Heat Shock Protein 27 Is a Substrate of cGMP-dependent Protein Kinase in Intact Human Platelets

PHOSPHORYLATION-INDUCED ACTIN POLYMERIZATION CAUSED BY HSP27 MUTANTS*

Elke ButtDagger §, Dorian Immler||, Helmut E. Meyer||, Alexey Kotlyarov**, Kathrin Laaß**, and Matthias Gaestel**

From the Dagger  Institute of Clinical Biochemistry and Pathobiochemistry, Medical University Clinic, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany, the   Bayer AG, ZF-ZAS/Q18, Leverkusen, Germany, and the || Institute of Physiological Chemistry, Protein Structure Lab, Ruhr University of Bochum, Universitätsstr. 150, D-44780 Bochum, Germany, and ** Institute of Pharmaceutical Biology, University of Halle, Hoher Weg 8, D-06120 Halle, Germany

Received for publication, October 10, 2000, and in revised form, November 22, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphorylation of heat shock protein 27 (Hsp27) in human platelets by mitogen-activated protein kinase-activated protein kinase (MAPKAP) 2 is associated with signaling events involved in platelet aggregation and regulation of microfilament organization. We now show that Hsp27 is also phosphorylated by cGMP-dependent protein kinase (cGK), a signaling system important for the inhibition of platelet aggregation. Stimulation of washed platelets with 8-para-chlorophenylthio-cGMP, a cGK specific activator, resulted in a time-dependent phosphorylation of Hsp27. This is supported by the ability of cGK to phosphorylate Hsp27 in vitro to an extent comparable with the cGK-mediated phosphorylation of its established substrate vasodilator-stimulated phosphoprotein. Studies with Hsp27 mutants identified threonine 143 as a yet uncharacterized phosphorylation site in Hsp27 specifically targeted by cGK. To test the hypothesis that cGK could inhibit platelet aggregation by phosphorylating Hsp27 and interfering with the MAPKAP kinase phosphorylation of Hsp27, the known MAPKAP kinase 2-phosphorylation sites (Ser15, Ser78, and Ser82) as well as Thr143 were replaced by negatively charged amino acids, which are considered to mimic phosphate groups, and tested in actin polymerization experiments. Mimicry at the MAPKAP kinase 2 phosphorylation sites led to mutants with a stimulating effect on actin polymerization. Mutation of the cGK-specific site Thr143 alone had no effect on actin polymerization, but in the MAPKAP kinase 2 phosphorylation-mimicking mutant, this mutation reduced the stimulation of actin polymerization significantly. These data suggest that phosphorylation of Hsp27 and Hsp27-dependent regulation of actin microfilaments contribute to the inhibitory effects of cGK on platelet function.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Urea (ultra pure), IPG strips, [gamma -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 Ialpha and the catalytic subunit of cAK type II were purified from bovine lung and bovine heart, respectively (17). cGK Ibeta 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 Ialpha , Ibeta , 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 beta -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 [gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

In Vitro Phosphorylation of Hsp27-- To determine whether cGK phosphorylates Hsp27 in vitro, purified, recombinant Hsp27 was incubated with the three cGK isoforms Ialpha , Ibeta , and II or the catalytic subunit of cAMP-dependent protein kinase in the presence of [gamma -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 Ialpha , Ibeta , 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.

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.

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 [gamma -32P]ATP in the presence of MAPKAP kinase 2 (0.1 units/20 µl), cGMP-dependent protein kinase (cGK Ibeta ) 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 Ibeta 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 [gamma -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 [gamma -32P]ATP in the presence of cGMP-dependent protein kinase (cGK Ibeta ) 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 Ibeta 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 [gamma -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.

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.



    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-delta at Ser15 and Ser86 (analogous to Ser82 of the human sequence). Earlier studies by Gaestel et al. (31) also identified both protein kinase C-alpha 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 PLCgamma 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.


    ACKNOWLEDGEMENTS

We thank Wiebke Hayen for help with actin purification and labeling and Nina Göttfert for preparation of the figures.


    FOOTNOTES

* This work was supported by Research Grant Bu 740/2-1 from the Deutsche Forschungsgemeinschaft (DFG).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: Institute of Clinical Biochemistry and Pathobiochemistry, Medical University Clinic, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany. Tel.: 49-931- 201-3154; Fax: 49-931-201-3153; E-mail: butt@klin-biochem.uni-wuerzburg.de.

Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M009234200


    ABBREVIATIONS

The abbreviations used are: Hsp, heat shock protein; MAPKAP kinase 2, mitogen-activated protein kinase-activated protein kinase 2; C, catalytic subunit; cAK, cAMP-dependent protein kinase; cGK, cGMP-dependent protein kinase; VASP, vasodilator-stimulated phosphoprotein; 8-pCPT-cGMP, 8-para-chlorophenylthio-cGMP; H2B, histone 2B; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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