From the Synaptic Function Unit, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4154
Received for publication, January 16, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Second messenger regulation of protein interactions within the exocytotic apparatus, for example, by protein phosphorylation or dephosphorylation, may be one mechanism by which cellular events could affect SNARE protein function and mediate synaptic plasticity (8). Although the time course between action potential arrival at the nerve terminal and the resultant vesicle fusion is too short for protein phosphorylation to exact a direct and acute effect during a single round of vesicle exocytosis, protein kinases, and phosphatases may have significant effects on subsequent neurotransmitter release events in an activity or Ca2+-dependent manner. Activation of protein kinases in presynaptic terminals, particularly CaMKII, PKA, and PKC, has been shown to correlate with transmitter release (1528). A number of SNAREs and their regulatory proteins have been reported as potential substrates in vitro for protein kinases (2943). For example, in vivo phosphorylation of synapsin, which is dependent on intracellular [Ca2+], has been shown to affect synaptic vesicle availability at release sites and control the kinetics of vesicle pool turnover (4446). It is reasonable to speculate that the phosphorylation/dephosphorylation state of synaptic proteins that mediate vesicle exocytosis is critical to the regulation of the biochemical pathways leading from vesicle docking to neurotransmitter release. Thus, identifying protein kinases and phosphatases that modulate the assembly/disassembly of the fusion machinery, particularly kinases and phosphatases functionally regulated by synaptic activity or intracellular [Ca2+], could offer valuable insights to the molecular mechanisms underlying synaptic transmission and plasticity.
In the current study, we have used the C-terminal half of syntaxin-1A as a
bait to screen a human brain cDNA library via the yeast two-hybrid selection
technique. We have isolated one cDNA encoding the C-terminal domain of DAP
kinase (47), a
calcium/calmodulin-dependent serine/threonine protein kinase
(48). DAP kinase was first
reported as a potential mediator of -interferon-induced cell death
(47). However, the native
substrates of DAP kinase and the molecular pathways underlying DAP
kinase-mediated signal transduction remain unclear. Examination of the tissue
distribution of DAP kinase mRNA demonstrated that it is predominantly
expressed in brain and lung. DAP kinase mRNA is widely distributed in the
embryonic brain and gradually declines in postnatum expression. Interestingly,
the adult expression of DAP kinase is restricted to particular neuronal
subpopulations that are important in memory and learning, such as the
hippocampus and cerebral cortex, suggesting that DAP kinase-mediated signal
transduction pathway may be implicated in neuronal functions related to
synaptic transmission or plasticity
(4950).
Our current yeast two-hybrid cloning results and biochemical studies
demonstrate that DAP kinase binds to and phosphorylates neuronal syntaxin-1A.
This phosphorylation event occurs both in vitro and in vivo
in a Ca2+-dependent manner. Phosphorylation of syntaxin-1A at
Ser-188, or its mutation to S188D, which mimics a state of complete
phosphorylation, significantly decreases syntaxin binding to Munc 18-1, a
syntaxin regulatory protein that controls SNARE complex formation and vesicle
docking
(5153).
Our results suggest that syntaxin is a Ca2+-dependent
phosphorylation target of DAP kinase and that syntaxin phosphorylation at
Ser-188 could be a mechanism for regulation of syntaxin binding properties in
response to elevated intracellular [Ca2+] and synaptic
activity.
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EXPERIMENTAL PROCEDURES |
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Fusion Protein Construction, Preparation, and
PurificationHA-DAP kinase cDNA was kindly provided by A. Kimchi
(47), and purified DAP kinase
catalytic domain (KD, amino acids 1285) by Dr. Martin Watterson
(54). DAP kinase (KD-CaM) was
produced by subcloning the region of the DAP kinase cDNA that encodes amino
acids 1320, corresponding to the catalytic domain and calmodulin
binding regulatory domain, into the pASK-IBA3 (Sigma) expression vector at the
BsaI sites. DAP kinase K42A and syntaxin S188A/S188D mutants were
generated using the QuikChangeTM XL site-directed mutagenesis kit
(Stratagene). Full-length syntaxin-1A (FL), syntaxin-1ATM(1264,
lacking the transmembrane domain), syntaxin-1A N-terminal-half (NT)
(2190), syntaxin-1A C-terminal-half (CT) (181264), SNAP-25,
VAMP-2, Munc18-1, and DAP kinase C-terminal (CT)(11571431) were
subcloned into GST fusion protein vector pGEX-4T-1 (Amersham Biosciences) or
His6-tagged fusion protein vector (pET28, Novagen). Fusion proteins
were prepared as crude bacterial lysates by mild sonication in
phosphate-buffered saline (50 mM sodium phosphate, pH 7.4, 140
mM NaCl, plus protease inhibitors of 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin)
with 1% Triton X-100. His-tagged fusion proteins were purified by binding to
Ni2+-charged nitrilotriacetic acid agarose columns (Qiagen) and
eluted with 500 mM imidazole in phosphate-buffered saline. GST
fusion proteins were purified by binding to glutathione-Sepharose beads
(Amersham Biosciences) in TBS buffer (50 mM Tris-HCl, pH 7.5, 140
mM NaCl, 0.1% Triton X-100, plus protease inhibitors), and eluted
by 15 mM reduced glutathione (in 50 mM Tris-HCl, pH 8.0)
or cleaved from the GST tag by incubation with biotinylated thrombin (Novagen)
at a final concentration of 0.1 units/µl at room temperature for 4 h.
Biotinylated thrombin was removed from the resulting cleavage reactions using
streptavidin-agarose beads (Novagen). The eluates were dialyzed in a
10,000-molecular weight cutoff dialysis cassette (Pierce) against TBS and
concentrated using Centriprep-10 filtration units (Amicon).
In Vitro BindingApproximately 1 µg of GST fusion proteins were bound to glutathione-Sepharose beads (Amersham Biosciences) in TBS buffer, incubated at 4 °C for 1 h with constant agitation, and washed with TBS to remove unbound proteins. Glutathione-Sepharose beads coupled with similar amounts of GST fusion proteins were added to the full-length HA-tagged DAP kinase lysates from transfected HEK293T cells or His-DAP-kinase-CT lysates from bacteria and incubated with gentle mixing for 3 h at 4 °C. The beads were washed three times with TBS buffer, and bound proteins were eluted in 30 µl of SDS-PAGE sample buffer and electrophoresed on 1020% SDS-Tricine gradient gel. Bound HA-DAP-kinase or His-DAP-kinase-CT was detected by anti-DAP kinase antibody (Sigma) or monoclonal anti-HisT7-tag antibody (Novagen), respectively, and GST fusion proteins were detected by anti-GST antibody (Amersham Biosciences). Horseradish peroxidase-conjugated secondary antibodies and ECL (Amersham Biosciences) were used to visualize the bands.
Transfection of HEK293T CellsHEK293T cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Invitrogen) and 0.5% L-glutamine. Syntaxin-1A cDNA was inserted into the HindIII site of the pcDNA3.1 vector (Invitrogen). HEK293T cells cultured in 100-mm dishes were transfected with 510 µg of cDNA using LipofectAMINE 2000 (Invitrogen), according to the instruction manual. After 48 h, the cells were harvested and solubilized in TBS buffer with 1% Triton X-100, 5 mM EDTA, and protease inhibitors. Cell lysates were centrifuged at 13,000 rpm for 20 min at 4 °C, and the supernatant was used for in vitro binding and immunoprecipitation studies.
Synaptosome PreparationRat brain synaptosomes were prepared by differential and discontinuous Percoll gradient centrifugation and solubilized as described (55). Synaptosome fractions were isolated as described (56). Briefly, after Percoll-sucrose gradient centrifugation, the synaptosomes were washed once in medium M (0.32 M sucrose, 1 mM K2HPO4, 0.1 mM EDTA, pH 7.5), resuspended and homogenized in medium L (1 mM K2HPO4, 0.1 mM EDTA, pH 8.0), and incubated at 4 °C for 1 h. The resulting suspension was layered over 5 ml of 1 M sucrose in medium L and centrifuged for 30 min at 96,300 x g in an SW27 rotor. The supernatant was mixed to homogeneity and centrifuged again for 14 h at 25,000 x g. The supernatant was collected as synaptosol, and the pellets were homogenized in medium L and applied to a gradient of 7 ml of 1.2, 1.0, 0.8, 0.6, and 0.4 M sucrose in medium L and centrifuged for 90 min at 68,000 x g in an SW27 rotor. Bands at each interface were collected and washed once with medium L in a Ti50 rotor (45 min at 106,500 x g). The synaptosol was dialyzed against medium L and centrifuged at 140,000 x g in a Ti50 rotor for 1 h to separate any remaining synaptic vesicles from soluble proteins. All pellets were then resuspended in 20 mM Tris-HCl (pH 7.5). The concentrations of total proteins in each fraction were determined by BCA protein assay (Pierce) against a bovine serum albumin standard.
Immunocytochemistry of Cultured Hippocampal CellsRat hippocampal neuron cultures were prepared at a density of 100,000 cells/ml on polyornithine-and fibronectin-coated glass chamber slides (Lab-Tek) from hippocampi dissected from E18 rat embryos as described (57). Cultures were plated on a glial bed in Neurobasal (Invitrogen) supplemented with 1x B27 and 100x L-glutamine, with half-feed changes every 34 days. Neurons were prepared for immunofluorescent confocal microscopy on DIV11 following fixation in 4% paraformaldehyde, solubilization in 0.1% Triton X-100, and blocking in 1% bovine serum albumin. Antibodies against syntaxin-1 (from M. Takahashi) and DAP kinase (from R.-H. Chen, Ref. 58) were detected using tagged fluorescent anti-mouse and anti-rabbit antibodies (Jackson).
CoimmunoprecipitationSolubilized proteins (100300 µg) from transfected HEK293T cell lysates or brain homogenates were incubated with either 3 µg of anti-syntaxin-1 (10H5) monoclonal antibody or 3 µg of non-immune mouse IgG (Zymed Laboratories Inc.) as control in 0.5 ml of TBS with 0.1% Triton X-100 and protease inhibitors, and incubated on a microtube rotator at 4 °C for 3 h. Protein A-Sepharose CL-4B resin (2.5 mg) (Amersham Biosciences) was added to each sample, and the incubation continued for an additional 1 h, followed by three washes with TBS/0.1% Triton X-100. Subsequent interaction assays of immunoprecipitated and immobilized protein complexes are described in the section on in vitro binding assays above. For multiple detection with different antibodies, blots were first stripped between applications of each antibody in a solution of 62.5 mM Tris-HCl (pH 7.5), 20 mM dithiothreitol, and 1% SDS for 20 min at 50 °C with agitation and then washed with TBS/0.1% Tween-20 for 30 min.
Phosphorylation ReactionsLysates of transfected HEK293T
cells with pcDNA-HA-DAP-kinase were immunoprecipitated with 1 µg of anti-HA
polyclonal antibodies (Clontech) in 0.5 ml of TBS/0.1% Triton X-100 with
protease inhibitors. After 3 h of incubation at 4 °C, protein A-Sepharose
CL-4B resin (2.5 mg) was added and incubated for 1 h. The immunoprecipitates
were washed three times with TBS/0.1% Triton X-100 and once with the kinase
assay buffer (50 mM HEPES pH 7.5, 8 mM MgCl2,
and 0.5 mM dithiothreitol). Isolated and immobilized DAP kinase
full-length or purified DAP kinase catalytic domain (KD) or KD-CaM domain were
then incubated for 30 min at 30 °Cin2550 µlof reaction buffer
containing 515 µCi [-32P]ATP (1.65 pmol)
(Amersham Biosciences), 100 µM ATP, 0.1 mg/ml bovine serum
albumin, and 50 pmol of purified syntaxin-1A. The reactions were terminated by
adding SDS-PAGE sample buffer and then heated at 95 °C for 5 min. Aliquots
of phosphorylated products were analyzed by 1020% Tricine SDS-PAGE,
stained with Coomassie Blue, and then dried and exposed to x-ray film for
autoradiography. For the Ca2+-buffering system, 10 mM
EGTA or 10 mM nitrolotriacetic acid was used to produce free
Ca2+ buffer (59) as
calculated using the max chelator software (version 6.63). For
back-phosphorylation studies, HEK293T cells co-transfected with syntaxin-1A
and HA-DAP-kinase wild type or K42A mutant were stimulated with 10
µM ionomycin at room temperature for 30 min and then solubilized
with 1% Triton X-100. Lysates (2 mg of total protein) were incubated with an
anti-stx1 antibody, and the immunoprecipitates were then back-phosphorylated
in vitro with anti-HA-immobilized full-length DAP kinase isolated
from transfected HEK293 cell lysates. Back-phosphorylation was performed in
small volumes (100 µl) with constant agitation throughout the incubation
time.
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RESULTS |
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Distribution and Subcellular Localization of DAP Kinase DAP kinase mRNA has been found to be present in brain tissue, and particularly in the hippocampus of adult rats (4950). To determine the expression of DAP kinase protein in brain, we performed immunoblot using a monoclonal anti-DAP kinase antibody on various homogenates of anatomically and functionally distinct areas of adult rat brain including cortex, hippocampus, olfactory bulb, mesencephalon, midbrain, cerebellum, and spinal cord. Only cortex, hippocampus, and olfactory bulb showed prominent expression of DAP kinase in the adult rat brain (Fig. 2A). To examine the subcellular distribution of DAP kinase in neurons, we prepared a subcellular fractionation assay from crude synaptosomal preparations. By sucrose density gradient centrifugation, rat cerebral synaptosomes were fractionated into cytosol (synaptosol), synaptic vesicle, and synaptic plasma membrane fractions and then analyzed by sequential immunoblotting with various antibodies as indicated (Fig. 2B). The relative purity of these subcellular fractions was confirmed by immunoreactivity corresponding to markers of synaptic vesicles (synaptophysin), plasma membrane (Na+/K+-ATPase), and cytosol (LDH). DAP kinase was present predominantly in the cytosolic fraction and, to a lesser extent, in the plasma membrane fraction, and was absent from the synaptic vesicle fraction, consistent with structural predictions given its lack of a hydrophobic transmembrane segment. The presence of DAP kinase in the plasma membrane fraction is significant given our description of its interaction with the plasma membrane protein syntaxin.
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To confirm that DAP kinase is present at neuronal processes and to examine whether its localization in neurons would allow it to interact with syntaxin-1 at the plasma membrane, we performed double-labeled immunocytochemistry in cultured hippocampal cells using antibodies against DAP kinase and syntaxin-1. As shown in Fig. 2C, syntaxin-1 staining was detected in a punctate pattern along the plasma membrane surface of neuronal cell bodies and processes. While some staining for DAP kinase was found in glia, DAP kinase signal was seen predominantly in neurons. Consistent with our immunoblot findings in subcellular synaptosomal fractions, the majority of DAP kinase staining was detected in the cytoplasmic space, but extended throughout neuronal processes to the plasma membrane, where it was found to colocalize with syntaxin-1. These data demonstrate that DAP kinase partially colocalizes with syntaxin-1A at the plasma membrane of intact neurons, and suggest that our findings that DAP kinase and syntaxin-1A are binding partners could be physiological relevant in vivo. The restricted distribution of DAP kinase in adult brain to the hippocampus, cortex, and olfactory bulb, its presence in both the cytosolic and plasma membrane fractions of synaptosomes, and its partial colocalization with syntaxin-1 in hippocampal neurons suggest that DAP kinase-mediated signal transduction pathway may be involved in neuronal functions related to synaptic transmission or plasticity.
In Vitro Phosphorylation of Recombinant Syntaxin-1A by DAP
KinaseDAP Kinase was first reported as a
calcium/calmodulin-dependent serine/threonine protein kinase that mediates
-interferon-induced cell death
(47); however, its potential
roles in mature neurons is still unknown. Given our finding of a specific
interaction between syntaxin-1A and DAP kinase, we wondered whether
syntaxin-1A might be a substrate for DAP kinase phosphorylation. Due to its
presence in synapses, we also speculated that DAP kinase activity might
represent a signal transduction pathway coupled to synaptic activity,
i.e. that syntaxin phosphorylation by DAP kinase might occur in a
Ca2+/CaM-dependent manner. First, we examined the ability of
recombinant syntaxin-1A to serve as a substrate of DAP kinase. We incubated 50
pmol of purified recombinant syntaxin-1A
TM cleaved from the GST tag
with a truncated DAP kinase (KD-CaM-(1320)) containing both the
catalytic domain and CaM-binding regulatory domain, and
[
-32P]ATP in buffer with 0, 0.1, 1, 10, or 100
µM free Ca2+. As shown in
Fig. 3A,
phosphorylation of syntaxin-1A is hardly observed in the absence of
Ca2+, is very weak at 0.1 and 1 µM [Ca2+],
and increases sharply between 1 and 10 µM free
[Ca2+]. As synaptic vesicle exocytosis requires elevated
intracellular free [Ca2+]
(60), it is reasonable to
speculate that increases in intracellular free [Ca2+] that occur
with opening of voltage-dependent calcium channels at the synapses could lead
to the activation of DAP kinase localized at or near active zones and
consequently, the phosphorylation of syntaxin-1. Interestingly, DAP kinase
(KD-CaM) was also found to be autophosphorylated in a
Ca2+-regulated manner (Fig.
3A). While increasing Ca2+ levels activate the
phosphorylation of syntaxin 1A by DAP kinase, DAP kinase autophosphorylation
was stronger in the absence than in the presence of free [Ca2+]. In
addition, DAP kinase autophosphorylation inhibited its phosphorylation of
syntaxin-1, an observation consistent with previous findings
(61) suggesting a mechanism
for Ca2+-mediated regulation of DAP kinase activity.
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To map potential DAP kinase phosphorylation sites in syntaxin-1A, we examined the capacity of syntaxin deletion mutants to serve as DAP kinase substrates. We incubated 50 pmol of purified His-tagged syntaxin-1A full-length, syntaxin-1A-NT-(2190), or syntaxin-1A-CT-(181264) with immobilized full-length DAP kinase and found that syntaxin-1A-CT fragment was efficiently phosphorylated in vitro by DAP kinase in a Ca2+/CaM-dependent manner (data not shown). As the consensus sequence for DAP kinase phosphorylation has not yet been illustrated, we used site-directed mutagenesis to generate seven syntaxin-1A mutants in which the serine, at positions 188, 200, 208, 249, or 259, or the threonine, at positions 197 or 251, was substituted with alanine. While T197A, S200A, S208A, S249A, T251A, and S259A mutants serve as efficient substrates for the purified DAP kinase KD (data not shown), the S188A mutation effectively eliminated 32P incorporation following incubation with DAP kinase KD (Fig. 3B), indicating that serine 188 of syntaxin-1A is the primary phosphorylation site for DAP kinase. The DAP kinase-mediated phosphorylation sequence of syntaxin-1A, X(S/T)(K/R)QAL, is conserved among the syntaxin-14 isoforms, but is unique from the consensus phosphorylation sequences of CaMKII, PKC, PKA, and casein kinases I and II.
To confirm that serine 188 is a dominant phosphorylation site of syntaxin
and argue against trace levels of syntaxin phosphorylation, we performed
stoichiometric analysis of phosphorylation of purified syntaxin-1A by DAP
kinase in vitro. The reactions included equal amounts (3.8 pmol) of
syntaxin-1A or myosin light chain (MLC) (10 pmol) and excess DAP kinase and
[-32P]ATP, and were terminated at various time points by the
addition of SDS sample buffer and boiling. The products were separated by
SDS-PAGE, stained with Coomassie Blue to verify equal amounts of protein
loading, the gel dried, and finally submitted to autoradiography. To quantify
total Pi incorporation, gel slices corresponding to phosphorylated
syntaxin bands were excised and scintillation counted. Stoichiometry values
were expressed as the ratio of moles of phosphate (Pi) incorporated per mole
of syntaxin-1A or MLC and plotted against reaction time. The maximal
stoichiometric ratio of
0.4 under our phosphorylation conditions
approximates that of MLC (Fig. 3,
C and D), a classic DAP kinase substrate with
one dominant phosphorylation site
(62); this is consistent with
the results of our mutagenetic studies, which identified one phosphorylation
site in syntaxin for DAPK. The relatively low stoichiometry for
phosphorylation of both syntaxin and MLC (
0.4) is probably a result of
our phosphorylation assay, which does not replicate the optional conditions
for DAP kinase activity or lacks a cofactor for maximal activation of the
recombinant DAP kinase in vitro.
In Vivo Phosphorylation of Syntaxin-1A by DAP Kinase While our biochemical experiments showed that DAP kinase incorporates 32P into syntaxin-1A in a Ca2+-dependent manner, the conditions used for in vitro phosphorylation may not reflect conditions found in the native cellular environment. In addition, treatment of proteins with detergent for solubilization may expose sites that are normally not available for phosphorylation in vivo. Therefore, to investigate in vivo phosphorylation, we performed back-phosphorylation assays (40) in HEK293T cells co-transfected with cDNAs of syntaxin-1A and DAP kinase. In this procedure, endogenous phosphate is incorporated in vivo into syntaxin-1A after stimulation with ionomycin, a Ca2+ ionophore that induces Ca2+ channel-independent Ca2+ influx. The cell lysates from stimulated and non-stimulated HEK293T cells are then processed for in vitro phosphorylation (back-phosphorylation) with immunoprecipitated full-length DAP kinase to incorporate [32P]ATP into syntaxin-1A that was left unphosphorylated in vivo after stimulation. In this protocol, a decrease in back-phosphorylation in vitro reflects in vivo phosphorylation of syntaxin-1A by DAP kinase in transfected HEK293T cells in response to Ca2+ influx.
It has been reported that the DAP kinase K42A mutant, in which the conserved lysine 42 in the kinase subdomain II is substituted with alanine, is catalytically inactive to phosphorylate MLC (48). We therefore generated a DAP kinase K42A mutant to use as a negative control in our in vivo phosphorylation studies. To confirm that K42A DAP kinase is catalytically inactive, both HA-tagged DAP kinase wild type and K42A mutant expressed in HEK293T cells were immunoaffinity purified with anti-HA antibody and then incubated with recombinant syntaxin-1A-CT in the presence of Ca2+, calmodulin, and [32P]ATP. Syntaxin-1A-CT was phosphorylated by DAP kinase wild type but not by the K42A mutant (Fig. 4A), consistent with previous findings that mutation of this site abolishes kinase activity (48). The K42A mutation does not affect binding of DAP kinase to syntaxin since equal amounts of DAP kinase was coimmunoprecipitated by anti-syntaxin-1 antibody from the lysates of the co-transfected HEK293T cells (Fig. 4, B and C).
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As expected, the amount of 32P incorporation during in vitro back-phosphorylation was significantly reduced after stimulation of DAP kinase/syntaxin-1A co-transfected cells with ionomycin. However, stimulation of cells co-transfected with DAP kinase K42A/syntaxin-1A caused no significant reduction in [32P]ATP incorporation in vitro when compared with the non-stimulation control (Fig. 4D). Coomassie Blue staining corroborated the identity of the phosphorylated band (35 kDa) as syntaxin-1A and was used to normalize back-phosphorylation to the amount of syntaxin-1A immunoprecipitated by the anti-syntaxin-1 antibody. Quantitative analysis of the results of seven independent experiments showed that ionomycin stimulation significantly reduced back-phosphorylation of syntaxin-1A from the cells transfected with the cDNAs of DAP kinase/syntaxin-1A to 82.1 ± 5.9% of the non-stimulated control value (mean ± S.E., n = 7, p < 0.05; Fig. 4E). In contrast, no significant decrease in back-phosphorylation of syntaxin-1A (93.5 ± 5.3%, mean ± S.E., n = 7, p > 0.05) from the cells transfected with the cDNAs of DAP kinase K42A/syntaxin-1A was observed. These data suggest that syntaxin-1A is a substrate for DAP kinase-mediated phosphorylation in vivo through a Ca2+-dependent pathway.
Biochemical Effects of DAP Kinase Phosphorylation of
Syntaxin-1ASince SNAP-25
(1) and Munc18-1, a regulatory
protein involved in synaptic vesicle docking and fusion
(53 and
6364),
are two of the most important binding partners of syntaxin-1A in
neurotransmitter release, we first tested whether syntaxin-1A phosphorylation
by DAP kinase effected the mutually exclusive interactions of syntaxin 1A with
these two proteins. We incubated immobilized DAPK-phosphorylated or
unphosphorylated GST-syntaxin-1ATM(1264), a mutant lacking the
C-terminal transmembrane segment or GST as a control with His-tagged Munc18-1
or SNAP-25. As shown in Fig. 5,
while phosphorylated syntaxin-1A did not affect its binding to SNAP-25, its
binding to Munc18-1 decreased dramatically to 54.80 ± 7.98% (n
= 8, p < 0.01) of Munc 18-1 binding by unphosphorylated
syntaxin-1A.
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Our mutagenesis studies demonstrated syntaxin-1A serine 188 to be the
primary site for DAP kinase phosphorylation. Ser-188 is located in the linker
region between N-terminal Habc and C-terminal H3 coil domains, adjacent to the
binding site for Munc18. To test whether introduction of a negatively charged
residue at this site has any effect on binding of syntaxin 1A to Munc18-1, we
attempted to mimic complete phosphorylation of serine 188 by mutating it to a
negatively charged aspartic acid residue (S188D), and then analyzed the
resultant binding properties of the mutated protein. His-tagged
syntaxin-1ATM wild type or S188D mutant was incubated with immobilized
GST-Munc18-1, GST-SNAP-25, or GST alone. We found that the binding of the
S188D mutant to GST-Munc18-1 decreased significantly compared with the binding
of wild-type syntaxin-1A to GST-Munc18-1, while wild-type and mutant
syntaxin-1A bound equally to GST-SNAP-25
(Fig. 6A).
Quantitative analysis showed that the S188D mutation decreases syntaxin 1A
binding to Munc18-1 to 48.7 ± 9.4% (n = 5, p <
0.01; Fig. 6B) of
wild-type binding, while binding of the mutant syntaxin 1A to SNAP-25 is not
significantly changed (n = 5, 1.01 ± 6.5%, p >
0.05). Our binding results suggest that phosphorylation of the end linker
region of syntaxin-1 has a significant effect on its binding to Munc18-1.
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As the syntaxin-Munc18-1 complex prevents formation of the SNARE complex
(51,
6566),
we further studied whether the formation and stability of the SNARE complex
are affected by this mutation. We incubated immobilized GST-VAMP-2 with 2
µg of His-tagged SNAP-25 and syntaxin-1A wild type or its S188D mutant to
examine the formation of recombinant SNARE complex in vitro. The
syntaxin-1A S188D mutation showed no significant effect on either the assembly
or heat stability of the recombinant SNARE complex in vitro
(Fig. 7A). To test the
effect of this mutation on SNARE complex formation in vivo, we
coimmunoprecipitated the SNARE complex with an anti-VAMP antibody from lysates
of PC12 cells transfected with the cDNA of either wild type or S188D mutant
syntaxin-1A. As in our in vitro study, the S188D mutation showed no
significant effect on the assembly and heat stability of the native SNARE
complex in transfected PC12 cells (Fig.
7B). The lack of effect of the syntaxin-1A S188D mutation
on its binding to other SNAREs further supports the notion that only the
C-terminal H3 coiled-coil domain, but not the syntaxin linker region, is
involved in SNARE core complex formation
(1,
51,
67).
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DISCUSSION |
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The relatively low quantities of endogenous DAPK associated with syntaxin in extracts derived from rat brain could be a consequence of the significant impact of detergents on the stability of a dynamic kinase-substrate complex. Furthermore, our immunocytochemical studies in cultured hippocampal neurons suggest a relative enrichment of DAPK in some restricted synapses; if this is the case, our brain homogenates, which are derived from a mixed population of neurons and glial cells, would not allow us to estimate the abundance of this complex in specific synapses. Finally, the conformation and thus the stability of the DAPK-syntaxin complex in solution with detergent may not reflect that of the complex in its in vivo membrane-bound environment. Thus, our coimmunoprecipitation data should not be considered as evidence of the relative enrichment of a native DAPK-syntaxin complex in intact synapses. Rather, the in vivo data provide evidence showing that endogenous DAPK indeed associates with syntaxin, and strengthens the conclusions of our in vitro studies.
Most significantly, we have found that the phosphorylation of syntaxin-1A by DAP kinase is Ca2+-dependent, and that this phosphorylation can exact a significant and selective effect on syntaxin binding with its regulatory protein Munc18-1, while maintaining its properties governing assembly and the binding stability of the SNARE complex. The sharp rise in intracellular calcium levels ([Ca2+]i) that accompanies the arrival of an action potential to the presynaptic terminal is critical not only as a trigger for synaptic vesicle fusion with the terminal plasma membrane, but also as a regulator of the multiple steps constituting the synaptic vesicle life cycle. Endocytosis is tightly regulated by [Ca2+]i levels during action potential firing at hippocampal synapses (68). Activity-dependent mobilization of synaptic vesicles is also elevated during stimulation (46, 69, 70). The priming stage of synaptic vesicles is also tightly regulated by [Ca2+]i, the supply of releasable vesicles is accelerated during high frequency stimulation in both chromaffin cells (71) and at different neuronal synapses (7274) in a Ca2+-dependent manner. One mechanism by which [Ca2+]i could affect multiple steps of synaptic vesicle trafficking is through protein phosphorylation/dephosphorylation by Ca2+-dependent kinases/phosphatases.
Although our back-phosphorylation experiments detected a relatively low ratio (about 20%) of in vivo phosphorylation of syntaxin by DAPK in co-transfected 293 cells, multiple repeats (n = 7) of these experiments showed that this in vivo phosphorylation event is consistently and statistically significant when compared with syntaxin phosphorylation by the control K42A DAPK mutant. Several factors could contribute to the low ratio of in vivo syntaxin phosphorylation seen in 293 cells. First, the level of syntaxin expression in co-transfected 293 cells was much higher than that of DAPK; thus, only a relatively small percentage of total syntaxin could be phosphorylated by limited amounts of DAPK in vivo. Second, the targeting mechanisms responsible for syntaxin co-localization with DAPK at a subset of synapses are not present in 293 cells. Third, DAPK-mediated phosphorylation requires free Ca2+ levels above 10 µM, which would highly restrict syntaxin phosphorylation to some regions within the cell following ionomycin treatment. Finally, DAPK function in vivo could be further modulated by cellular cofactors. We lack knowledge of any potential activator or inhibitor of this newly discovered kinase, which could be limiting our capability to fully activate this kinase in vivo except by elevating intracellular Ca2+.
As our data suggest that syntaxin-1A could be phosphorylated by DAP kinase when synaptic [Ca2+]i is elevated during action potential stimulation, it seems rational to ask whether phosphosyntaxin-1A is able to affect the formation of the SNARE complex and the syntaxin-Munc18-1 complex. We found that phosphorylation of syntaxin-1A or its S188D mutation reduces its association with Munc18-1 by 50%, while no significant effect is detected on its binding with SNAP-25 in vitro (Figs. 5 and 6). Furthermore, syntaxin-1A S188D mutation affects neither the assembly nor stability of the SNARE complex in vitro (Fig. 7). The formation of the trans-SNARE complex is thought to bring lipid membranes in close apposition, perhaps even resulting in merging of the two bilayers (10). Unlike VAMP-2 and SNAP-25, in which most of the protein sequence participates in core complex formation, only the C-terminal third of the cytoplasmic region of syntaxin is involved in formation of the SNARE core complex. The N-terminal half of syntaxin forms an independently folded domain (75) and is involved in binding to several SNARE regulatory proteins, Munc13 (76) and Munc18-1 (77). Particularly, Munc18-1 was reported to bind syntaxin with high affinity, and this binding is mutually exclusive with SNARE complex formation (51, 6566). In the three-dimensional crystal structure of the neuronal-Sec1(nSec1, also know as Munc18-1)-syntaxin-1A complex (78), the N-terminal half Habc domain of syntaxin-1A is folded back onto the C-terminal H3 domain, representing a "closed" conformation, and the linker region (residues 145188) between the N-terminal half and C-terminal coiled-coil domain is structured in the favorable environment provided by the lower part of domain 3 of Munc18-1. For this reason, the linker region of syntaxin-1A is critical both as a structural switch for syntaxin to transform from a closed conformation to an open one, and for its binding affinity to Munc18 and other SNARE proteins, which was confirmed by an earlier finding that residue mutations in the linker region of syntaxin-1A abolishes its binding to Munc18-1 and consequently inhibits secretion in PC12 cells (51). Likewise, phosphorylation in the linker region would be predicted to have biochemical import for syntaxin conformation and syntaxin interactions with other binding partners. We speculate that DAP kinase phosphorylation of syntaxin-1A at serine 188, which is located in the end of the linker region, would induce conformational changes in syntaxin-1A and thereby affect its binding to Munc18-1. Our in vitro binding studies, which demonstrate that the phosphorylation of syntaxin-1A or its S188D mutant decreases its binding to Munc18-1 by about 50% without affecting the assembly and stability of the SNARE complex, supports this hypothesis.
Munc18 has been proposed to play both activating (63, 64) and inhibitory (79) roles in synaptic vesicle exocytosis by acting at different steps in the release pathway. One of the most well characterized roles for Munc18-1 is to sequester syntaxin from binding to SNAP-25 and inhibit formation of the SNARE complex. However, recent work from knockout animals showed that Munc18 could also function upstream of SNARE complex formation and promote large dense-core vesicle (LDCV) docking in chromaffin cells (53). Thus, the existence of cellular signal pathways to regulate the switch between the assembly/disassembly of the Munc18-syntaxin complex in response to synaptic activity could be of physiological import. Our present study provides a novel signal transduction pathway by which the formation of the syntaxin-Munc18 complex could be regulated via syntaxin phosphorylation in response to intracellular [Ca2+] and synaptic activity.
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FOOTNOTES |
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To whom correspondence should be addressed: Synaptic Function Unit, NINDS,
National Institutes of Health, Bldg. 36, Rm. 5A23, 36 Convent Dr., Bethesda,
MD 20892-4154. Tel.: 301-435-4596; E-mail:
shengz{at}ninds.nih.gov.
1 The abbreviations used are: VAMP, vesicle-associated membrane protein;
SNARE, soluble N-ethylmaleimide sensitive fusion protein attachment
protein receptor; PKA, cAMP-dependent protein kinase; CaMKII, calcium, and
calmodulin-dependent protein kinase type II; PKC, phospholipid-dependent
protein kinase; MLC, myosin light chain; DAP, death-associated protein; DAPK,
DAP kinase; HA, hemagglutinin; TBS, Tris-buffered saline; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; GST, glutathione
S-transferase; TM, transmembrane; stx, syntaxin; WT, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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