Analysis and Regulation of Vasodilator-stimulated Phosphoprotein
Serine 239 Phosphorylation in Vitro and in Intact Cells
Using a Phosphospecific Monoclonal Antibody*
Albert
Smolenski,
Christiane
Bachmann,
Kathrin
Reinhard,
Petra
Hönig-Liedl,
Thomas
Jarchau,
Heinz
Hoschuetzky
, and
Ulrich
Walter§
From the Medizinische Universitätsklinik, Institut für
Klinische Biochemie und Pathobiochemie, Josef-Schneider Strasse 2,
97080 Würzburg, and
nanoTools
Antikörpertechnik, Ferdinand Porsche Strasse 5/1,
D-79211 Denzlingen, Germany
 |
ABSTRACT |
The development and functional analysis of
a monoclonal antibody (16C2) are reported; the antibody recognizes
vasodilator-stimulated phosphoprotein (VASP; an established substrate
of both cAMP- and cGMP-dependent protein kinase) only when
serine 239 is phosphorylated. VASP serine 239 represents one of the
best characterized cGMP-dependent protein kinase
phosphorylation sites in vitro and in intact cells. Experiments with purified, recombinant human VASP and various VASP
constructs with mutated phosphorylation sites (S157A, S239A, T278A) and
experiments with intact cells (human/rat platelets and other cells)
treated with cyclic nucleotide-elevating agents demonstrated the
specificity of the monoclonal antibody 16C2. Quantitative analysis of
the VASP shift from 46 to 50 kDa (indicating VASP serine 157 phosphorylation) and the appearance of VASP detected by the 16C2
monoclonal antibody (VASP serine 239 phosphorylation) in human
platelets stimulated by selective protein kinase activators confirmed
that serine 239 is the VASP phosphorylation site preferred by
cGMP-dependent protein kinase in intact cells.
Immunofluorescence experiments with human platelets treated with cGMP
analogs showed that the 16C2 monoclonal antibody also detects VASP
serine 239 phosphorylation in situ at established
intracellular localization sites. Analysis of VASP serine 239 phosphorylation by the 16C2 antibody appears to be the best method
presently available to measure cGMP-dependent protein
kinase activation in intact cells. Also, the 16C2 antibody promises to
be an excellent tool for the evaluation of VASP function in intact
cells.
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INTRODUCTION |
Although cGMP-dependent protein kinases
(cGPKs)1 have
been recognized as important components of major signal transduction pathways (1-3), quantitative analysis of cGPK activation in intact cells has been very difficult (1-4). This is because of the relatively low expression of cGPK in most cell types compared with the relatively high expression of its closest functional homolog, the
cAMP-dependent protein kinase (cAPK), and the scarcity of
specific cGPK substrates. Unfortunately, the mediating role of cGPK for
a given effect/function is often implied or excluded by the use of cGPK
activators and/or inhibitors alone, which is clearly insufficient to
establish or rule out functional roles of cGPKs (1-4). One of the few
established cGPK substrates is the 46-kDa/50-kDa
vasodilator-stimulated
phosphoprotein (VASP), which was initially discovered and
characterized as a substrate of both cAPK and cGPK in human platelets
(5-8). VASP phosphorylation in response to cyclic
nucleotide-regulating vasodilators (i.e. cAMP-elevating
prostaglandins and cGMP-elevating nitric oxide donors) closely
correlates with platelet inhibition and in particular with the
inhibition of fibrinogen binding to the integrin
IIb
3 of human platelets (9-11).
Molecular cloning of human, canine, and mouse VASP predicted highly
homologous proteins and revealed a proline-rich protein that is
organized into three structural segments of different sequence
complexity (12, 13). VASP is the founding member of a new family of
proline-rich proteins, which includes Enabled (Ena), a
dose-dependent suppressor of Drosophila Abl- and
Disabled-dependent phenotypes, its mammalian
homolog Mena, and the Ena-VASP-like protein Evl (14-16). These
proteins all share an overall domain organization consisting of highly homologous NH2-terminal and COOH-terminal domains (Ena-VASP
homology domains 1 and 2, EVH1 and EVH2), which are separated by a
proline-rich central domain of low complexity (12-16). In platelets
and many other cells including vascular smooth muscle cells,
endothelial cells, and fibroblasts, VASP has been found to be
associated with stress fibers, focal adhesions, cell-cell contacts, and
highly dynamic membrane regions (16, 17). VASP colocalizes with
profilins and binds directly to their poly(L-proline)
binding site (18), binds to and colocalizes with zyxin and vinculin
(16, 19), and also directly binds to Listeria monocytogenes
surface protein ActA, which is essential for the actin
polymerization-based intracellular motility of this pathogen (20).
Functional evidence indicates that VASP is a crucial factor involved in
the enhancement of spatially confined actin filament formation (16, 20,
21).
Three distinct phosphorylation sites were biochemically identified in
VASP (serine 157, serine 239, and threonine 278) which are used
in vitro and in intact human platelets by both cAPK and cGPK
and by the serine/threonine protein phosphatases 2A and 2B with
overlapping selectivity (8, 22). Phosphorylation of serine 157, the
site preferred by the cAPK, leads to a marked shift in apparent
molecular mass of VASP in SDS-PAGE from 46 kDa to 50 kDa (6, 8). In
experiments with well characterized protein kinase activators, analysis
of the shift in the apparent molecular mass of VASP from 46 to 50 kDa
in SDS-PAGE has been helpful in analyzing the preferential activation
of both cAPK and cGPK in intact cells (3, 7-9). However, as already
indicated, this shift is caused by the phosphorylation at serine 157, a
VASP phosphorylation site used by both cGPK and cAPK but actually
preferred by cAPK. In contrast, VASP phosphorylation at serine 239, a
sequence representing one of the best in vitro
cGPK-preferred phosphorylation sites (8), has been very difficult to
assess in intact cells. Here, we describe the development and
experimental use of a monoclonal antibody that recognizes VASP only
when serine 239 is phosphorylated. The data suggest that this
monoclonal antibody allows the quantitative assessment of cGPK
activation in intact cells, at least for those systems containing both
cGPK and VASP. In addition, the antibody allows the detection of the
serine 239-phosphorylated form of VASP in distinct subcellular
sites.
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EXPERIMENTAL PROCEDURES |
Materials--
All standard chemicals were obtained from Merck
and Sigma. Restriction enzymes were from New England Biolabs and
oligonucleotides from Amersham Pharmacia Biotech.
Generation of a Monoclonal Antibody Recognizing VASP
Phosphorylated at Position Ser239--
Dephospho- and
phosphopeptides (RKVS(239)KQE or the RKVpS(239)KQE) representing the
VASP serine 239 phosphorylation site (8, 12) were synthesized on an
Applied Biosystems model 431A peptide synthesizer using Fmoc chemistry.
Phosphoserine was incorporated during peptide synthesis using
Fmoc-Ser(PO(OBzl)OH)-OH (Calbiochem). Peptides were purified to >98%
purity by reversed phase chromatography using a Vydac 18TP column. The
peptides were conjugated to thiolated keyhole limpet hemocyanin
(nanoTools) after activation with bromoacetic acid-N-hydroxysuccinimide ester (Sigma). Female BALB/c mice
(6 weeks old) were immunized subcutaneously with the keyhole limpet hemocyanin phosphopeptide immunogen (10 µg/animal and injection) four
times at 14-day intervals with the first injection in complete Freund's adjuvant and the following injections in incomplete Freund's adjuvant. 2 weeks later, animals were given booster injections (10 µg
of immunogen in PBS) on 3 consecutive days. 1 day after the last
booster injection, animals were killed, and their spleens were excised.
Spleen cells were fused with nonproducer myeloma cells. Hybridoma cell
lines secreting antibodies specific for phosphorylated VASP were
identified initially by differential screening using phosphorylated and
nonphosphorylated peptides covalently bound to enzyme-linked
immunosorbent assay plates (DNA-BIND, Costar). Subsequently,
immobilized recombinant His6-VASP or His6-VASP phosphorylated in vitro by cGPK was used. Monoclonal
antibodies were purified from serum-free cell culture supernatants by
thiophilic adsorption chromatography (thiophilic support based on POROS
50-OH, nanoTools).
Construction of VASP Mutants--
pVSV-VASP is based on the
expression vector pcDNA3 (Invitrogen) and encodes human VASP (12)
NH2-terminally tagged with an epitope of vesicular
stomatitis virus (VSV) glycoprotein G (23). The tag (amino acids
YTDIEMNRLGK, preceded by methionine) was fused via a proline to the
second amino acid of VASP. In this vector, site-directed mutagenesis of
the phosphorylation sites (serines 157 and 239, threonine 278) to
alanines was performed using splice overlap extension polymerase chain
reaction. For the mutation of serine 157 to alanine (S157A), the
sequence of the mutagenic primer was 5'-GCGCCGGGTCgCgAATGCAGGAGGC-3'
(the mismatches with the wild-type VASP sequence are printed in
lowercase). Together with the corresponding reverse primer and primers
from the NH2- or the COOH-terminal region of VASP a mutant
VASP fragment was amplified and cloned back into the VASP plasmid
pVSV-VASP using unique restriction sites. The sequence of the mutagenic primer for the S239A mutant was 5'-CAAACTCAGGAAAGTCgcgAAGCAGGAGGAGG-3' and for the T278A mutant, 5'-GGAGAAGGAAAGCtgCGCAAGTTGGGG-3'. All mutations and the polymerase chain reaction-amplified fragments were
checked by sequencing.
Purification of His6-VASP--
The
Escherichia. coli strain BL21 (DE3) pREP4pQEVASP1 was used
for the expression of biologically active VASP as hexahistidine fusion
protein of 420 amino acids having a 30-amino acid spacer between the
purification tag and the authentic VASP NH2 terminus. Bacterial cell pellets (250-ml cultures) corresponding to a mass of one
A600 × liter were suspended in 20 ml of
ice-cold 50 mM sodium phosphate buffer (pH 8.0) containing
0.5 mM EDTA, 5% (v/v) glycerol, 50 mM NaCl, 2 µg/ml leupeptin, 20 units/ml aprotinin, 5 mM benzamidine,
1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme;
they were incubated on ice for 30 min and lysed by ultrasonic disintegration in the presence of 5 µg/ml DNase I and 10 µg/ml RNase. The efficiency of lysis was monitored by phase-contrast microscopy. After adding 1 M imidazole buffer (pH 8.0) to a
final concentration of 50 mM and adding Triton X-100 to
0.5% the lysate was extracted by stirring on ice for 20 min. The
supernatant after centrifugation at 16,500 × g for 10 min was adjusted in NaCl to a final concentration of 300 mM, recentrifuged, and added to a settled bed volume of
1.25 ml of 50% nickel-nitrilotriacetic acid-agarose (Qiagen;
equilibrated previously in 50 mM sodium phosphate buffer (pH 8.0), containing 5% (v/v) glycerol, 300 mM NaCl, 50 mM imidazole buffer (pH 8.0))/1 A600 × liter of lysed cell mass. After overnight incubation at 4 °C with
shaking, the agarose beads were collected by centrifugation at
2,000 × g for 10 min. The loaded
nickel-nitrilotriacetic acid-agarose corresponding to a bed volume of
1.25 ml of a 50% slurry was then washed five times with 6 ml each of
ice cold 50 mM sodium phosphate buffer (pH 8.0) containing
300 mM NaCl, 50 mM imidazole buffer (pH 8.0),
0.5% (v/v) Triton X-100, including protease inhibitors. The beads were
carefully drained dry and then batch eluted with 50 mM
sodium phosphate buffer (pH 7.0) with 300 mM NaCl, 100 mM EDTA in three steps each of a 10-min incubation at room
temperature in a total elution volume of 1.25 ml. The eluate was then
dialyzed in the cold against 1 liter of 25 mM HEPES buffer
(pH 7.0) with 75 mM NaCl, 5% (v/v) glycerol, and 1 mM dithiothreitol for 24 h with three buffer changes.
The sample was finally concentrated.
Phosphorylation of Recombinant VASP by cAPK and cGPK--
cGPK
and the catalytic subunit of cAPK were purified as described (24, 25).
Purified recombinant His6-VASP (25 µg/ml) was
phosphorylated by a purified catalytic subunit of cAPK (11 µg/ml) or
purified cGPK (24 µg/ml) at 30 °C in a 10 mM HEPES
buffer (pH 7.4) containing 5 mM MgCl2, 1 mM dithioerythritol, 0.2 mM EDTA, 100 µM ATP, and (only with cGPK phosphorylation conditions) 20 µM cGMP. Aliquots were taken at various time points,
mixed immediately with an SDS-containing stop solution (66.6 mM Tris-HCl buffer (pH 6.7) containing 2% SDS, 5%
glycerol, 10%
-mercaptoethanol, and a trace of bromphenol blue),
boiled, and separated on 9% SDS-polyacrylamide gels. Gels were stained
with Coomassie Blue or immunoblotted as described below.
Transfection of PtK2 Cells--
PtK2 cells were grown in minimal
essential medium with 10% fetal calf serum (Life Technologies, Inc.)
until they were about 50% confluent. Then plasmids containing the
cDNA of epitope-tagged wild-type and mutated VASP under the control
of a cytomegalovirus promoter (pVSV-VASP) were cotransfected together
with an expression vector for cGPK I
as described (26) using the
calcium phosphate precipitation method. Two days after transfection,
the cells were incubated with 30 µM 8-pCPT-cGMP for 30 min at 37 °C to activate the cGPK I
and stimulate VASP
phosphorylation. Then, cells were washed and harvested immediately for
immunoblotting.
Isolation and Washing of Platelets--
Venous blood from human
volunteer donors was mixed with citrate buffer (20 mM
sodium citrate, 1.4 mM citric acid, 28 mM
glucose (pH 6.7)), centrifuged for 20 min at 18 °C and 200 × g. The platelet-rich plasma (PRP) was removed, and apyrase
(0.1 unit/ml) was added. The PRP was centrifuged for 10 min at 18 °C
and 500 × g, the platelet pellet was resuspended in
resuspension buffer (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM HEPES (pH 7.4), 10 mM glucose, 5 mM NaOH), and cells were adjusted
to a final concentration of 0.7 × 109/ml. Rat
platelets were prepared essentially as described for the human
platelets with minor modifications: male Sprague-Dawley rats were
anesthetized, blood was taken from the vena cava, mixed with citrate
buffer (18 mM citrate), and centrifuged for 10 min at
250 × g to obtain the rat PRP.
Analysis of VASP Phosphorylation in Washed Platelets--
Washed
platelets (0.7 × 109 cells/ml) were incubated with
prostacyclin (prostaglandin (PG) I2; Wellcome, Beckenham,
U. K.), PGE1, sodium nitroprusside (SNP) (both from
Sigma), 8-pCPT-cGMP or 5,6-DCL-cBIMPS (Biolog, Bremen, Germany) at
37 °C. Aliquots (2.8 × 107 cells) were taken at
various time points, mixed immediately with an SDS-containing stop
solution, boiled and subsequently analyzed.
Immunoblotting--
Samples were separated on 9%
SDS-polyacrylamide gels. Proteins were then blotted onto nitrocellulose
filters and labeled with the VASP antiserum M4 (1:1,500, v/v) as
described (9) followed by 125I-protein A (ICN) for
radiolabeling or peroxidase-labeled anti-rabbit antibody detection by
ECL (Amersham Pharmacia Biotech) or with the monoclonal antibody
prepared against VASP phosphorylated at position Ser239
(16C2, 0.5 µg/ml) followed by 125I-labeled sheep
anti-mouse antibody or peroxidase-labeled anti-mouse antibody. Figs. 4
and 7 are autoradiograms of 125I-labeled blots; Figs. 2 and
3 show ECL-labeled blots.
Immunofluorescence of Human Platelets--
PRP was diluted 1:10
in PBS, and platelets were allowed to spread on glass coverslips for 45 min at room temperature as described (17). Then the cells were
incubated with PBS alone or with 100 µM 8-pCPT-cGMP in
PBS for 15 min at room temperature. The cells were washed once with PBS
and fixed immediately with 4% (w/v) paraformaldehyde in PBS for 15 min
at 4 °C, washed once with PBS, and subsequently permeabilized for 10 min with 0.2% (v/v) Triton X-100 in PBS at room temperature. The
samples were then incubated with the M4 VASP antiserum (1:1,000; v/v)
and with the antibody 16C2 (3.3 µg/ml) for 1 h at 37 °C
followed by labeling with fluorescein isothiocyanate-anti-rabbit
antibody (Sigma) and Texas Red-anti-mouse antibody (Molecular Probes)
for 1 h at 37 °C. Photographs were taken with a Leitz
Aristoplan microscope on Kodak Tri-X-400 film.
 |
RESULTS AND DISCUSSION |
Three phosphorylation sites have been identified previously in
VASP (serine 157, serine 239, and threonine 278) which are phosphorylated by both cAPK and cGPK in vitro and in intact
cells with overlapping specificity. As shown in Fig.
1, the serine 157 and serine 239 VASP
phosphorylation sites are preferred by cAPK and cGPK, respectively.
However, with respect to VASP phosphorylation in intact cells, only
serine 157 phosphorylation could be monitored effectively by analyzing
the electrophoretic shift of VASP from the 46- to the 50-kDa species in
SDS-PAGE (6-9). To obtain a possible tool for the detection of
VASP Ser239 phosphorylation and thereby also a
possible sensitive marker for cGPK activation in intact cells, a
monoclonal antibody (16C2) against a phosphorylated peptide
(RKVpS(239)KQE)) representing the VASP Ser239
phosphorylation site was developed and purified as outlined under "Experimental Procedures."

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Fig. 1.
VASP structure and phosphorylation
sites. VASP contains three phosphorylation sites specific for the
Ser/Thr cAMP- or cGMP-dependent protein kinases (cAPK,
cGPK). The sites are located within the proline-rich region
(PRR) of VASP (serine 157) or within the Ena-VASP homology
domain 2 (EVH2) (serine 239, threonine 278). Data indicate
that Ser157 is phosphorylated preferentially by cAPK,
whereas Ser239 is the cGPK preferred site (8).
Phosphorylation of Ser157, but not of Ser239 or
Thr278, alters the electrophoretic mobility of VASP in
SDS-PAGE, resulting in an apparent shift of the molecular mass from 46 to 50 kDa.
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The specificity of this 16C2 antibody was tested by analyzing the time
course of the cGPK- and cAPK-catalyzed phosphorylation of purified
recombinant VASP (expressed as hexahistidine-tagged protein in E. coli). Coomassie Blue staining revealed an initial shift/complete
shift of VASP from 46 to 50 kDa (indicating Ser157
phosphorylation) after a 0.3-min/5-min incubation time with cAPK versus 0.6 min/15 min with cGPK, which was also observed by
immunoblot analysis using the polyclonal antibody (M4) against VASP
(Fig. 2). In contrast, the newly
developed monoclonal antibody 16C2 did not recognize VASP at all as
unphosphorylated protein (0-min aliquot in Fig. 2), but a strong signal
appeared after VASP had been incubated with cAPK and cGPK using
phosphorylation conditions (Fig. 2). However, a striking time course
difference with respect to the appearance of a 16C2-recognized VASP
signal was observed. With cGPK, a clear signal was detected at the
earliest time point tested (0.3 min) which reached its maximum at about
the 5-min incubation time with no apparent VASP shift (46-Da
50-kDa
form) at 0.3 min. With cAPK, a 16C2-recognized signal only appeared after 15 min and increased thereafter, whereas the VASP shift was
complete within 5 min of the incubation time (Fig. 2). These data
demonstrate that the 16C2 monoclonal antibody does not recognize the
phosphorylation site responsible for the VASP mobility shift in
SDS-PAGE (serine 157) and indicate that the 16C2-recognized phosphorylation site (serine 239) is preferred by the cGPK in vitro in agreement with our published data (6-9). Further
evidence was sought to demonstrate the specificity of the 16C2 for the phosphorylated VASP sequence around serine 239. Therefore, VASP mutants
lacking each of the three phosphorylation sites (mutants S157A, S239A,
T278A) were constructed. Together with cGPK, these constructs and
wild-type VASP were expressed in PtK2 cells, which contain very little
if any endogenous cGPK (data not shown). Incubation of these
transfected PtK2 cells with 30 µM 8-pCPT-cGMP resulted in
the phosphorylation of VASP as indicated by the appearance of the
50-kDa VASP form and a 16C2-recognized signal (Fig.
3, wild type; first lane).
Without 8-pCPT-cGMP treatment, the M4 VASP antiserum and the 16C2
antibody detected in extracts of these cells only the 46-kDa form of
VASP or no signal at all, respectively (data not shown). Mutation S157A
prevented the stimulus-induced shift of VASP from 46 to 50 kDa but not
the appearance of a 16C2-recognized VASP signal that had the mobility
of the 46-kDa species (Fig. 3). Mutation S239A abolished any reactivity
of the 16C2 antibody with VASP but did not prevent the stimulus-evoked
shift of VASP from the 46- to the 50-kDa species (Fig. 3). Mutation
T278A had no effect on the stimulus-induced VASP shift (serine 157 phosphorylation) or the appearance of a 16C2-recognized signal (serine
239 phosphorylation). These data confirm our previous conclusions
(6-9) that serine 157 (but not serine 239) is responsible for the
phosphorylation-induced VASP mobility shift in SDS-PAGE (46
50 kDa)
and demonstrate that the VASP immunoreactivity recognized by the
monoclonal antibody 16C2 requires VASP serine 239 phosphorylation.
These data also suggest that the three point mutations analyzed (S157A,
S239A, T278A) have no gross effects on the overall VASP protein because the properties and functions of the other phosphorylation sites remained essentially unchanged.

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Fig. 2.
Analysis of cAPK- and cGPK-catalyzed
phosphorylation of recombinant VASP by the polyclonal antibody M4 and
the monoclonal antibody 16C2. Purified recombinant
hexahistidine-tagged VASP (25 µg/ml) was phosphorylated by purified
catalytic subunit of cAPK (11 µg/ml; left panels) or by
purified cGPK (24 µg/ml; right panels). Aliquots were
removed at the time points indicated, mixed immediately with an
SDS-containing stop solution, and boiled. VASP phosphorylation was
analyzed by Coomassie Blue staining (Coom., upper
panel) and by immunoblots with either the polyclonal VASP antibody
M4 (middle panel) or with the monoclonal antibody (16C2)
prepared against the phosphorylated VASP phosphorylation site 239 (lower panel). The shift in the electrophoretic mobility of
VASP from a 46- to a 50-kDa form (as detected by Coomassie Blue
staining and immunoblots) indicates the phosphorylation of VASP at
position Ser157. The bands below or above the 46/50-kDa
VASP in the Coomassie Blue-stained lanes represent the added catalytic
subunit of the cAPK (40 kDa) or cGPK (74 kDa), respectively.
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Fig. 3.
Phosphorylation of VASP and VASP mutants in
transfected PtK2 cells. Wild-type (wt) VASP and VASP
mutants with altered phosphorylation sites (S157A, S239A, T278A) were
cotransfected with cGPK I into PtK2 cells, which contain very low
amounts of endogenous VASP. 2 days after transfection, cells were
incubated with 30 µM 8-pCPT-cGMP for 30 min and then
lysed immediately with an SDS-containing stop solution and boiled.
Aliquots of the lysates were analyzed by immunoblots using a polyclonal
antiserum against native VASP (M4) or a monoclonal antibody (16C2)
prepared against the (phosphorylated) VASP phosphorylation site
Ser239.
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The experiments with purified proteins and proteins/protein mutants
overexpressed in PtK2 cells already indicated that the VASP shift (46 kDa
50 kDa) and 16C2 immunoreactivity represent VASP serine 157 and
239 phosphorylation, respectively, and that the 16C2 antibody may also
be used to detect VASP serine 239 phosphorylation and cGPK activity in
intact cells. The latter issue was addressed more systematically in
VASP phosphorylation studies with intact human platelets. Established
conditions (27, 28) also confirmed by experiments with cGPK-deficient
human platelets (29) for potent and specific activation of either
platelet cAPK (incubation with 1 µM prostacyclin or 0.5 mM 5, 6-DCl-cBIMPS) or cGPK (10 µM sodium
nitroprusside or 1.0 mM 8-pCPT-cGMP) were chosen. In extracts of washed, unstimulated human platelets the M4 VASP antiserum detected only the 46-kDa VASP species, whereas the 16C2 antibody detected no specific signal at all (Fig.
4). These data confirm that VASP serine
157 is not phosphorylated in basal, unstimulated human platelets (7-9)
and demonstrate this for the first time also with respect to VASP
serine 239. PGI2 (1 µM) and SNP (10 µM) induced transient and reversible phosphorylation of
VASP at serine 157 (shift analysis) and serine 239 (16C2
immunoreactivity) as shown in Figs. 4 and
5. The reversibility of VASP
phosphorylation was particularly apparent with PGI2 most
likely because of the short half-life of PGI2 in solution.
With SNP, however, the stimulated Ser239 phosphorylation
declined significantly already after 10 min and thereafter, whereas
Ser157 remained elevated, perhaps indicating differential
dephosphorylation of VASP serines 157 and 239. Incubations of intact
human platelets with the membrane-permeable cAPK and cGPK activators,
5,6-DCl-cBIMPS and 8-pCPT-cGMP, respectively, produced the expected and
essentially irreversible extent of VASP serine 157 (shift analysis)
phosphorylation and serine 239 (16C2 immunoreactivity) phosphorylation.
The results shown in Figs. 4 and 5 indicate that activation of either
cAPK or cGPK results in the phosphorylation of VASP serines 157 and 239. In intact human platelets, cAPK-mediated phosphorylation of VASP
serines 157 and 239 is very similar with respect to extent and time
course, although serine 157 phosphorylation appears to precede serine
239 phosphorylation (Figs. 4 and 5). Activation of cGPK in intact human
platelets by SNP or 8-pCPT-cGMP produces the maximal extent of VASP
serine 239 phosphorylation but only 50% of VASP serine 157 phosphorylation compared with the cAPK effect (Figs. 4 and 5). In all
experiments performed, i.e. extracts of untreated human
platelets or platelets/cells treated with cAPK or cGPK activators, the
monoclonal antibody 16C2 did not specifically label or detect any
protein other than VASP (see Figs. 3, 4, and 7; other data not
shown).

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Fig. 4.
Analysis of VASP phosphorylation in intact
human platelets by the polyclonal antibody M4 and the monoclonal
antibody 16C2. Washed human platelets (0.7 × 109
cells/ml) were incubated with 1 µM PGI2, 0.5 mM 5,6-DCl-cBIMPS, 10 µM SNP, or 1 mM 8-pCPT-cGMP. Aliquots (2.8 × 107
cells) were removed at the time points indicated, mixed immediately
with an SDS-containing stop solution, and boiled. VASP phosphorylation
was analyzed by immunoblots using either the polyclonal VASP antibody
(M4) or the monoclonal antibody (16C2) prepared against the
(phosphorylated) VASP phosphorylation site Ser239.
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Fig. 5.
Time course of the effects of
PGI2, SNP, 5,6-DCl-cBIMPS, and 8-pCPT-cGMP on
VASP serine 157 and serine 239 phosphorylation. Washed human
platelets were incubated with 1 µM PGI2
(A), 0.5 mM 5,6-DCl-cBIMPS (B), 10 µM SNP (A), or 1 mM 8-pCPT-cGMP
(B) and processed as described in the legend to Fig. 4.
Immunoblots were performed with 125I-protein A, and labeled
bands were cut out from the nitrocellulose filters and counted in a
-counter. Phosphorylation of VASP at position Ser157
(closed boxes) was quantitated as described previously using
the polyclonal antibody (M4) and is expressed as amount of VASP
phosphorylated at serine 157 (50-kDa protein) as percent of total VASP
(46-kDa + 50-kDa protein). Phosphorylation of VASP at position
Ser239 (open circles) was analyzed using the
monoclonal antibody (16C2) prepared against the (phosphorylated) VASP
phosphorylation site Ser239. The serine 239 phosphorylation
(the sum of 46-kDa and 50-kDa labeled bands by the 16C2 antibody) of
each condition is expressed as percent of maximally detectable serine
239-phosphorylated VASP for each stimulus tested (the relatively
highest amount of Ser239 phosphorylation was obtained after
a 60-min incubation with 1 mM 8-pCPT-cGMP; the relative
order of potency of the other stimuli was: PGI2 > SNP > 5,6-DCl-cBIMPS). The data shown represent the means (±S.E.) of two
separate experiments.
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The results obtained with the 16C2 antibody presented in Figs. 2-5
suggested the use of this antibody to detect and analyze VASP serine
239 phosphorylation in situ, i.e. at the sites of the cellular localization of VASP. Previous immunofluorescence studies
with human platelets spread on glass showed VASP to be localized on the
rim of the lamella and associated primarily with radial microfilament
bundles representing the contact sites of the platelets with the
extracellular matrix (17; see also Fig. 6, A and B). Now,
the localization of VASP phosphorylated at serine 239 protein was
investigated using the 16C2 monoclonal antibody. In untreated
platelets, no specific signal was detected by the 16C2 antibody (Fig.
6C), whereas a signal overlapping the one detected by the M4
antiserum clearly appeared once the glass-attached human platelets had
been incubated with the cGPK activator 8-pCPT-cGMP (Fig. 6,
B and D). The staining of VASP with the 16C2
antibody almost completely overlapped the staining of the polyclonal
antibody M4 in double labeling experiments (not shown). The only
difference observed was a somewhat weaker and less complete staining of
the radial microfilaments by the phospho-VASP antibody compared with the staining with the polyclonal M4 antiserum. Also, the VASP signals
detected by the 16C2 antibody by Western blot analysis (Figs. 2, 3, 4,
and 7) or immunofluorescence analysis
were completely prevented if the 16C2 monoclonal antibody was
preincubated with the phosphopeptide RKVpS(239)KQE (data not
shown).

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Fig. 6.
Immunofluorescence analysis of VASP and VASP
phosphorylation in human platelets. Human platelets (PRP) were
allowed to spread on glass for 45 min followed by an incubation for 15 min without (panels A and C) or with
100 µM 8-pCPT-cGMP (panels B and
D). Then, the cells were immediately fixed and
double-labeled as described under "Experimental Procedures."
Panels A and B show the immunofluorescence
staining obtained with the polyclonal VASP antiserum (M4), panels
C and D the staining obtained with the monoclonal
antibody (16C2) prepared against the VASP phosphorylation site
Ser239 (bar = 10 µM).
|
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Fig. 7.
Analysis of VASP phosphorylation in rat
platelets. Rat platelets (0.7 × 109 cells/ml of
PRP) were incubated with 10 µM PGE1 or with
100 µM SNP. Aliquots were taken at the times indicated
and centrifuged, the cell pellet was lysed immediately in an
SDS-containing stop solution and boiled. VASP phosphorylation was
analyzed by immunoblots using either the polyclonal VASP antiserum (M4)
or the monoclonal antibody (16C2) prepared against the (phosphorylated)
VASP phosphorylation site Ser239.
|
|
The results presented indicate that monoclonal antibody 16C2 detects
the phosphorylation of VASP at position serine 239 in human cells.
Considering the fact that the sequence representing the serine 239 phosphorylation site is highly conserved among human, canine, and
rodent VASP (12, 13) we predicted that the 16C2 antibody also detects
VASP serine 239 phosphorylation in non-human cells. This was indeed
demonstrated by experiments with intact rat platelets (Fig. 7).
Treatment of these cells with SNP and PGE1 resulted in the
phosphorylation of VASP at positions serine 157 (shift analysis) and
serine 239 (appearance of a 16C2-recognized signal) as shown in Fig. 7.
The extent and regulation of rat VASP phosphorylation were similar to
the effects seen with human platelets, and, importantly, the 16C2
antibody did not specifically recognize any rat platelet protein other
than phosphorylated VASP.
 |
Conclusions |
The results of this study demonstrate that monoclonal antibody
16C2 is able to detect and quantitate VASP serine 239 phosphorylation with purified proteins and, most importantly, in crude cell extracts (Western blots) and in fixed, permeabilized cells (immunofluorescence analysis). The results demonstrate for the first time that serine 239 of VASP is essentially dephosphorylated in basal, untreated human
platelets and other cell types and rapidly and reversibly phosphorylated when the cGPK or cAPK are activated. Although VASP serines 157 and 239 are the phosphorylation sites preferred by the cAPK
and cGPK, respectively, using purified proteins (8, this study) and
intact cells (this study) cGPK as well as cAPK clearly use both VASP
phosphorylation sites in intact human platelets. This overlapping
phosphorylation may be caused by the particularly high concentration of
VASP, cGPK, and cAPK known to be present in human cells (7) because a
more selective cAPK-mediated VASP serine 157 phosphorylation and
cGPK-mediated VASP serine 239 phosphorylation were observed in other
human cell types (data not shown). Both VASP serine 157 and 239 phosphorylation can be used as parameters indicating the activation of
cAPK, cGPK, or both in intact cells. In experiments with
membrane-permeable cGMP analogs and selective activators of the cGPK
(i.e. 8-pCPT-cGMP, used at concentrations that do not
activate the cAPK) the analysis of cGMP-stimulated VASP serine 239 phosphorylation allows the sensitive detection and quantitation of cGPK
activation in intact cells. In fact, we observed (data not shown) that
the 8-pCPT-cGMP-induced VASP serine 239 phosphorylation detected in
cultured human umbilical vein endothelial cells that do not contain
endogenous cGPK (30) indicates a contamination of those cells with a
small number of fibroblasts, which are known to contain both VASP and
cGPK (17)). Also, the decline of 8-pCPT-cGMP-induced VASP serine 239 phosphorylation in primary cell cultures and cGPK-transfected cells
indicates the loss of endogenous or transfected cGPK (30; other data
not shown). Because VASP is expressed in most if not all mammalian cells, albeit at different cellular concentrations (16, 17), the
analysis of VASP serine 239 phosphorylation can be performed with most
mammalian cells. However, VASP and/or the VASP serine 239 phosphorylation site can also be introduced into cells containing very
low amounts of endogenous VASP (see Fig. 3).
Previously, the analysis of cGPK activation in intact cells has been
very difficult because of the biochemical properties and the relatively
low expression of this kinase in most cell types (1-3, 31). Sensitive
methods for the analysis of cGPK activity in intact cells are very much
needed. Unfortunately, the mediating role of cGPK for a given
physiological function/effect is often implied or excluded by the use
of cGPK inhibitors claimed to be specific, which is insufficient to
establish or rule out functional roles of cGPKs in intact cells. For
example, the compound KT 5823 (an indole carbazole and potent inhibitor
of the cGPK in vitro (32)) has never been evaluated with
respect to cGPK inhibition in intact cells despite its widespread
pharmcological use. In our hands, KT 5823 inhibited purified cGPK (as
expected) but potentiated 8-pCPT-cGMP-induced VASP serine 239 phosphorylation in intact human
platelets,2 indicating that
cGPK inhibition by this compound in intact cells is not established.
Perhaps the analysis of cGMP-induced VASP serine 239 phosphorylation
will be helpful to search for more potent and selective cGPK activators
and inhibitors useful for intact cell studies.
Finally, the 16C2 monoclonal antibody allows the detection of VASP
serine 239 phosphorylation at sites of the endogenous, intracellular
VASP localization (i.e. focal contacts, cell-cell contacts,
microfilaments) as demonstrated in Fig. 6. Of particular interest would
be the analysis of VASP phosphorylation at sites of accelerated actin
polymerization, i.e. at the leading edge of lamellipodia and
in the tails of L. monocytogenes (16, 17, 20, 21). Although
the precise functional role(s) of the VASP phosphorylation sites remain
to be elucidated, increasing evidence, also obtained with the
VASP-related Drosophila protein Ena, suggests that
phosphorylation of Ena-VASP family members alters the interaction of
these proteins with their binding partners and their in vivo function (11, 15, 16, 33).3
Hopefully, the 16C2 monoclonal VASP antibody will be a useful tool to
analyze cGPK activation and VASP serine 239 phosphorylation in intact
cells and thereby help to define the physiological function of these
two proteins in intact cells.
 |
ACKNOWLEDGEMENTS |
We thank Drs. M. Eigenthaler and S. Lohmann
for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the Interdisziplinäres
Klinisches Forschungszentrum Würzburg and by the Deutsche
Forschungsgemeinschaft.The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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: Institut für
Klinische Biochemie und Pathobiochemie, Josef-Schneider Strasse 2, D-97080 Würzburg, Germany. Tel.: 49-931-201-5144/5479; Fax: 49-931-201-3153. E-mail: uwalter{at}klin-biochem.uni-wuerzburg.de.
The abbreviations used are:
cGPK(s), cGMP-dependent protein kinase(s); cAPK, cAMP-dependent protein kinase; VASP, vasodilator-stimulated
phosphoprotein; PAGE, polyacrylamide gel electrophoresis; Fmoc, N-(9-fluorenyl)methoxycarbonylPBS, phosphate-buffered
salineVSV, vesicular stomatitis virusPRP, platelet-rich plasmaPGI2, prostaglandin I2 (prostacyclin)PGE2, prostaglandin E2SNP, sodium
nitroprusside8-pCPT-cGMP, 8-(4-chlorophenylthio)-cGMP5, 6DCL-cBIMPS,
Sp-5,6-dichloro-1-
-D-ribofuranosylbenzimidazole-3',5'- monophosphorothioate.
2
A. M. Burkhardt, U. Walters, A. Smolenski, and
S. M. Lohmann, unpublished data.
3
A. Smolenski, C. Bachmann, W. Poller, U. Walter,
and S. M. Lohmann, manuscript in preparation.
 |
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