Ca2+-Dependent Phosphorylation of Syntaxin-1A by DAP-kinase

Ca2+-Dependent Phosphorylation of Syntaxin-1A by DAP-kinase

Regulates its Interaction with Munc-18*

Jin-Hua Tian, Sunit Das and Zu-Hang Sheng#

From Synaptic Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of

Health, Building 36, Room 5A23, 36 Convent Drive, Bethesda, Maryland 20892-4154, USA.

Running title: Ca2+-dependent phosphorylation of syntaxin-1

Number of text pages with references and figure legends: 34

Number of words: Summary, 179; Introduction, 609; Discussion, 1349.

Number of Figures: 7

* This work was supported by the intramural research program of NINDS, NIH. The costs of

publication of this article were defrayed in part by the payment of page charges. This article must

therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

# To whom correspondence should be addressed: Synaptic Function Unit, National Institute of

Neurological Disorders and Stroke, National Institutes of Health, Building 36, Room 5A23, 36 Convent

Drive, Bethesda, Maryland 20892-4154, USA. Tel: 301-435-4596; E-mail: [email protected].

1 The abbreviations used are: SNARE, soluble N-ethylmaleimide sensitive fusion protein attachment

protein receptor; PKA, cAMP-dependent protein kinase; CaMKII, calcium and calmodulin-dependent

Tian et al (M3:00492)

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protein kinase type II; PKC, phopholipid-dependent protein kinase; MLC, myosin light chain; DAPK,

Death Associated Protein kinase.

Key words: DAP-kinase/ syntaxin / SNARE / Ca2+-dependent phosphorylation / exocytosis /

Munc18-1 / synaptic vesicle

Summary

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with

VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding

properties by protein kinases could be critical to the control of neurotransmitter release. Using yeast

two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal

domain of DAP-kinase (Death Associated Protein kinase), a calcium/calmodulin-dependent

serine/threonine protein kinase. Expression of DAP-kinase in adult rat brain is restricted to particular

neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies

demonstrate that DAP-kinase binds to and phosphorylates syntaxin-1 at serine-188. This

phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A

phosphorylation by DAP-kinase or its S188D mutant, which mimics a state of complete

phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that

regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest

that syntaxin is a DAP-kinase substrate, and provide a novel signal transduction pathway by which

syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.


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INTRODUCTION

Neurotransmitter release from presynaptic nerve terminals requires calcium-triggered

exocytosis of synaptic vesicles, a process that involves a series of protein-mediated docking and fusion

events between the membranes of synaptic vesicles and the presynaptic terminal (1, 2). The synaptic

vesicle-associated protein synaptobrevin (VAMP, v-SNARE) interacts with two plasma membrane-

associated proteins (t-SNAREs), SNAP-25 and syntaxin, to form a stable SNARE core complex (4-7).

Considerable evidence indicates that the SNARE complex is a biochemical intermediate essential for a

late step in the membrane fusion process (2, 8-11). In neurons, the final stages of vesicle priming and

membrane fusion are strictly Ca2+-dependent (12). Presynaptic exocytosis is characterized by a fast

response with a very short delay between excitation and secretion (13), and by limited release with only

a small percentage of morphologically docked vesicles completing fusion upon Ca2+ influx (14). These

properties suggest that the molecular bases of the SNARE protein-protein interactions that mediate the

final steps of synaptic vesicle docking/fusion are tightly and finely regulated.

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 (15-28). A

number of SNAREs and their regulatory proteins have been reported as potential substrates in vitro for

protein kinases (29-43). For example, in vivo phosphorylation of synapsin, which is dependent on


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intracellular [Ca2+], has been shown to affect synaptic vesicle availability at release sites and control the

kinetics of vesicle pool turnover (44-46). 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 Death Associated Protein kinase (DAP-kinase) (47), a

calcium/calmodulin-dependent serine/threonine protein kinase (48). DAP-kinase was first reported as a

potential mediator of γ-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 post-natum 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

pathways may be implicated in neuronal functions related to synaptic transmission or plasticity (49-50).

Our current yeast two-hybrid cloning results and biochemical studies demonstrate that DAP-kinase

binds to and phosphorylates neuronal syntaxin-1A. Our results suggest that syntaxin is a Ca2+-

dependent phosphorylation target of DAP-kinase and that syntaxin phosphorylation at S188 could be a


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mechanism for regulation of syntaxin binding properties in response to elevated intracellular [Ca2+] and

synaptic activity.

EXPERIMENTAL PROCEDURES

Isolation of DAP-Kinase----The yeast two-hybrid system was used to isolate proteins that interact

with syntaxin-1A. The C-terminal half (CT, 181-288) of rat syntaxin-1A cDNA was inserted in-

frame into a pGBT9 bait vector containing the GAL4 DNA-binding domain (Clontech). Yeast two-

hybrid screens of a human brain cDNA library in vector pACT1 (Clontech) containing the GAL4

activation domain were performed and evaluated according to the protocols described for the MATCH-

MAKER yeast two-hybrid system (Clontech). Positive clones were selected on plates lacking leucine,

tryptophan, and histidine with 50 mM 3-aminotriazole and confirmed by filter assay for β-galactosidase

activity.

Fusion Protein Construction, Preparation and Purification----HA-DAP-kinase cDNA was kindly

provided by A. Kimchi (47), and purified DAP-kinase catalytic domain (KD, aa 1-285) by Dr. Martin

Watterson (54). DAP-kinase (KD-CaM) was produced by subcloning the region of the DAP-kinase

cDNA that encodes amino acids 1-320, corresponding to the catalytic domain and calmodulin-binding

regulatory domain, into the pASK-IBA3 (Sigma) expression vector at the Bsa I 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-1ADTM (1-264, lacking the

transmembrane domain), syntaxin-1A amino-terminal half (NT)(2-190), syntaxin-1A carboxy-

terminal half (CT)(181-264), SNAP-25, VAMP-2, Munc18-1 and DAP-kinase carboxy-terminal

(CT)(1157-1431) were subcloned into GST-fusion protein vector pGEX-4T-1 (Pharmacia) or


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hexahistidine-tagged fusion protein vector (pET28, Novagen). Fusion proteins were prepared as crude

bacterial lysates by mild sonication in PBS (50 mM sodium phosphate, pH 7.4, 140 mM NaCl, plus

protease inhibitors of 1 mM PMSF, 1 mg/ml Leupeptin, 2 mg/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 PBS. GST-fusion proteins were purified by

binding to glutathione Sepharose beads (Pharmacia) in TBS buffer (50 mM Tris-HCl, pH 7.5, 140 mM

NaCl, 0.1% TX-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 per ml at room temperature for 4 h. Biotinylated thrombin was removed

from the resulting cleavage reactions using strepiavidin-agarose beads (Novagen). The eluates were

dialyzed in a 10,000 MW cutoff dialysis cassette (Pierce) against TBS and concentrated using

Centriprep-10 filtration units (Amicon).

In vitro Binding----Approximately 1 µg of GST fusion proteins were bound to glutathione-Sepharose

beads (Pharmacia) in TBS buffer, incubated at 4°C for 1 hr 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 HEK

293 T cells or His-DAP-kinase-CT lysates from bacteria and incubated with gentle mixing for 3 hr at

4°C. The beads were washed three times with TBS buffer, and bound proteins were eluted in 30 ml

SDS-PAGE sample buffer and electrophoresed on 10–20% 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 (Pharmacia). HRP-conjugated secondary antibodies and ECL (Amersham) were

used to visualize the bands.


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Transfection of HEK 293 T Cells----HEK 293 T cells were maintained in DMEM with 10% fetal

bovine serum (Gibco) and 0.5% L-glutamine. Syntaxin-1A cDNA was inserted into the Hind III site of

pcDNA3.1 vector (Invitrogen). HEK 293 T cells cultured in 100-mm dishes were transfected with 5–10

µg of cDNA using Lipofectamine 2000 (Life Technologies), according to the instruction manual. After

48 hr, the cells were harvested and solubilized in TBS buffer with 1% TX-100, 5mM 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 Preparation----Rat 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 hr.

The resulting suspension was layered over 5 ml of 1 M sucrose in medium L and centrifuged for 30 min

at 96,300 g in an SW27 rotor. The supernatant was mixed to homogeneity and centrifuged again for 14

hr at 25,000 g. The supernatant was collected as synaptosol, and the pellets were homogenized in

medium L and applied to a gradient of 7 ml 1.2 M, 1.0 M, 0.8 M, 0.6 M, and 0.4 M sucrose in medium L

and centrifuged for 90 min at 68,000 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 g). The synaptosol was dialyzed against

medium L and centrifuged at 140,000 g in a Ti 50 rotor for 1 hr 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 BSA standard.

Immunocytochemistry of Cultured Hippocampal Cells----Rat hippocampal neuron cultures were


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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 (Gibco) supplemented with 1X B27 and 100X L-glutamine, with half-feed

changes every 3-4 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, 58) were detected using tagged fluorescent anti-mouse and anti-rabbit antibodies (Jackson).

Coimmunoprecipitation----Solubilized proteins (100–300 µg) from transfected HEK 293 T cell lysates

or brain homogenates were incubated with either 3 µg of anti-syntaxin-1 (10H5) monoclonal antibody

or 3 µg non-immune mouse IgG (Zymed) as control in 0.5 ml TBS with 0.1% TX-100 and protease

inhibitors, and incubated on a microtube rotator at 4°C for 3 hr. Protein A-Sepharose CL-4B resin (2.5

mg) (Pharmacia) was added to each sample, and the incubation continued for an additional 1 hr,

followed by three washes with TBS/0.1% TX-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 DTT, and 1% SDS

for 20 min at 50°C with agitation and then washed with TBS/0.1% Tween-20 for 30 min.

Phosphorylation Reactions----Lysates of transfected HEK 293 T cells with pcDNA-HA-DAP-kinase

were immunoprecipitated with 1 µg anti-HA polyclonal antibodies (Clontech) in 0.5 ml TBS/0.1% TX-

100 with protease inhibitors. After 3 hr of incubation at 4°C, Protein A-Sepharose CL-4B resin (2.5

mg) was added and incubated for 1 hr. The immunoprecipitates were washed three times with

TBS/0.1% TX-100 and once with the kinase assay buffer (50 mM HEPES at pH 7.5, 8 mM MgCl2 and

0.5 mM DTT). Isolated and immobilized DAP-kinase full-length or purified DAP-kinase catalytic


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domain (KD) or KD-CaM domain were then incubated for 30 min at 30°C in 25-50 ml of reaction

buffer containing 5-15 mCi [g-32P] ATP (1.6-5 pmol) (Amersham), 100 mM ATP, 0.1 mg/ml BSA,

and 50 pmol of purified syntaxin-1A. The reactions were terminated by adding 30 µl SDS–PAGE

sample buffer and then heated at 95 °C for 5 min. Aliquots of phosphorylated products were analyzed

by 10–20% Tricine SDS–PAGE, stained with Coomassie blue, and then dried and exposed to X-ray

film for autoradiography. For the Ca2+-bufferring system, 10 mM EGTA or 10 mM NTA was used to

produce free Ca2+ buffer (59) as calculated using the MAX CHELATOR software (version 6.63). For back-

phosphorylation studies, HEK 293 T cells co-transfected with syntaxin-1A and HA-DAP-kinase wild

type or K42A mutant were stimulated with 10 µM ionomycin at RT for 30 min and then solubilized with

1% Triton X-100. Lysates (2 mg 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 HEK 293 cell lysates. Back-phosporylation was performed in

small volumes (100 µl) with constant agitation throughout the incubation time.

RESULTS

Isolation of DAP-kinase and its Association with Syntaxin-1A----To elucidate the molecular

mechanisms underlying neurotransmitter release and its modulation, we have used the carboxy-terminal

half (181-288) of syntaxin-1A as a bait to screen a human brain cDNA library via the yeast two-hybrid

selection technique. Screening of ~2 x 106 colonies led to the isolation of six classes of cDNAs

encoding α-SNAP (10 clones), β-SNAP (13 clones), SNAP-25 (3 clones), syntaphilin (2 clones),

syntaxin-1A (5 clones), SNAP-29 (2 clones), and one cDNA encoding the carboxyl terminal domain

(1386-1431) of DAP-kinase (Fig. 1A) (47). As the yeast two-hybrid system may identify low-affinity

interactions that may not normally occur either in vitro or in vivo, we conducted four lines of


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experiments to confirm the selective and direct interaction between DAP-kinase and syntaxin-1A.

First, we performed in vitro binding assays with recombinant proteins. While HA-tagged full-length

DAP-kinase selectively bound to GST-syntaxin-1A in a Ca2+/calmodulin-independent manner, no

binding was detectable to GST-VAMP2 or GST alone (Fig. 1B). Next, while DAP-kinase-CT (1157-

1431, Fig. 1A) bound to both GST-syntaxin-1A full length and GST-syntaxin-1A-CT (181-288), no

binding was found to GST-syntaxin-1A-NT (2-190), GST-VAMP2 or GST alone (Fig. 1C),

consistent with a specific association of DAP-kinase with syntaxin-1A via a direct interaction of their

carboxyl terminal domains. Furthermore, we sought to confirm the DAP-kinase-syntaxin-1A

interaction in a mammalian expression system. A cDNA encoding His-tagged full-length DAP-kinase

was co-transfected into HEK 293 T cells with the cDNA encoding syntaxin-1A. The association of

endogenous DAP-kinase with syntaxin-1A was then confirmed by immunoprecipitation with anti-

syntaxin-1 antibody (Fig. 1D, E). DAP-kinase was co-immunoprecipitated with syntaxin-1A from rat

brain homogenate by anti-syntaxin-1 antibody but not by non-immune mouse IgG (Fig. 1F). Finally,

we performed a Coomassie-based estimation of the stoichiometry of the syntaxin-DAPK-CT

interaction using purified recombinant proteins. Our studies show that, under our experimental

conditions, the interaction of His-tagged syntaxin-1A with GST-DAPK-CT occurs at relative

concentrations approaching a 1:1 molar ratio, as determined by semi-quantification using purified

recombinant proteins as standards (Fig. 1G). The results from our yeast two-hybrid selection, in vitro

binding assays, and immunoprecipitation studies indicate that DAP-kinase directly interacts with

syntaxin-1A.

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 (49-50). To determine the

expression of DAP-kinase protein in brain, we performed immunoblot using a monoclonal anti-DAP-


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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.

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


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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 pathways may be involved in neuronal functions related to synaptic

transmission or plasticity.

In vitro Phosphorylation of Recombinant Syntaxin-1A by DAP-kinase----DAP-kinase was first

reported as a calcium/calmodulin-dependent serine/threonine protein kinase that mediates γ-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-1ADTM cleaved from the GST-

tag with a truncated DAP-kinase (KD-CaM 1-320) containing both the catalytic domain and

Ca2+/CaM-binding regulatory domain, and [γ-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


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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.

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 (2-190), or syntaxin-1A-CT (181-264)

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, XS(T)K(R)QAL, is conserved among the syntaxin-1-4 isoforms, but is unique from the

consensus phosphorylation sequences of CaMKII, PKC, PKA and Casein kinase 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


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syntaxin-1A or myosin light chain (MLC) (10 pmol) and excess DAP-kinase and γ-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. 3C 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 co-factor 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 HEK 293 T 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 HEK 293 T

cells are then processed for in vitro phosphorylation (back-phosphorylation) with immunoprecipitated


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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 HEK 293 T 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 HEK 293 T cells were immuno-affinity 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 co-immunoprecipitated by anti-syntaxin-1 antibody from the lysates of

the co-transfected HEK 293 T cells (Fig. 4B and C).

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 to 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-


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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 ± SEM, n=7, P<0.05; Fig. 4E). In contrast,

no significant decrease in back-phosphorylation of syntaxin-1A (93.5±5.3%, mean ± SEM, 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-1A----Since SNAP-25 (1) and

Munc18-1, a regulatory protein involved in synaptic vesicle docking and fusion (53 and 63-64), 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 (1-264, a mutant lacking the carboxyl 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.

Our mutagenesis studies demonstrated syntaxin-1A serine-188 to be the primary site for

DAP-kinase phosphorylation. S188 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-


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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.

As the syntaxin-Munc18-1 complex prevents formation of the SNARE complex (51, 65-

66), we further studied whether the formation and stability of the SNARE complex are affected by this

mutation. We incubated immobilized GST-VAMP-2 with approximately 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 co-immunoprecipitated 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).

DISCUSSION


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Our current findings suggest that syntaxin-1 could serve as a substrate for DAP-kinase, a

Ca2+/CaM-dependent protein kinase. Using the yeast two-hybrid selection approach with the carboxyl

terminal half of syntaxin-1A as bait, we have isolated the cDNA encoding DAP-kinase. We then

confirmed that syntaxin-1 and DAP-kinase bind to each other directly via their carboxyl terminal

domains. We have found that DAP-kinase is relatively enriched in hippocampus and cortex and present

in synaptosomal preparations, and partially co-localizes with syntaxin-1 in the plasma membrane along

the processes of cultured hippocampal neurons. Furthermore, we have demonstrated that syntaxin-1A is

phosphorylated by DAP-kinase both in vitro and in vivo in a Ca2+-dependent manner, with maximal

phosphorylation occurring when [Ca2+] reaches 10 µM. PCR-based site-directed mutagenesis

pinpoints the syntaxin-1A phosphorylation site to serine-188. Finally, we show that phosphorylation of

syntaxin-1A or its S188D mutant, which mimics a state of complete phosphorylation, significantly

decreases the interaction of syntaxin-1A with Munc18-1, a protein that regulates the synaptic vesicle

docking/priming process by forming a syntaxin-Munc18 complex (53, 63-64). DAP-kinase was first

reported to be a mediator of apoptosis (47); however, its downstream targets in apoptosis or otherwise

have not yet been identified. Because of its restricted expression in adult rat hippocampus, cortex and

olfactory bulb, its presence in the cytosolic fraction of synaptosomes, its partial colocalization with

syntaxin-1 in neuronal processes, its direct association with syntaxin-1A, and its Ca2+-dependent

phosphorylation of syntaxin-1A, we propose that DAP-kinase plays a non-apoptotic role in modulating

synaptic transmission.

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


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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 co-

immunoprecipitation 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 (72-74) 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%)


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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 co-factors. 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 suggests 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

phospho-syntaxin-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 (Fig. 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


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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, 65-66). 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 145-188) between the amino-

terminal half and carboxyl 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


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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.

Acknowledgments----- We thank the following people for their critical help: G. Lao and Q. Su for

initiating yeast two-hybrid screening; R. Mehta for preparations of synaptosomal fractions; C. Gerwin

for hippocampal culture; A. Kimchi for HA-DAP-kinase cDNA; M. Watterson and A. Velentza for

purified DAP-kinase catalytic domain; R.-H. Chen for polyclonal anti-DAP-kinase antibody; M.

Takahashi for SNARE antibodies; J.W. Nagle for DNA sequencing.

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LEGENDS TO FIGURES

Fig. 1. DAP-kinase (DAPK) structure and its specific interaction with syntaxin-1A. A, Domain

structure of DAPK and its mutants. Human DAPK is a 1431 residue protein that contains an NH2-

terminal kinase domain followed by a CaM binding sequence and a COOH-terminal syntaxin-binding

domain. The K42A and carboxy-terminal truncated (1157-1431) mutants are illustrated schematically.

B, Ca2+-independent binding of GST-syntaxin-1A to DAPK in vitro. GST or GST fusion proteins

(~1 µg each) were immobilized on glutathione-Sepharose beads and then incubated with HEK 293 cell

lysates transfected with a construct encoding HA-tagged DAPK-full length construct in the presence of

either Ca2+ (500 µM)/CaM (1 µM) or EGTA (2 mM) as indicated. Bound protein complexes were


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eluted from the matrix, separated by SDS-PAGE, and immunoblotted with anti-DAPK antibody (lower

panel). Membranes were then stripped and reprobed with anti-GST antibody (upper panel). C, Binding

of both full-length (FL) and COOH-terminal half (CT) but not NH2-terminal half (NT) of syntaxin-1A

(stx1A) to the DAPK COOH-terminal truncated mutant (DAPK-CT). Immobilized GST or GST fusion

proteins (~1 µg) were incubated with His-tagged DAPK-CT and binding was analyzed by SDS-PAGE

followed by immunoblot with anti-HisT7-tag (lower panel) and anti-GST (upper panel) antibodies. D,

HEK 293 T cells were co-transfected with DAPK-FL and stx1A-FL constructs. The expression of

both proteins was confirmed by immunoblotting of co-transfected or untransfected cell extracts with

antibodies as indicated. E, The DAPK-stx1A complex was co-immunoprecipitated from co-

transfected cell extracts by anti-stx1 antibody, and then detected by immunoblot with anti-DAPK

antibody. Non-immune mouse IgG (Ms-IgG) was used as a control. 10% input of 293 cell lysates for

Co-IP was loaded as an indicator of relative amounts of proteins used. F, The DAPK-stx-1 complex

was immunoprecipitated from TX-100 solubilized rat brain homogenates by anti-stx-1 antibody.

Homogenates were incubated with anti-stx-1 antibody or control Ms-IgG, and the isolated

immunoprecipitates probed with anti-DAPK. 10% input of brain homogenates for Co-IP was loaded as

an indicator of relative amounts of proteins used. G, Stoichiometric binding between DAPK and stx-

1A. Immobilized GST-DAPK-CT (~250 ng) was incubated with increasing amounts of His-stx-1A as

indicated. Bound proteins were visualized by Coomassie blue staining. The degradation band of GST-

DAPK-CT is marked with “*”.

Fig. 2. Regional and subcellular distribution of DAPK. A, Regional expression of DAPK in adult rat

brain. 20 µg of homogenates isolated from different regions of rat adult brain were analyzed by SDS-

PAGE and sequential immunoblotting on the same membrane with antibodies against DAPK and tubulin


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as indicated. B, Subcellular localization of DAPK in rat brain synaptosome fractions. 1 µg of rat brain

synaptosome fractions enriched in synaptosol (SS), synaptic vesicles (SV), and plasma membrane (PM)

were analyzed by SDS-PAGE and sequentially immunoblotted with antibodies as indicated.

Membranes were stripped and re-blotted between applications of each antibody. The subcellular

localization of DAPK was determined by comparison with markers for synaptic vesicles

(synaptophysin), plasma membrane (Na/K-ATPase), and synaptosol (lactate dehydrogenase, LDH). C,

Co-immunostaining of DAPK and stx1 in cultured hippocampal cells. Rat hippocampal neurons were

co-labeled with antibodies against DAPK (red) and stx1 (green) on DIV11. Syntaxin-1 staining was

detected in a punctate pattern along the plasma membrane surface of neuronal cell bodies and processes.

DAP-kinase staining was detected in the cytoplasmic space, but extended throughout neuronal

processes to the plasma membrane, where it partially co-localized with syntaxin-1.

Fig. 3. Ca2+/CaM-dependent phosphorylation of syntaxin-1A by DAPK in vitro. A, Phosphorylation

of stx-1A by DAPK is Ca2+/CaM-dependent. 50 pmol of purified stx1A-DTM was incubated with

γ32P-ATP and purified DAPK KD-CaM in a 25-µl reaction in 0, 0.1, 1, 10 or 100 µM free Ca2+ buffer.

Phosphorylation reactions were terminated by boiling in SDS sample buffer, the products separated by

SDS-PAGE, and the gels stained with Coomassie blue (lower panel), and dried and exposed to x-ray

film for autoradiography (upper panel). B, Serine-188 is a prominent DAPK-phosphorylation site in

stx1A. His-tagged stx1A-DTM or its S188A mutant (50 pmol each) were incubated with purified

DAPK catalytic domain (3 µg/ml) under conditions described in Methods, and the products separated by

10-20% SDS-PAGE; gels were then stained with Coomassie blue (left panel), dried and exposed to x-

ray film for autoradiography (right panel). (*:degradation product of stx1A-DTM). C and D,

Stoichiometry of syntaxin phosphorylation by DAPK. Stx1A-DTM (3.8 pmol) or MLC (10 pmol) was


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incubated with DAPK catalytic domain (KD)(3 µg/ml) for phosphorylation under conditions described

in Methods for the indicated time periods. Additional DAPK-KD was added every 60 min. Reactions

were terminated by the addition of SDS sample buffer and boiling, the products separated by SDS-

PAGE, and the gel exposed to film for 16 or 5 hours for stx1A-DTM or MLC, respectively. Gel slices

containing labeled stx1A or MLC were excised, scintillation levels were counted, and the moles of Pi

incorporated per mole of stx1A or MLC were calculated and plotted as a function of time.

Fig. 4. Ca2+-dependent phosphorylation of syntaxin-1A by DAPK in vivo. A, HA-tagged DAPK wild

type (WT) or its catalytically inactive K42A mutant expressed in HEK 293 T cells were immobilized on

protein A Sepharose beads with anti-HA antibody and then incubated with recombinant stx-CT (181-

264) in the presence of Ca2+, CaM and γ32P-ATP. Phosphorylation reactions were terminated by

boiling in SDS sample buffer, the products separated by SDS-PAGE, and the gels stained with

Coomassie blue (lower panel), dried and exposed to x-ray film for autoradiography (upper panel).

Equal loading of DAPK WT or K42A for the phosphorylation was verified by directly loading DAPK

immunoprecipitates on a 10-20% gradient gel followed by immunoblotting with anti-DAPK antibody

(data not shown). B, DAPK WT or K42A mutant were co-expressed with stx1A-FL in transfected

HEK 293 T cells, and expression was confirmed by immunoblotting with anti-stx1 or anti-DAPK

antibodies as indicated. C, Both DAPK WT and K42A mutant were co-immunoprecipitated with stx1A

from co-transfected HEK 293 T cells. Non-immune mouse IgG (Ms-IgG) was used as a control. D,

HEK 293 T cells co-transfected with stx1A and DAPK wild type (WT) or K42A mutant, as indicated,

were stimulated with ionomycin (10 µM) at RT for 30 minutes and then solubilized with 1% Triton X-

100. 2 mg total protein was incubated with an anti-stx1 antibody. Immunoprecipitates were then back-

phosphorylated in vitro with immobilized DAPK. The proteins were resolved on SDS-PAGE and


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stained with Coomassie blue (lower panel) followed by autoradiography (upper panel). E, Normalized

percentage of back-phosphorylation from unstimulated versus stimulated HEK 293 T cells. Bar,

mean±SEM. Back-phosphorylation of syntaxin-1A in lysates from stimulated cells co-transfected with DAPK

WT was decreased to 82.1±5.9% (P<0.05) of the unstimulated co-transfected control.

Fig. 5. Phosphorylation of syntaxin-1A decreases its binding to Munc18-1 but not to SNAP-25. A,

Phosphorylation of GST-stx-1A-TM (residues 2-264) by the DAP-kinase catalytic domain decreases

its binding to Munc18-1, but not to SNAP-25. 2 pmol of DAPK-phosphorylated (+P) or

unphosphorylated (-P) GST-stx1A-TM or GST alone were incubated with either His-tagged Munc18-

1 or SNAP-25 for 3 hours. The resulting complexes were separated by SDS-PAGE and sequentially

blotted with anti-stx-1 (lower panel) and anti-GST antibodies (upper panel). B, Binding signal

intensities were quantified using NIH Image analysis. The relative levels of binding of phosphorylated

or unphosphorylated GST-stx1-A-TM to Munc18-1 or SNAP 25 were calculated using a linear

standard curve of stx1-A-TM blots as a standard. Semi-quantitative analysis revealed that

phosphorylation of GST-stx-1A-TM by DAPK elicited a nearly 45 % (n=8, P<0.01) decrease in

binding to Munc18-1, but did not affect binding to SNAP-25 relative to that of unphosphorylated

GST-stx1A-TM.

Fig. 6. Mutation of serine-188 of syntaxin-1A to aspartic acid (S188D) decreases its binding to

Munc18-1 but not to SNAP-25. A, His-tagged syntaxin-1ATM (residues 2-264) wild type or S188D

mutant were incubated with immobilized GST-Munc18-1, GST-SNAP-25 or GST alone for 2 h at

4°C. The resulting complexes were separated by SDS-PAGE and blotted with anti-syntaxin-1 antibody

(lower panel). Membranes were then stripped and reprobed with anti-GST antibody (upper panel). B,


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Binding signal intensities were quantified using NIH Image. The relative binding levels of wild type

and S188D syntaxin-1A to GST-Munc18-1 or GST-SNAP-25 were calculated using a linear standard

curve of syntaxin-1A blot. Semi-quantitative analysis revealed that the S188D mutation elicited a

nearly 50% (n=5, P<0.05) decrease in binding to Munc18-1, but did not affect binding to SNAP-25

relative to that of wild-type syntaxin-1A.

Fig. 7. The S188D mutation of syntaxin-1A has no effect on SNARE complex assembly. A,

Recombinant SNARE core complex was constituted by incubation of GST-VAMP-2 (1 µg)-bound

glutathione Sepharose beads with purified His-tagged wild type or S188D mutant syntaxin-1A and

SNAP-25 (2 µg each) at 4°C for 2 hr. B, Native SNARE complexes were isolated from the lysates of

PC12 cells transfected with cDNA of wild-type or S188D mutant syntaxin-1A using anti-VAMP

antibody. Expression of endogenous SNARE components and exogenous syntaxin-1A was confirmed

by western blot analysis (left panel). After washing to remove unbound SNARE proteins, samples were

heated at temperatures as indicated, analyzed by SDS-PAGE, and immunoblotted with antibody against

syntaxin-1. Appearance at 70/75°C of an approximately 100 kDa band for the recombinant SNARE

complex, or of a band at 80 kDa for native SNARE complexes, and disappearance of these bands above

80°C are indicative of formation of SDS-resistant SNARE complex when blotted with anti-syntaxin

antibodies.


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Jin-Hua Tian, Sunit Das and Zu-Hang Shenginteraction with Munc-18

Ca2+-dependent phosphorylation of syntaxin-1A by DAP-kinase regulates its

published online May 2, 2003J. Biol. Chem.

10.1074/jbc.M300492200Access the most updated version of this article at doi:

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Ca2+-Dependent Phosphorylation of Syntaxin-1A by DAP-kinase