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INTRODUCTION |
The activation of human platelets and vessel wall-platelet
interactions are processes tightly regulated under physiological conditions and often impaired in thrombosis, arteriosclerosis, hypertension, and diabetes. Agonists such as thrombin, thromboxane, vasopressin, and ADP activate platelets and cause shape change, aggregation, and degranulation. Platelet activation is inhibited by a
variety of agents, including aspirin and Ca2+ antagonists
as well as cGMP- and cAMP-elevating agents such as NO and prostaglandin
I2, respectively (for review, see Ref. 1). The inhibitory
effects of cGMP and cAMP are principally mediated by cGMP- and
cAMP-dependent protein kinases (cGK and cAK, respectively), with some cross-talk existing between the two systems. For example, cGMP stimulates the hydrolysis of cAMP via cGMP-regulated
phosphodiesterases (2, 3). The molecular mechanisms of platelet
inhibition by cGMP signaling distal to cGK activation are only
partially understood (4). Studies using cGK-deficient mice demonstrated defective cGMP-mediated inhibition of platelet aggregation (5). Several
proteins have been reported to be phosphorylated in response to cGK
activation either in vitro or in intact cells, including cGMP-specific phosphodiesterase (6), myosin light chain kinase (7), the
inositol 1,4,5-trisphosphate receptor (8), an inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (9),
G-substrate (10), Na+/K+-ATPase (11), and
endothelial NO synthase (Ref. 12, for review, see Ref. 13). None of
these proteins, however, could be established as a downstream effector
of cGK in platelets. Recently, it was assumed that at least part of the
inhibitory response mediated by cGK depends on the phosphorylation of
the thromboxane receptor (14). These experiments, however, were
performed using HEL cells. The only known in vivo substrates
of cGK involved in platelet inhibition are the vasodilator-stimulated
phosphoprotein VASP, associated with focal adhesion (15), and the small
GTP-binding protein rap 1b (16).
To identify additional intracellular targets for cGK, we used
two-dimensional gel electrophoresis of radiolabeled human platelets in
combination with nano-electrospray ionization mass spectrometry (nano-ESI-MS). By applying this method, we identified heat shock protein 27 (Hsp27)1 as a
substrate of cGK I in intact platelets. Additionally, we suggest that
phosphorylation of Hsp27 may contribute to the inhibitory actions of
cGMP by regulating actin polymerization.
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EXPERIMENTAL PROCEDURES |
Materials--
Urea (ultra pure), IPG strips,
[
-32P]ATP, and ECL detection kit were purchased from
Amersham Pharmacia Biotech (Braunschweig, Germany), QuickChange
Site-directed Mutagenesis Kit was from Stratagene (La Jolla, CA),
trypsin was from Promega (Heidelberg, Germany), 8-pCPT-cGMP
was from BioLog (Bremen, Germany), goat anti-rabbit IgG, goat
anti-mouse IgG, and nonfat dry milk were from Bio-Rad (München,
Germany), p38 antibody was from New England Biolabs (Schwalbach,
Germany), Hsp27 human polyclonal antibody and recombinant human active
MAPKAP kinase 2 were from Biozol (Eching, Germany), N-(1-pyrenyl)iodoacetamide was from Molecular Probes
(Leiden, Netherlands), [32P]orthophosphate (HCl-free) was
from PerkinElmer Life Sciences, polyvinylidene difluoride
membrane was from Millipore (Eschborn, Germany), and nitrocellulose was
obtained from Schleicher and Schuell (Kassel, Germany). All
other chemicals, reagents and solvents of the highest purity available
were purchased from Sigma (Deisenhofen, Germany).
cGK I
and the catalytic subunit of cAK type II were purified from
bovine lung and bovine heart, respectively (17). cGK I
and cGK II
were expressed in and purified from the baculovirus-Sf9 cell
system (18).
Isolation of Platelets--
Freshly donated blood from healthy
volunteers (50 ml) was collected in acid-citrate dextrose and
centrifuged for 10 min at 300 × g to yield
platelet-rich plasma. Platelet-rich plasma was centrifuged for 20 min
at 500 × g and the pellet was resuspended and washed
once in an isotonic buffer containing 10 mM Hepes (pH 7.4),
137 mM NaCl, 2.7 mM KCl, 5.5 mM
glucose, and 1 mM EDTA at a density of 1 × 109 cells/ml. After resuspension, platelets were allowed to
rest at 37 °C for 15 min.
32P Labeling of Platelets--
Platelet preparation
was carried out essentially as described above. After washing, 1 ml of
platelets at a concentration of 1 × 109/ml was
incubated with 500 µCi of [32P]orthophosphate
(HCl-free) for 1.5 h at 37 °C. Platelets were then centrifuged
at 500 × g for 7 min and resuspended in 1 ml of
isotonic buffer. Aliquots of 100 µl (corresponding to 200 µg of
protein) were used for activation with 500 µM
8-pCPT-cGMP for 30 min at 37 °C. After stimulation,
platelets were briefly centrifuged (500 × g) to yield
a pellet.
Two-dimensional Gel Electrophoresis--
Isoelectric focussing
for two-dimensional gel electrophoresis was performed using the
Multiphor II system from Amersham Pharmacia Biotech (Uppsala, Sweden)
according to the instructions of the manufacturer. The platelet pellet
(about 200 µg of protein) was solubilized for 15 min by sonication in
220 µl of lysis buffer containing 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 15 mM
dithiothreitol (electrophoresis grade), 0.5% carrier ampholytes, pH
3-10. Pellet homogenate was loaded on a 13-cm immobilized IPG strip,
pH 3-10, using a reswelling cassette (custom-built). Focussing was
carried out for 1 h at 150 V, 1 h at 600 V, and 25 h at
3500 V.
After equilibration in 50 mM Tris, pH 8.9, 6 M
urea, 30% glycerol, 2% SDS, strips were immediately applied to a
vertical 10% SDS gel without stacking gel. Electrophoresis was carried
out at 8 °C with a constant current of 30 mA per gel. The gels of radioactively labeled platelet proteins were fixed in 30% ethanol, 10% acetic acid and exposed. Radioactive spots were cut out,
collected, and concentrated in a Pasteur pipette according to Gaevert
et al. (19).
Mass Spectrometry--
The concentrated gel piece was washed
sequentially for 10 min in tryptic digestion buffer (10 mM
NH4HCO3) and digestion buffer:acetonitril, 1:1.
These steps were repeated three times and led to a shrinking of the
gel. It was reswollen with 2 µl of protease solution (trypsin at 0.05 µg/µl) in digestion buffer and incubated overnight at 37 °C. The
supernatant was collected and dried down to 1 µl.
Electrospray ionization mass spectrometry (ESI-MS) was carried out
using a TSQ 7000 triple quadrupole mass spectrometer (Finnigan MAT,
Bremen, Germany) equipped with a nanospray source of 0.6 to 1.1 kV
constructed in-house. Mass spectra were acquired with a scan speed of
1000 Da/s. Argon at a pressure of 3 mTorr was used as collision gas.
For the fully automated interpretation of fragment ion spectra, the
SEQUESTTM algorithm (version B22) was employed.
Western Blot Analysis of p38 and Hsp27--
Washed, intact human
platelets (100 µl) at a concentration of 1 × 109
cells/ml were incubated at 37 °C by adding 2 units/ml thrombin for 2 min or by adding 500 µM 8-pCPT-cGMP for the
times indicated in the figures. After treatment, platelets were briefly
centrifuged (500 × g) to yield a pellet, which was
immediately boiled in Laemmli SDS stop solution and separated by
SDS-PAGE on a 10% gel. After blotting on polyvinylidene difluoride
membrane and blocking with 3% nonfat dry milk in 10 mM
Tris (pH 7.5), 100 mM NaCl, 0.1% Tween 20, the membrane
was first incubated with a polyclonal antibody against dual
phosphorylated p38 (1:500) followed by incubation with horseradish
peroxidase-coupled goat anti-rabbit IgG (1:5000) and detection by ECL.
For Hsp27 detection, a two-dimensional SDS gel was blotted on
nitrocellulose, blocked with 1% hemoglobin in phosphate-buffered saline, and incubated first with an anti-Hsp27 rabbit polyclonal antibody (1:1000) followed by incubation with horseradish
peroxidase-coupled goat anti-rabbit IgG (1:5000) and detection by ECL.
Site-directed Mutagenesis--
Mutagenesis of pAK3038-Hsp27 (20)
and pAK3038-Hsp27-S15D,S78D,S82D (21) was performed using the
QuickChange Site-directed Mutagenesis Kit and the two
corresponding oligonucleotides 5'-CACGCGGAAATACGAGCTGCCCCCCGGTG-3'and 5'-GTGGCCCCCCGTCCTCCATAAAGGCGCAC-3' by changing the codon for threonine 143 to glutamate, producing the plasmids pAK3038-Hsp27-T143E and pAK3038-Hsp27-S15D,S78D,S82D,T143E,respectively. The constructs for pAK3038-Hsp27-S15D, pAK3038-Hsp27-S78D,S82D, and
pAK-Hsp27-S15D,S78D,S82D have been described earlier (21). All mutants
were verified by sequencing.
In Vitro Phosphorylation of Hsp27--
Hsp27 and its mutants
S15D, S78D,S82D, S15D,S78D,S82D, S15D,S78D,S82D,T143E, and T143E (each
0.5 µM) were incubated at 30 °C in a total volume of
20 µl with 10 mM Hepes (pH 7.4), 5 mM MgCl2, 1 mM EDTA, 0.2 mM
dithiothreitol, and the C subunit of cAK or cGK I
, I
, or II (each
0.05 µM) and 5 µM cGMP. Alternatively, Hsp27 and its mutants were incubated with 5 mM MOPS (pH
7.2), 6.25 mM
-glycerol phosphate, 1.25 mM
EGTA, 0.25 mM sodium orthovanadate, 0.25 mM
dithiothreitol, 20 mM MgCl2, and 0.1 unit of
MAPKAP kinase 2. Reactions were started by the addition of 50 µM ATP containing 0.5 µCi of
[
-32P]ATP, and terminated after 30 min or at the times
indicated in the figures by the addition of 10 µl of Laemmli SDS stop
solution. Proteins were separated by SDS-PAGE on 10% gels.
Incorporation of 32P was visualized by autoradiography.
Preparation of Pyrene Actin--
G-actin was prepared from pig
skeletal muscle according to the procedure of Pardee and Spudich (22).
For labeling with N-(1-pyrenyl)iodoacetamide, G-actin was
dialyzed 3 times for 12 h against G-buffer (2 mM Tris, pH 8.0, 0.2 mM ATP, 0.2 mM CaCl2).
To polymerize G-actin to F-actin, 100 mM KCl and 1 mM MgCl2 was added for 1 h at room
temperature. N-(1-Pyrenyl)iodoacetamide (at a 2-fold molar
excess) was dissolved in dimethyl sulfoxide and added slowly with
gentle stirring to the F-actin solution. The solution was kept at room
temperature in the dark for 20 h. After labeling, F-actin was
dialyzed 5 times against G-buffer with 0.5 mM
dithioerythritol at 4 °C to form G-actin. To remove any residual
F-actin, the solution was centrifuged for 1 h at 100,000 × g in a swing-bucket rotor and the supernatant was used in
the polymerization experiments. The degree of labeling was determined
by UV spectroscopy at 344 nm assuming an extinction coefficient of
2.2 × 104 M
1
cm
1, and was found to be 70-80%.
Fluorometric Measurement of Actin Polymerization--
For
standard assays, pyrene-labeled G-actin in G-buffer at a final
concentration of 2 µM and various amounts of Hsp27 were mixed in a total volume of 500 µl in a solution of 20 mM
Tris (pH 7.6), 0.05 mM NaN3, 0.002 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithioerythritol, 10 mM MgCl2, 30 mM NH4Cl. The solutions were mixed with 1 µl
of 1 M MgCl2 and 12.5 µl of 2 M
KCl to start actin polymerization. Polymerization was measured by the
enhancement of pyrene-actin fluorescence using the luminescence
spectrophotometer LS50 (PerkinElmer Life Sciences). Excitation was
measured at 365 nm with a 2.5-mm slit width, and emission was detected
at 407 nm with 2.5-mm slit width.
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RESULTS |
Phosphorylation of Hsp27 in Intact Human Platelets Treated with the
cGK-specific Stimulus 8-pCPT-cGMP--
To identify substrates of cGK
in intact human platelets, cells were labeled with
[32P]orthophosphate, stimulated with 500 µM
of the specific cGMP-dependent protein kinase activator
8-pCPT-cGMP, and proteins of the resulting platelet lysate
were separated by two-dimensional gel electrophoresis. Fig.
1 shows low basal phosphorylation of
three proteins with an approximate molecular mass of 27 kDa in resting
platelets (control). Phosphorylation of the two more acidic protein
spots was significantly increased after stimulation with
8-pCPT-cGMP. To identify these proteins, the three spots
were excised from several two-dimensional gels, concentrated, digested
with trypsin, and the resulting peptides were analyzed by electrospray
ionization-tandem mass spectrometry. All spots contained Hsp27,
suggesting that the three spots either represent the mono-, bis-, and
tris-phosphorylated isoforms of the protein with a
8-pCPT-cGMP-induced increase in the amount of the bis- and
tris-phosphorylated forms or indicate some different post-translational
modifications (23). To confirm the identification of Hsp27, human
platelets were labeled with [32P]orthophosphate,
stimulated with 500 µM 8-pCPT-cGMP for 10 min, and proteins of the homogenate were separated by two-dimensional gel
electrophoresis. The proteins were transferred to nitrocellulose and
positions of the phosphoproteins were determined by autoradiography (Fig. 2, lower panel). The
membranes were probed with a rabbit polyclonal antibody against Hsp27.
Two radioactive protein spots that demonstrated increases in
phosphorylation after cGK activation were immunoreactive with
anti-Hsp27 antibody (Fig. 2, upper panel). Three more basic
proteins, most likely representing additional nonphosphorylated or
weakly phosphorylated isoforms of Hsp27, were also immunoreactive and
decreased in amount during stimulation.

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Fig. 1.
Phosphorylation of a 27-kDa protein in intact
human platelets. Human platelets were incubated in the
presence of [32P]orthophosphate and treated with buffer
alone (Control) or with 500 µM cGK activator
8-pCPT-cGMP for 30 min (Stimulation). Platelet
homogenate proteins were separated by two-dimensional gel
electrophoresis and an autoradiogram was obtained. The pH gradient is
indicated at the top of the gels. The blots are
representative of five separate experiments.
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Fig. 2.
The 27-kDa protein is immunoreactive with
anti-Hsp27 antibody. Intact human platelets were labeled with
[32P]orthophosphate and treated with 500 µM
8-pCPT-cGMP for 10 min. Platelet homogenate proteins were
separated by two-dimensional gel electrophoresis and proteins were
transferred to nitrocellulose. The autoradiogram (lower
panel) reveals phosphorylation of two proteins. The corresponding
anti-Hsp27 immunoblots (upper panel) demonstrate
immunoreactive protein corresponding to the phosphoprotein. In
addition, three unphosphorylated immunoreactive proteins were
identified. The location of the unphosphorylated isoforms and the
phosphorylated spots are indicated at the top of the figure.
The blots are representative of two separate experiments.
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In Vitro Phosphorylation of Hsp27--
To determine whether cGK
phosphorylates Hsp27 in vitro, purified, recombinant Hsp27
was incubated with the three cGK isoforms I
, I
, and II or the
catalytic subunit of cAMP-dependent protein kinase in the
presence of [
-32P]ATP. An autoradiogram of a
representative SDS-PAGE gel is shown in Fig.
3. Incorporation of phosphate was
observed after 30 min with all of the four kinases, albeit at different
levels, with cGK causing less phosphate incorporation than the C
subunit. In a control experiment, VASP, a well known substrate for cAK
and cGK (15), was equally phosphorylated by all four kinases.

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Fig. 3.
In vitro phosphorylation of Hsp27
by cGMP- and cAMP-dependent protein kinase. Purified
recombinant Hsp27 or VASP (each 1 µM) were phosphorylated
by cGK type I , I , II, or the C subunit of cAK (each 0.05 µM) in a total volume of 20 µl for 30 min as described
under "Experimental Procedures." The bands for autophosphorylated
cGK are indicated at the left. The autoradiogram shown is
representative of three separate experiments.
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No Stimulatory Effect of cGK on p38 MAPK Phosphorylation--
It
is known that Hsp27 is phosphorylated in human platelets directly by
MAPKAP kinase 2 after stimulation of the platelets with thrombin and
subsequent activation of the p38 MAPK cascade (24-28). To exclude any
direct stimulation of p38 MAPK or MAPKAP kinase 2 by
8-pCPT-cGMP and any indirect effect of cGK on Hsp27 phosphorylation via p38 MAPK, human platelets were stimulated with 500 µM 8-pCPT-cGMP or, as a positive control, with
2 units/ml thrombin, and p38 MAPK activation was monitored by a
specific antibody that recognizes the active, bis-phosphorylated form
of p38 MAPK. In contrast to the control experiment with thrombin treatment, where p38 MAPK was rapidly phosphorylated and activated in
platelets after 2 min, the stimulation with 8-pCPT-cGMP did not lead to increased p38 phosphorylation at any of the times analyzed
(Fig. 4). Similar negative results were
obtained when we investigated the ability of cGK to directly
phosphorylate and activate MAPKAP kinase 2 (data not shown).

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Fig. 4.
Detection of p38 MAPK in platelets by Western
blotting. Intact human platelets were stimulated with 2 units/ml
thrombin or 500 µM 8-pCPT-cGMP for the times
indicated and lysed. Platelet lysate proteins were separated by SDS-gel
electrophoresis, transferred to nitrocellulose, and incubated with
monoclonal p38 MAPK antisera as described under "Experimental
Procedures." The figure shown is representative of two independent
experiments.
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Identification of Phosphorylation Sites--
It has been shown
that Hsp27 is phosphorylated in vitro and in vivo
by MAPKAP kinase 2 at Ser15, Ser78, and
Ser82, with this latter residue being the most prominent
in vitro phosphorylation site (29, 30). Experiments with
cAMP-dependent protein kinase revealed phosphorylation of
Ser15 and Ser86 of mouse Hsp25 in
vitro, albeit with low efficiency (31). Interestingly, our
sequence analysis of Hsp27 identified an additional putative phosphorylation site of Hsp27 for cAK and cGK at threonine 143 (Arg-Lys-Tyr-Thr143-Leu). To study this
potential phosphorylation site, we constructed two mutants in which
threonine 143 was replaced by a phosphate-mimicking glutamic acid:
Hsp27-T143E and Hsp27-S15D,S78D,S82D,T143E. In addition, we
investigated three Hsp27 mutants reported previously: Hsp27-S15D,
Hsp27-S78D,S82D, and Hsp27-S15D,S78D,S82D (21). Analysis of these
earlier mutants by in vitro phosphorylation experiments
confirmed the results obtained previously with MAPKAP kinase 2 showing
complete absence of phosphate incorporation after substitution of all
three known serine phosphorylation sites in mutant Hsp27-S15D,S78D,S82D
(Fig. 5). In contrast, this mutant was
still phosphorylated by both cGK and cAK (8 ± 0.8 and 20 ± 0.5% of wild-type phosphorylation, respectively). Only after mutation of threonine 143 to glutamic acid (Hsp27-S15D,S78D,S82D,T143E), phosphate incorporation was abolished (Fig. 5, Table
I). To confirm this result, the threonine
phosphorylation by cGK and cAK was further analyzed by phosphorylating
wild type Hsp27 and Hsp27-T143E with the two kinases. A 50% reduction
in phosphate incorporation was observed for the threonine mutant
providing further evidence that threonine 143 represents an important
phosphorylation site for cGK and cAK in Hsp27 (Fig.
6A). For quantification, the
areas of the gel corresponding to the autoradiogram in Fig. 5 were
collected for liquid scintillation counting. These data are summarized
in Table I. Ser15 is probably not phosphorylated by cGK
since the mutants Hsp27-S78D,S82D and Hsp27-S15D,S78D,S82D showed
similar phosphate incorporation. Interestingly, cAK also appears not to
phosphorylate Ser15, although mimicking Ser15
phosphorylation (Hsp27-S15D) increased incorporation of phosphate by
cAK about 2-fold compared with wild-type Hsp27. This enhanced phosphate
incorporation after Ser15 mutation was also observed with
MAPKAP kinase 2, albeit to a lesser extent: S15D phosphorylation
increased to 118 ± 9% of wild-type phosphorylation (Fig. 5,
Table I). In the presence of cGK, wild-type Hsp27 is phosphorylated
24.8 ± 3% with respect to wild-type phosphorylation by MAPKAP
kinase 2 (Table I).

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Fig. 5.
Phosphorylation of wild-type and mutant
Hsp27. Purified recombinant wild-type Hsp27 and the mutants
Hsp27-S15D, Hsp27-S78D,S82D, Hsp27-S15D,S78D,S82D, and
Hsp27-S15D,S78D,S82D,T143E (0.5 µM each) were incubated
with [ -32P]ATP in the presence of MAPKAP kinase 2 (0.1 units/20 µl), cGMP-dependent protein kinase (cGK I )
and the C subunit of cAMP-dependent protein kinase (C)
(each 0.05 µM) for 30 min as described under
"Experimental Procedures." Proteins were resolved by SDS-PAGE and
the phosphorylated proteins visualized by autoradiography. In addition
to Hsp27 phosphorylation (bold-faced arrow),
autophosphorylation of cGK I is observed (scalloped
arrow). The results shown are representative of three independent
experiments.
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Table I
Phosphate incorporation of wild-type and mutant Hsp27
Wild-type Hsp27 and the mutants S15D,S78D,S82D, S15D,S78D,S82D and
S15D,S78D,S82D,T143E (each 0.5 µM) were incubated with
0.2 units of MAPKAP kinase 2 or 0.05 µM cGK and C-subunit
in the presence of [ -32P]ATP at 30 °C for 30 min in a
total volume of 20 µl. The proteins were resolved by SDS-PAGE and
visualized by autoradiography. The corresponding areas of the gel were
excised for liquid scintillation counting. Values presented are
mean ± S.E. from triplicate studies. Wild-type phosphorylation
(set at 100%) corresponds to 0.6 mol of phosphate/mol of Hsp27.
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Fig. 6.
Phosphorylation of wild-type Hsp27 and
Hsp27-T143E. A, purified recombinant wild-type Hsp27
and mutant Hsp27-T142E (each 0.5 µM) were incubated with
[ -32P]ATP in the presence of
cGMP-dependent protein kinase (cGK I ) and the C subunit
of cAMP-dependent protein kinase (cAK) (each 0.05 µM) for 30 min as described under "Experimental
Procedures." Proteins were resolved by SDS-PAGE and the
phosphorylated proteins visualized by autoradiography. In addition to
Hsp27 phosphorylation (bold-faced arrow),
autophosphorylation of cGK I is observed (scalloped
arrow). The results shown are representative of three independent
experiments. B, purified recombinant wild-type Hsp27 and
mutant Hsp27-T143E (each 0.5 µM) were incubated with
[ -32P]ATP in the presence of MAPKAP kinase 2 (0.1 units/20 µl). At the time points indicated, aliquots were taken,
proteins therein resolved by SDS-PAGE and visualized by
autoradiography. Incorporation of phosphate was not significantly
different between wild-type and mutant. The results shown are
representative of three independent experiments.
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We next examined whether the phosphorylation of Hsp27 at threonine 143 (Hsp27-T143E) by cGK and cAK might influence MAPKAP kinase 2 phosphorylation. However, neither the kinetics of MAPKAP kinase 2 phosphorylation nor the overall phosphate incorporation by MAPKAP
kinase 2 was influenced by the T143E mutation (Fig. 6B).
Actin Polymerization--
It has been suggested that small heat
shock proteins are important regulatory components of the actin-based
cytoskeleton (32) and that phosphorylation of Hsp27 might be implicated
in regulating actin polymerization (33, 34). Therefore, we compared the stimulatory effects on actin polymerization of the unphosphorylated wild-type Hsp27 with the phosphorylation-mimicking mutants. When preincubated with labeled G-actin, wild-type recombinant Hsp27 at all
concentrations tested (up to 1 µM) did not alter
the polymerization of actin (Fig.
7A). In contrast, wild-type
Hsp27 phosphorylated by MAPKAP kinase 2, as well as the MAPKAP kinase 2 phosphorylation-mimicking mutant Hsp27-S15D,S78D,S82D, both revealed a
faster and stronger actin polymerization (124 ± 6 and 124 ± 1.8% of the control, respectively; Fig. 7B). This
polymerization was significantly reduced (115 ± 2.6% of control)
by introducing the fourth cGK/cAK phosphorylation-mimicking site at
threonine 143 (Hsp27-S15D,S78D,S82D,T143E) (Fig. 7C). Analysis of the single and double mutants Hsp27-S15D
and Hsp27-S78D,S82D showed a slight decrease in actin polymerization (90 ± 2 and 87 ± 4% of wild-type, respectively) (Fig.
7D; curve for S78D,S82D not shown). The single mutant
Hsp27-T143E, however, did not affect the formation of actin filaments
(96 ± 7% of wild-type) (Fig. 7D). The data of the
actin polymerization experiments are summarized numerically in Table
II. Attempts to study actin
polymerization with cGK-phosphorylated wild-type Hsp27 were not
successful because the reaction buffer itself interfered with the actin
polymerization assay.

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Fig. 7.
Influence of Hsp27 mutants on actin
polymerization analyzed by fluorescence spectroscopy.
A, actin polymerization of labeled G-actin (2 µM) in the absence (curve 2) or presence of
0.4 µM wild-type Hsp27 (curve 1) or 1 µM wild-type Hsp27 (curve 3). B,
actin polymerization of labeled G-actin (2 µM) in the
absence (curve 3) or presence of 0.5 µM mutant
Hsp27-S15D,S78D,S82D (curve 2) or 0.5 µM
wild-type Hsp27 previously phosphorylated by 0.05 unit/20 µl of
MAPKAP-kinase 2 for 30 min (curve 1) as described under
"Experimental Procedures." C, actin polymerization of
labeled G-actin (2 µM) in the presence of 0.5 µM wild type HSP27 (curve 3), 0.5 µM Hsp27-S15D,S78D,D82D,T143E (curve 2), or
0.5 µM HSP27-S15D,S78D,S82D (curve 3).
D, actin polymerization of labeled G-actin (2 µM) in the presence of 0.5 µM wild-type
Hsp27 (curve 1), 0.5 µM Hsp27-T143E
(curve 2), or 0.5 µM Hsp27-S15D (curve
3). The results shown are representative of three to eight
independent experiments with 2 different lots of purified actin.
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Table II
Influence of Hsp27 mutants on actin polymerization
Actin polymerization was measured by fluorescence spectroscopy of
pyrene-labeled G-actin (2 µM) in the presence of various
Hsp27 mutants (0.5 µM) as described under "Experimental
Procedures." Experiments were performed with 2 different lots of
purified actin.
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DISCUSSION |
Hsp27 was first identified as a substrate of the p38 MAPK/MAPKAP
kinase 2 pathway (24-28, 35). In human platelets, activation of the
p38/MAPKAP kinase 2 pathway after thrombin stimulation leads to a
marked shift from the 27-kDa unphosphorylated form to at least three
major phosphorylated forms (24, 28). The sites phosphorylated by MAPKAP
kinase 2 in vivo were identified as Ser15,
Ser78, and Ser82 (29, 30). The present study
characterizes Hsp27 as a substrate additionally of cGK in
vivo, a protein involved in platelet inhibition (5). Analysis of
phosphate incorporation of different Hsp27 mutants after
phosphorylation by cGK in vitro revealed an unknown phosphorylation site at threonine 143, as well as phosphorylation of
residues Ser78 and Ser82. Phosphorylation of
human Hsp27 and its mouse homologue Hsp25 by different protein kinases
has been addressed in several studies. Recently, Maizel et
al. (36) showed that recombinant Hsp25 is efficiently
phosphorylated by protein kinase C-
at Ser15 and
Ser86 (analogous to Ser82 of the human
sequence). Earlier studies by Gaestel et al. (31) also
identified both protein kinase C-
and cAMP-dependent
protein kinase as being capable of phosphorylating Hsp25 in
vitro at Ser15 and Ser86.
(Ser78, representing the third phosphorylation site in
human Hsp27, is not conserved in rodent species.) In addition, another
small heat shock protein in muscle (Hsp20) is phosphorylated on
Ser16 during activation of the cAMP-dependent
pathway and could be phosphorylated in vitro by both cAK and
cGK (37, 38). The phosphorylation is associated with changes in the
macromolecular association of Hsp20 (39).
Since the cGMP- and cAMP-dependent protein kinase pathways
are involved in platelet inhibition, it was tempting to speculate that
the phosphorylation at threonine 143 might negatively influence Hsp27
phosphorylation by MAPKAP kinase 2. In contrast to our hypothesis, however, we could not detect any changes in the rate or extent of
Hsp27-T143E phosphorylation by MAPKAP kinase 2 in comparison with
wild-type Hsp27 phosphorylation.
In our experiments concerning the influence of Hsp27 and its mutants on
actin filament organization, we demonstrate that recombinant wild-type
Hsp27 and the Hsp27-T143E mutant had no effect on actin polymerization.
Interestingly, recombinant Hsp27, phosphorylated by MAPKAP kinase 2, and the phosphorylation-mimicking mutant Hsp27-S15D,S78D,S82D both
increased actin polymerization by ~23%. In general, these results
show the same tendency as the data described by Benndorf et
al. (34), where phosphorylation of native Hsp25 leads to a
transition from inhibition to neutral effects, while in our experiments
phosphorylation of recombinant Hsp27 shifts from neutral to stimulating
effects. Most interestingly, additional mimicry of phosphorylation by
changing threonine 143 to glutamic acid in the Hsp27-S15D,S78D,S82D
mutant significantly reduces the stimulation of actin polymerization
and could explain in part the inhibitory effect of cGK on
thrombin-induced platelet activation since actin polymerization is
required for agonist-induced shape change.
The single- and double-mutated Hsp27 forms S15D and S78D,S82D induced a
10% decrease in actin polymerization. This is probably due to the
different oligomerization of the mutants. Hsp27-S15D and
Hsp27-S78D,S82D both showed large, round particles (6 tetramers), whereas wild-type Hsp27 phosphorylated by MAPKAP-kinase 2 as well as
the mutant Hsp27-S15D,S78D,S82D formed mostly single tetramers, which
are thought to be responsible for stabilization of actin filaments
(21).
Apart from this, phosphorylation of Hsp27 by cGK might influence its
chaperone function. For example, Zhue et al. (32, 40) observed that thrombin activation leads to co-precipitation of platelet
factor XIII and two yet unidentified proteins with Hsp27. This
association between any of these protein and Hsp27 might be influenced
by cGK phosphorylation. Heat shock protein 60 (Hsp60) has been shown to
function as a molecular chaperone for histone 2B (H2B) when both
proteins are in their dephosphorylated form. Phosphorylation by cAK of
both Hsp60 and H2B causes dissociation of H2B from Hsp60 and loss of
H2B from the plasma membrane (41).
Certainly, phosphorylation of Hsp27 by cGK is not the only
cGK-dependent pathway involved in platelet inhibition. For
example, phosphorylation of Rap 1B by cGK (and cAK) in intact
platelets is associated with the inhibition of thrombin-induced PLC
activity (16, 42). Phosphorylation of the major cGK substrate in human platelets, the 50-kDa protein VASP, is assumed to be involved in the
regulation of the fibrinogen receptor glycoprotein IIb-IIIa (43).
Recent experiments with VASP mutants imitating defined phosphorylation
states revealed that phosphorylation of VASP down-regulates its
in vitro F-actin binding and actin polymerization promoting activity (44). The mechanism of cGK action also includes inhibition of
inositol 1,4,5-trisphosphate-mediated Ca2+ mobilization
from intracellular stores and the secondary, store-related calcium
influx (45, 46). Phosphorylation of the inositol
1,4,5-trisphosphate-receptor, however, is well established in
vitro (8, 47) but remains to be demonstrated for human platelets.
In summary, the present findings demonstrate that Hsp27 is an important
substrate for cGK in intact human platelets. The different effects of
mutating the phosphorylation sites for MAPKAP kinase 2 and cGK on the
stimulation of actin polymerization could explain the stimulatory role
of MAPKAP kinase 2 and in part the inhibitory role of cGK in platelet
activation. This further supports the notion that different enzymes can
phosphorylate Hsp27 in human platelets at different sites, and, by this
mechanism, positively or negatively regulate platelet activation.