic acetylcholine receptor agonist
1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Analysis of release
from transfected cells was carried out by measurement of human growth
hormone that was co-transfected with the test vector (i.e.
pcDNA-p25). Human growth hormone appearing in the medium was
measured with a luminometric assay kit from Nichols Institute (San Juan
Capistrano, CA).
Electrophysiology--
Whole-cell patch clamp methods were used
to evoke and record Ca2+ currents and measure the changes
in membrane capacitance (
Cm) from single bovine chromaffin cells
using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA).
Patch pipettes were constructed out of 1.5-mm outer diameter capillary
glass (AM Systems), coated with Sylgard elastomer (Dow Corning, Midland
MI), and fire-polished. The patch pipettes had tip resistances of 2-5
megohms and were filled with a pipette solution that contained 140 mM CsMeSO3, 1 mM MgCl2,
0.25 mM EGTA, 2 mM ATP, 0.5 mM
Li-GTP, and 10 mM HEPES with pH adjusted to 7.1 with NaOH.
For recording, the cells were placed in a solution containing 130 mM tetraethylammonium chloride, 10 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose with pH adjusted to
7.15 with Tris. The whole-cell capacitance and 60-70% of the series
resistance were compensated electronically. High resolution
measurements of changes in membrane capacitance reflecting net of
exocytotic and endocytotic activity were performed using a modified
phase-tracking method with a software-based (Pulse Control)
phase-sensitive detector (33). A 19.1-kHz sampling rate was used to
compute 1 Cm point for each 13 ms. Calibration pulses of 100 femtofarads and 500 k
were generated and placed at the beginning of
each Cm data record.
 |
RESULTS |
Cdk5 Regulation of Munc18a-Syntaxin 1a Interaction--
To
demonstrate that Cdk5 was capable of phosphorylating Munc18a, Cdk5 was
immunoprecipitated from rat brain lysate and incubated in a
[32P]ATP solution with recombinant Munc18a
fusion protein. The obtained autoradiograph shows that
immunoprecipitated Cdk5 causes 32P incorporation into
Munc18a (Fig. 1A). A 23-kDa
upward shift of the radiolabeled signal in the GST-Munc18a sample
corresponds to the additional mass of the GST moiety and verifies that
Munc18a is the substrate for this reaction. That Cdk5 is the
phosphorylating kinase is demonstrated by inhibition of phosphate
incorporation by the specific Cdk inhibitor olomoucine (50 µM). Olomoucine, a purine analogue, has been demonstrated
to exhibit little or no inhibition of many other protein kinases
including protein kinase C, protein kinase A, and protein tyrosine
kinases (34). As a control, protein A-linked agarose beads which had
been incubated with the rat brain lysate for a commensurate period, but
without pretreatment of the lysate with Cdk5 antibody, failed to
demonstrate radiotracer incorporation.

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Fig. 1.
Cdk5 phosphorylation of Munc18a and effects
on assembled Munc18a·syntaxin heterodimer complex. A,
Cdk5 immunoprecipitated from rat brain lysate was incubated with
bacterially expressed and purified Munc18a or GST-Munc18a in
phosphorylation buffer containing [ -32P]ATP at
30 °C for 30 min. After centrifugation, equivalent amounts of the
supernatants were subjected to SDS-PAGE and autoradiography. Control
reactions included 50 µM olomoucine or protein A beads
that had been incubated with rat brain lysate in the absence of the
immunoprecipitating Cdk5 antibody. B, autoradiograph
demonstrating radiolabeling of Munc18a, which was prebound to
GST-syntaxin 1a prior to incubation with immunoprecipitated Cdk5 and
[ -32P]ATP. Following kinase treatment GST-syntaxin 1a
and Munc18a remaining bound were pelleted by centrifugation after
addition of glutathione-Sepharose beads. Munc18a appearing in the
supernatant was radiolabeled. C, preformed
Munc18a·GST·syntaxin 1a heterodimer complexes were incubated with
Cdk5 immunoprecipitated from rat brain lysate or with purified protein
kinase C in appropriate phosphorylation buffers containing ATP. Samples
were then centrifuged and pellets washed extensively. Equivalent
aliquots of the pellet and supernatant of each sample were then
subjected to SDS-PAGE, Western-blotted, and probed for Munc18a
immunoreactivity. Samples containing olomoucine (50 µM)
or where ATP was omitted were included as kinase activity
controls.
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To determine whether Cdk5 could phosphorylate Munc18a when bound to
syntaxin 1a, we utilized a pre-assembled Munc18a·GST·syntaxin 1a
heterodimer complex that was subsequently incubated with Cdk5 immunoprecipitate in a [32P]ATP-containing solution.
Following incubation, the GST-syntaxin 1a was pelleted by
centrifugation following addition of Sepharose 4B-glutathione beads and
then washed extensively. The GST-syntaxin 1a pellet and the supernatant
from the reaction were then probed for Munc18a radiolabeling. The
autoradiograph showed labeling of a 67-kDa protein in the supernatant
fraction alone, demonstrating that Munc18a bound to syntaxin is a
substrate for Cdk5 phosphorylation and that phosphorylation induces
disassembly of the complex (Fig. 1B). To determine the
extent of Munc18a dissociation from GST-syntaxin 1a, the experiments
were repeated and the fractions analyzed for Munc18a by immunoblotting.
The resulting immunoblot reveals that conditions supporting Cdk5 kinase
activity induce considerable dissociation (approximately 30-50%) of
Munc18a from syntaxin 1a (Fig. 1C). This dissociation was
found to be ATP-dependent and olomoucine-sensitive. In
addition, comparable dissociation could not be achieved by protein
kinase C, another putative regulator of the Munc18a·syntaxin 1a complex.
The Munc18a amino acid sequence possess two consensus phosphorylation
sequences for Cdk5 at residues 158-161 (SPHK) and residues 574-577
(TPQK). Analysis of Munc18a homologues revealed a high degree of
preservation of the Cdk5 phosphorylation sequence, which includes the
Thr574 residue but not that of the Ser158
residue (Table I). To determine the
importance of the Ser158 and the Thr574
sites to the Cdk5-induced phosphorylation and subsequent
dissociation of Munc18a binding from syntaxin 1a, single site directed
mutations (alanine substitution) of each site were generated (S158A and T574A). Initially, the S158A and T574A Munc18a mutants along with the
wild type Munc18a were tested in a kinase assay as substrates for
32P incorporation by Cdk5 immunoprecipitated from rat brain
lysate (Fig. 2A). The
Thr574 mutant failed to act as substrate for Cdk5, whereas
the S158A mutant served in a manner statistically indistinguishable
from the wild type Munc18a (6.3 ± 0.4% for T574A
versus 90 ± 9% for S158A versus 100 ± 10% for wild type Munc18a; n = 6). To verify that
the radiotracer incorporated was within the Munc18a protein, additional
reactions were run, and the protein was then separated by SDS-PAGE. As
shown by the resulting autoradiograph, although the S158A mutant was
phosphorylated similarly to the wild type protein, the T574A mutant
showed incorporation at a level no higher than the background control
(Fig. 2B). The GST-Munc18a and 50 µM
olomoucine controls demonstrated the substrate and kinase specificity of the reactions. An iso-olomoucine (200 µM) control was
also included to demonstrate the specificity of olomoucine.
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Table I
Comparison of Cdk5 consensus phosphorylation sequences across Munc18a
homologues
Amino acid residues in brackets correspond to the two consensus
sequences in Munc18a for Cdk5. Underlined residues indicate presence of
key amino acid residues.
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Fig. 2.
Effect of site-directed mutations of Munc18a
on Cdk5-mediated phosphate incorporation. A,
recombinant Munc18a and mutant Munc18a protein were incubated with Cdk5
immunoprecipitated from rat brain in [ -32P]ATP
containing phosphorylation buffer. Quantification of radiotracer
incorporated was performed as described under "Experimental
Procedures." Background reaction (Bkgrnd.) contains
immunoprecipitated Cdk5 with no Munc18a. Inclusion of olomoucine (50 µM) with Munc18a demonstrates specificity of
32P incorporation to Cdk5 activity. B,
specificity of radiotracer incorporation into Munc18a and mutant
Munc18a protein. Aliquots of phosphorylation reactions performed as in
A were subjected to SDS-PAGE followed by visualization with
phosphorimaging. Olomoucine (Olom.)- (50 µM)
and iso-olomoucine (Iso-olom.) (200 µM)-treated reactions were included as controls for
kinase specificity.
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A set of further experiments attempted to determine whether
phosphorylation at the Thr574 residue was responsible for
the Cdk5 induced dissociation of Munc18a from syntaxin 1a. First, we
examined the effect of the S158A and T574A mutations on recognition by
the Munc18a antibody and on binding to GST-syntaxin 1a. Western blots
of wild type versus the mutant Munc18a-expressed proteins
showed no effect of the mutations on the strength of the immunoreactive
signal. To evaluate protein interactions, Munc18a or mutant Munc18s
containing the GST moiety were incubated with syntaxin 1a. Binding was
then determined by collection and extensive washing of the glutathione Sepharose 4B beads to which GST bound followed by elution and analysis
by SDS-PAGE and Western blotting of bound syntaxin 1a. Binding of
syntaxin 1a was found to be saturable for each Munc18a protein
construct, and although the T574A Munc18a construct showed approximately 20-fold less total binding than the wild type or S158A
Munc18a protein, no significant difference was found in the 50%
effective concentration (EC50) for binding of the three Munc18a proteins (Fig. 3A).
Next, to assess the ability of the kinase to induce dissociation of the
heterodimer complexes, both the wild type and mutant Munc18a proteins
were bound to GST-syntaxin 1a and treated with immunoprecipitated Cdk5
in an ATP-containing solution. The GST-syntaxin 1a was pelleted, washed
thoroughly, and, along with the retained supernatant, probed for
Munc18a immunoreactivity. The obtained immunoblot revealed that whereas
Cdk5 was capable of inducing dissociation of both wild type and the
S158A mutant from syntaxin 1a, it could not disassemble the T574A
mutant from syntaxin 1a (Fig. 3B). A reaction containing
olomoucine with wild type Munc18a run as a control further demonstrated
the specificity of the reaction to Cdk5 activity.

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Fig. 3.
Binding of recombinant Munc18a and mutant
Munc18a protein to syntaxin 1a and effect of Cdk5 phosphorylation on
disassembly of resulting heterodimer complexes. A,
Munc18a or mutant Munc18a recombinant GST fusion proteins (300 nM except Munc18a T574A, which was 600 nM)
linked to glutathione-Sepharose 4B beads were incubated in binding
buffer with given concentrations of syntaxin 1a. After incubation, the
samples were centrifuged, pellets washed, and then subjected to
SDS-PAGE followed by Western blotting. Blots were probed for syntaxin
1a immunoreactivity, and the signal was quantitated by phosphorimaging.
Syntaxin 1a bound to the recombinant Munc18a proteins was normalized
for each reaction as a percent of the signal obtained at a saturating
value of 1 µM syntaxin 1a ( ± S.E. for each
point, n = 4 wild type Munc18a; n = 3 S158A and T574A). Solid line represents least squares fit of
combined data. B, preformed Munc18a-GST or mutant Munc18a
protein-syntaxin 1a heterodimer complexes were incubated with Cdk5
immunoprecipitated from rat brain lysate in phosphorylation buffers
containing ATP. Following centrifugation, equivalent aliquots of pellet
and supernatant fractions of each sample were subjected to SDS-PAGE,
Western-blotted. and probed for Munc18a immunoreactivity. Samples
containing olomoucine (50 µM) were included as kinase
activity controls.
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Calcium-dependent Cdk5 Translocation and
Activation--
Dynamic regulation of secretory activity by Cdk5
necessitates that the kinase itself be strictly regulated. As secretion
from excitable cells is triggered by membrane depolarization and
activation of Ca2+ influx, these effects on Cdk5 activity
were investigated on neuroendocrine nerve endings isolated from the rat
pituitary neural lobe, as well as on bovine adrenal chromaffin cells
and on the neuroendocrine PC-12 cell line. Membrane depolarization with
elevated extracellular concentrations of K+ was found in
each case to induce translocation of Cdk5 from a cytosolic to a
particulate cellular compartment (Fig.
4A) and to be accompanied by
an activation of Cdk5 kinase activity (Fig. 4B). The
translocation resulted in approximately a doubling of particulate Cdk5
content. In addition, the depolarization-induced Cdk5 translocation was
observed to be Ca2+-dependent in the nerve
endings and PC-12 cells, while considerable variation was observed for
this parameter for chromaffin cells. The variation in chromaffin cells
may result from a higher degree of cell heterogeneity retained in the
isolation and culture of these cells. The enhancement of Cdk5 kinase
activity was found in each of the cell preparations, however, to be
dependent on the presence of extracellular Ca2+ during the
period of membrane depolarization.

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Fig. 4.
Translocation and activation of Cdk5 by a
secretory stimulus. A, Cdk5 protein present in
cytosolic and particulate fractions was determined from quantitation of
immunoblots following SDS-PAGE under control
(Ca2+-containing physiological saline) and
membrane-depolarizing (Depol.) conditions (elevated
extracellular [K+] ± [Ca2+]o).
Determinations were performed for isolated nerve endings from the
neural lobe of the pituitary (NL), from primary cultures of
bovine chromaffin cells (CC), and from PC-12 cells
(PC-12). Depolarizing treatments were as follows: neural
lobe, 100 mM [K+], 10 min; chromaffin cells
and PC-12, 50 mM [K+], 5 min. Results are
expressed as a percentage of the total Cdk5 protein present in the
particulate fraction ( ± S.E.; neural lobe,
n = 11; chromaffin cells, n = 6; PC-12
cells, n = 3). B, Cdk5 kinase activity of
neural lobe (NL), chromaffin cells (CC), and
PC-12 cells were analyzed following indicated treatments (as in
A). Kinase activity is given as a percentage of the activity
( ± S.E., neural lobe, n = 4; chromaffin
cells, n = 6; and PC-12 cells, n = 3)
determined under control conditions for each preparation.
Asterisks indicate significant difference (p < 0.05) from control in A and B by Student's
t test or Wilcoxon signed rank test.
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Cdk5 Regulation of Neuroendocrine Secretion--
Since conditions
that activate cellular secretion were observed to alter Cdk5
translocation and Cdk5 kinase activity, the effects of Cdk5 activity on
neuroendocrine secretion from chromaffin cells were investigated. Cells
were exposed to the Cdk inhibitor olomoucine, the less active analogue
iso-olomoucine or the drug carrier (Me2SO) in culture
medium for 16 h prior to stimulation of
[3H]norepinephrine secretion by the nicotinic
acetylcholine receptor agonist DMPP. Olomoucine treatment resulted in
an average 30% decrease in DMPP-stimulated secretion with respect to
that of control (Fig. 5A). No
significant effects of either olomoucine or iso-olomoucine were
observed on basal (i.e. non-stimulated) [3H]norepinephrine secretion. Secretion was also
investigated on digitonin-permeabilized chromaffin cells to evaluate
further the effects of Cdk5 inhibition on secretion and to determine if
Cdk5 alters the secretory response after Ca2+ influx.
Permeabilization of the cells was carried out under low calcium
conditions (5 mM EGTA) in the presence of the Cdk5
inhibitor or its analogue, and secretion was subsequently stimulated
with a free Ca2+ concentration of 30 µM.
Olomoucine exhibited a dose-dependent inhibition of
Ca2+-induced secretion with respect to the iso-olomoucine
control (Fig. 5B). At 300 µM, olomoucine
inhibition averaged 28%. Iso-olomoucine itself demonstrated no
inhibitory effects on secretion over the concentration range tested. As
Ca2+ has free access to the cell interior in
digitonin-permeabilized cells, the observed inhibition of secretion by
olomoucine suggests that Cdk5 action on secretion is not via alteration
of Ca2+ influx.

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Fig. 5.
Effects of Cdk5 inhibitor olomoucine on
[3H]norepinephrine secretion from intact
(A) and permeabilized (B) bovine
chromaffin cells. Chromaffin cells were preloaded with
[3H]norepinephrine for 3 h, rinsed, and incubated a
further 30 min prior to measurement of basal and evoked
[3H]norepinephrine release. Release responses were evoked
from intact cells by exposure to the nicotinic acetylcholine receptor
agonist DMPP (20 µM) for 4 min and from permeabilized
cells by raising the free Ca2+ concentration of the medium
to 30 µM for 15 min. Intact cells were pretreated with
olomoucine (Olom., 167 µM), iso-olomoucine
(Iso-olom., 167 µM), or drug carrier
(i.e. Me2SO 0.5%, control) for 16 h in
Dulbecco's modified Eagle's medium, and the drug concentrations were
maintained throughout the [3H]norepinephrine loading and
release portions of the experiments. In each experiment
n = 3 wells/group A, and asterisk indicates
significant difference (p < 0.05) for olomoucine
versus control; B, p < 0.05 for
olomoucine versus iso-olomoucine at concentrations greater
than 100 µM.
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The effects of Cdk5 inhibition on secretion were examined further
at the single chromaffin cell level under whole-cell voltage clamp.
Membrane capacitance changes were measured in response to depolarizing
stimuli to provide highly time-resolved measurements of exocytotic and
endocytotic activity and to evaluate changes in Ca2+
sensitivity of secretion. Repetitive depolarization of the membrane from a holding potential of
90 mV to a step potential of +20 mV
(50-ms duration, 200-ms interpulse interval) was used to activate voltage-dependent calcium channels and allow
Ca2+ influx. This stimulation resulted in a rapid increase
in membrane capacitance which was followed by a slow recovery on
cessation of stimulation. Representative data comparing changes in
membrane capacitance for an olomoucine and an iso-olomoucine-treated
cell are shown in Fig. 6A.
Olomoucine inhibited the stimulated
membrane capacitance increase, despite a
very similar level of time-integrated evoked Ca2+ influx
between the cells (Fig. 6B). Furthermore, the olomoucine inhibition of secretory responses was observed over a range of Ca2+ influx values, with differences most prominent in
cells that demonstrated higher influx (Fig. 6C). Averaged
changes in membrane capacitance normalized to total time-integrated
Ca2+ influx (femtofarads/picocoulombs) under control
conditions (n = 4) or following iso-olomoucine
(n = 8) and olomoucine (n = 8) treatment are shown in Fig. 6D. Chromaffin cells treated
with olomoucine gave significantly (p < 0.5, n = 8) smaller stimulated increases in membrane
capacitance than iso-olomoucine-treated cells.

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Fig. 6.
Effects of olomoucine on temporally resolved
membrane capacitance changes ( Cm) evoked by
membrane depolarization under voltage clamp of single chromaffin
cells. A, representative Cm of chromaffin cells
incubated with olomoucine (Olom.) or iso-olomoucine
(Iso-olom., 167 µM, 16 h) in response to
12 repetitive step depolarizations ( 90 mV holding potential to +10
step potential for 50 ms repeated at 5 Hz). Breaks in records indicate
periods of membrane depolarization. B, cumulative
time-integrated Ca2+ influx in response to the step
depolarizations of the records shown in A. C,
relationship of the total time-integrated Ca2+ influx in
response to repetitive step depolarizations (as in A) to the
peak Cm for olomoucine- (filled symbols) and
iso-olomoucine (open symbols)-treated cells (167 µM, 16 h). Each value represents a determination
from a separate cell in response to the first series of applied step
depolarizations. D, Cm normalized to total integrated
Ca2+ influx ( ± S.E.) for control
(n = 3), iso-olomoucine- (n = 8), and
olomoucine (n = 8)-treated chromaffin cells.
p < 0.05 for olomoucine versus
iso-olomoucine, Student's t test.
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Fig. 7.
Transient transfection with plasmid encoding
p25 increases secretion of co-transfected and expressed growth hormone
(hGH). hGH secretion was determined from
transfected chromaffin cells that were co-transfected with a plasmid
encoding either bovine p25 or a parent pCMV plasmid (neo).
Basal and DMPP (10 µM, 5 min)-induced secretions of hGH
were determined 4-6 days following transfection. In each experiment
n = 4 wells/group. p < 0.05 for DMPP
stimulated secretion of hGH from p25 versus neo.
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Cdk5, like other members of the family of cyclin-dependent
kinases, are not active as monomeric proteins but rather require binding of specific proteins to form an active heterodimeric holoenzyme (14, 15). Although cyclins represent the activator of most Cdks, neural
Cdk5 is activated by a brain-specific protein termed p35 (also termed
p35nck5a) that is highly expressed in post-mitotic neurons
and a p35 proteolytic cleavage product termed p25 (19, 35, 36). To
determine whether p35/Cdk5 kinase activity participates in regulation
of neurosecretion, we altered the endogenous levels of p25 in
chromaffin cells by transfection with plasmid DNAs. Analysis of release
from transfected cells was carried out by measurement of human growth
hormone, which was expressed in the chromaffin cells by co-transfection of a hGH vector with the p25 expression vector. Initial investigations confirmed that transfection and expression of the p25 protein in HEK
293 cells led to greatly increased (>10-fold) Cdk5 kinase activity.
Assessment of the effect of p25 transfection and expression on Cdk5
activity in primary cultures of chromaffin cells was precluded by low
transfection efficiency (approximately 1-2%). In two experiments, DMPP-induced hGH secretion was increased by 45 ± 2% following transfection and expression of p25, over the control pCMV
neo-transfected chromaffin cells. No effect of p25 was observed on
basal secretion. Thus, the results with the p25 transfection support an
important function for Cdk5 in the secretory mechanism.
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DISCUSSION |
It has previously been shown that Munc18a copurifies via a direct
protein-protein interaction with Cdk5 from rat spinal cord (14). We
previously reported that Munc18a is a phosphoprotein in situ
and that it is subject to phosphorylation by Cdk5 in vitro (30). The present report demonstrates that a preformed
Munc18a·syntaxin 1a heterodimer complex can be disassembled by
addition of catalytically active Cdk5. We further identify a threonine
residue 20 amino acids from the carboxyl terminus of Munc18a
(Thr574) as being the phosphorylation site. A conservative
mutation of this site to alanine blocked both 32P
radiotracer labeling of Munc18a by Cdk5 in vitro and
disassembly of the preformed Munc18a·syntaxin 1a complex. As the
binding of Munc18a with syntaxin 1a requires the full length of Munc18a
sequence and also requires the complete cytoplasmic domain of syntaxin 1a (37-39), a conformational or charge change caused in this region by
the phosphorylation of Thr574 may underlie the normal
disassembly of the Munc18a-syntaxin 1a heterodimer. A second putative
phosphorylation site (Ser158) defined by Cdk5
phosphorylation consensus sequence is not subject to phosphorylation
in vitro by active Cdk5. Mutation of either site
(Thr574 or Ser158) had no significant effect on
the EC50 for binding of Munc18a to syntaxin. Treatment of
the Munc18a·syntaxin 1a complex with protein kinase C under
conditions conducive to phosphorylation failed to affect the stability
of the complex, suggesting that although protein kinase C can
phosphorylate Munc18a that is not bound to syntaxin 1a, it cannot
induce disassembly of the already formed heterodimer complex.
The identification of Thr574 as the phosphorylation site is
consistent with the relatively high degree of conservation at the Thr574 site and the lack of conservation at the
Ser158 site among Munc 18 homologues. In both Sec1p (9) and
Unc-18 (40), which show 26 and 59% homology, respectively, at the
amino acid level, the Thr574 site is 100% conserved,
whereas the Ser158 site is not. Although these organisms do
not have Cdk5, they do possess Cdc2, which recognizes the
Thr574 site as a phosphorylation sequence and so could
function similarly to Cdk5. A comparison of Munc18a to its mammalian
homologues Munc18b and Munc18c (41) also point to importance of the
Thr574 site. Munc18b, which is distributed in tissues other
than brain, and Munc18c, which is distributed in brain as well as other
tissues, again show conservation of the Thr574 but not the
Ser158 site. In comparison, the Drosophila
Munc18a homologue Rop (42) does not possess the (S/T)PX(K/R)
sequence present in most of the other proteins. Although
Drosophila Cdk5 shares 77% homology with human Cdk5, there
are no reports of p35 in Drosophila, and the carboxyl
sequence (SPEL) may be sufficiently preserved that it may be recognized
by Cdk8, a Cdk unique to Drosophila (43). Therefore, it
remains possible that in Drosophila both a Cdk and the
carboxyl sequence of Rop are of importance to the process of secretion.
Indeed, in the same way that the SNARE hypothesis holds in only a
generalized sense across all membrane fusion events, the regulation of
SNARE-interacting proteins may also be only generalized. For example,
even though Munc18c possess the TPXK carboxyl sequence that
would target it for Cdk5-induced disassembly from syntaxin 1a, Munc18c
has only been found to bind syntaxin 5 (41). In addition, Munc18a has
been reported to bind to other proteins including DOC2 (44) and MINTs
(45) that are likely to affect Munc18a phosphorylation, as well as
syntaxin 1a binding and function. An additional consideration is
whether all the actions of Munc18a and its homologues are restricted to
their interactions with the target SNARE syntaxins. Munc18a, SNAP-25,
and syntaxin are not restricted to the synaptic region of neurons but
are distributed throughout the axon and soma, suggesting the
possibility of additional actions of Munc18a (46).
Substantial genetic and biochemical evidence exists to support an
essential role of members of the Sec1 protein family in the secretory
process. For example, SEC1 was identified as one of 10 genes in
S. cerevisiae required for the final stages of secretion of
protein to the cell exterior (9, 47); mutation in the unc-18
gene in Caenorhabditis elegans caused abnormal accumulation of acetylcholine (40, 48), and Rop overexpression in
Drosophila reduced spontaneous vesicle fusion and
significantly decreased evoked responses to repetitive stimulation
(49). Additional studies have demonstrated that loss of function
mutations in the Drosophila rop gene results in a
reduction of neurotransmitter release in adult photoreceptor cells (50)
and that levels of Rop expression regulate evoked neurotransmission at
the neuromuscular junction via an interaction with syntaxin (13). Yet
the precise nature of the role of Munc18a in secretion is less clear.
For example, exogenous addition of Munc18a to permeabilized chromaffin cells had no effect on calcium-induced secretion and overexpression of
Munc18a in transiently transfected PC-12 cells did not affect the
extent of evoked exocytosis (51). In contrast, microinjection of a
squid neuronal homologue of Sec1 protein into the squid giant synapse
inhibited evoked neurotransmitter release but did not alter the
distribution of synaptic vesicles at active zones (52).
Another important consideration is whether Cdk5 regulation of the
secretory pathway is itself regulated. That is whether Cdk5 phosphorylation of Munc18a is a persistent tonic affect or a dynamic component of the secretory response that modifies the level of vesicles
available for priming. Our present data are largely supportive of the
latter possibility. For example, we have demonstrated that Cdk5
translocates from a cytosolic to a particulate fraction in response to
membrane depolarization and activation of calcium influx. Membrane
depolarization alone is an insufficient stimulus for this translocation
event. Furthermore, the data demonstrate that induction of
translocation produces a corresponding increase in the level of Cdk5
kinase activity. This increase in activity occurs over a period of
minutes and, thus, likely represents activation of existing Cdk5 rather
than long term regulation by transcriptional/translational activation
and an increase in the Cdk5 activator protein p35 or the active
proteolytic cleavage product p25. Consistent with this observation, a
marked increase in Cdk5 activity occurring over minutes in response to
ischemic brain injury in rats has been reported, the increased Cdk5
activity being found to occur without a corresponding increase in Cdk5
protein levels (53).
The mechanism through which rapid regulation of Cdk5 may be achieved is
unknown, although certain parallels to the regulated activation of
other cell cycle kinases may be drawn. Other Cdks are regulated by
binding of specific cyclins, by cellular accumulation of cyclin and Cdk
protein, by the phosphorylation state of each of three sites on Cdks,
and by direct binding of a number of inhibitory proteins
(e.g. Kip1 and p21). Direct evidence to support these mechanisms in rapid Cdk5 regulation is, however, limited. For example,
regulation of Cdk5 activity by phosphorylation is also poorly
supported, although the regulatory phosphorylation sites Thr14 and Tyr15 of Cdc2 are entirely conserved
in Cdk5, and Thr160/161 is substituted by serine. Whereas
phosphorylation/dephosphorylation of these sites may modulate Cdk5
activity in vitro, it certainly is not required, as
activation of recombinant Cdk5 occurs upon binding recombinant neural
specific activator p35 or p25 in the absence of other kinase or
phosphatase activity (17-19, 35, 36). Rapid regulation of Cdk5
activity could occur by increased binding of free p35 to monomeric
Cdk5, particularly as the generally low cytoplasmic levels of p35 (30,
54) are under strict regulation, with rapid p35 turnover (half-life
20-30 min) controlled by a ubiquitin-proteasome pathway (27).
Moreover, a GTP-dependent association between p35 and Rac
has been recently reported to which Cdk5 can complex and be
catalytically activated (55). Interestingly, the present findings
showing Cdk5 translocation and activation in chromaffin cells, PC-12
cells and isolated peptidergic nerve endings suggests a general
conservation of a rapid regulatory mechanism across neuroendocrine cells.
Based on our findings that catalytically active Cdk5 can prompt
disassembly of a Munc18a·syntaxin 1a complex in vitro
through phosphorylation of Munc18a and that Cdk5 is activated by
conditions that stimulate secretion, we have attempted to determine if
the level of Cdk5 activity correlated to secretory responsiveness. The
results provide evidence for the importance of Cdk5 activity in the
secretory pathway, as pretreatment of intact chromaffin cells with the
Cdk inhibitor olomoucine inhibited DMPP and membrane depolarization
evoked secretion. The inhibition was not observed with the analogue
iso-olomoucine, which differs from olomoucine only in the location of a
methyl group on the imidazole ring of the purine backbone. The
inhibitory effects of olomoucine were not mediated by effects on
calcium influx. Overall, however, the secretory inhibition by
olomoucine treatment, while statistically significant, was modest and
thus allows several interpretive possibilities. For example, it is
possible that the olomoucine treatment failed to inhibit completely
Cdk5 activity, that Cdk5 phosphorylation of Munc18a is redundant with
other mechanisms to disassemble the Munc18a-syntaxin heterodimer, or
that assembly/disassembly of a Munc18a·syntaxin complex is not
rate-limiting in acute secretory responses. To avoid potential problems
of the specificity of the Cdk inhibitor olomoucine to experimental
interpretation, we also transiently transfected and overexpressed the
Cdk5 activator p25 in chromaffin cells and examined effects on evoked
secretion. Overexpression of p25 dramatically increased evoked
secretion consistent with increased Cdk5 activity leading to secretory effects.
The present data are supportive of a model whereby phosphorylation of
Munc18a by Cdk5 mediates disassembly of preformed Munc18a·syntaxin 1a
complexes. That these complexes form in situ is supported by genetic linkage of Sec-1 and Sso1p and Sso2p in yeast (56), by yeast
two-hybrid screens for Munc18-interacting proteins (44), by genetic
evidence in Drosophila (13), by immunocytochemical overlap
of a portion of cellular Munc18 and syntaxin (46), and by repeated
demonstration of high affinity binding of Munc18a with syntaxin 1a
in vitro (8, 57). However, co-immunoprecipitation from
cellular lysates has proved difficult, and both syntaxin 1a and Munc18
proteins show a distribution in neurons that includes axonal and
somatic regions (46). Thus, although there may be additional functions
for Munc18, genetic studies from yeast, C. elegans, and
Drosophila clearly establish an essential requirement of
Sec1 protein and its homologues to the secretory pathway. The Cdk5-mediated dissociation of Munc18a from syntaxin 1a may be important
in making available competent sites for vesicle SNARE interaction with
target SNAREs, as this SNARE interaction is blocked in vitro
when Munc18 is bound to syntaxin. The rapid increase in the level of
Cdk5 activity during secretory conditions suggests a mechanism by which
the rate of SNARE interactions can be dynamically regulated and which
would ultimately lead to changes in secretory responsiveness.