Heat Shock Protein 27 Is a Substrate of cGMP-dependent Protein Kinase in Intact Human Platelets
2001; Elsevier BV; Volume: 276; Issue: 10 Linguagem: Inglês
10.1074/jbc.m009234200
ISSN1083-351X
AutoresElke Butt, Dorian Immler, Helmut E. Meyer, Alexey Kotlyarov, Kathrin Laaß, Matthias Gaestel,
Tópico(s)Entomological Studies and Ecology
ResumoPhosphorylation 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 Thr143alone 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. 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 Thr143alone 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. heat shock protein mitogen-activated protein kinase-activated protein kinase 2 catalytic subunit cAMP-dependent protein kinase cGMP-dependent protein kinase vasodilator-stimulated phosphoprotein 8-para-chlorophenylthio-cGMP histone 2B 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 4-morpholinepropanesulfonic acid polyacrylamide gel electrophoresis 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. 1von Bruchhausen F. Walter U. Handb. Exp. Pharmacol. 1997; 126: 181-208Crossref Google Scholar). 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 (2Haslam R.J. Dickinson N.T. Jang E.K. Thromb. Haemost. 1999; 82: 412-423Crossref PubMed Scopus (150) Google Scholar, 3Butt E. Walter U. Adv. Mol. Cell. Biol. 1997; 18: 311-333Crossref Scopus (4) Google Scholar). The molecular mechanisms of platelet inhibition by cGMP signaling distal to cGK activation are only partially understood (4El-Daher S.S. Eigenthaler M. Walter U. Furuichi T. Miyawaki A. Mikoshiba K. Kakkar V.V. Authi K. Tromb. Haemost. 1996; 76: 1063-1071Crossref PubMed Scopus (18) Google Scholar). Studies using cGK-deficient mice demonstrated defective cGMP-mediated inhibition of platelet aggregation (5Corbin J.D. Turko I.V. Baesley A. Francic S.H. Eur. J. Biochem. 2000; 267: 2760-2767Crossref PubMed Scopus (228) Google Scholar). Several proteins have been reported to be phosphorylated in response to cGK activation either in vitro or in intact cells, including cGMP-specific phosphodiesterase (6Massberg S. Sausbier M. Klatt P. Bauer M. Pfeifer A. Diess W. Fassler R. Ruth P. Krombach F. Hofmann F. J. Exp. Med. 1999; 189: 1255-1264Crossref PubMed Scopus (194) Google Scholar), myosin light chain kinase (7Nishikawa M. de Lanerolle P. Lincoln T.M. Adelstein R.S. J. Biol. Chem. 1984; 259: 8429-8436Abstract Full Text PDF PubMed Google Scholar), the inositol 1,4,5-trisphosphate receptor (8Komalavilas P. Lincoln T. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), an inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate (9Schlossmann J. Ammendola A. Ashman K. Zong X. Huber A. Neubauer G. Wang G.-X. Allescher H.-D. Korth M. Wilm M. Hofmann F. Ruth P. Nature. 2000; 404: 197-201Crossref PubMed Scopus (386) Google Scholar), G-substrate (10Hall K.U. Collins S.P. Gamm D.M. Massa E. Depaoli-Roach A.A. Uhler M.D. J. Biol. Chem. 1999; 274: 3485-3495Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), Na+/K+-ATPase (11Fotis H. Tatjanenko L.V. Vasilets L.A. Eur. J. Biochem. 1999; 260: 904-910Crossref PubMed Scopus (37) Google Scholar), and endothelial NO synthase (Ref. 12Butt E. Bernhard M. Smolenski A. Kotsonis P. Fröhlich L.G. Sickmann A. Meyer H.E. Lohmann S.M. Schmidt H.H.H.W. J. Biol. Chem. 2000; 275: 5179-5187Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, for review, see Ref. 13Hofmann F. Ammendola A. Schlossmann J. J. Cell Sci. 2000; 113: 1671-1676Crossref PubMed Google Scholar). 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 (14Wang G.-R. Zhu Y. Halushka P.V. Lincoln T.M. Mendelsohn M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4888-4893Crossref PubMed Scopus (225) Google Scholar). 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 (15Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), and the small GTP-binding protein rap 1b (16Reep B.R. Lapetina E.G. Biochem. Biophys. Res. Commun. 1996; 219: 1-5Crossref PubMed Scopus (10) Google Scholar). 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. Urea (ultra pure), IPG strips, [γ-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 Iα and the catalytic subunit of cAK type II were purified from bovine lung and bovine heart, respectively (17Walter U. Miller P. Wilson F. Menkes D. Greengard P. J. Biol. Chem. 1980; 255: 3757-3762Abstract Full Text PDF PubMed Google Scholar). cGK Iβ and cGK II were expressed in and purified from the baculovirus-Sf9 cell system (18Pöhler D. Butt E. Meißner J. Müller S. Lohse M. Walter U. Lohmann S.M. Jarchau T. FEBS Lett. 1995; 374: 419-425Crossref PubMed Scopus (52) Google Scholar). 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 mmglucose, 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. 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 μm8-pCPT-cGMP for 30 min at 37 °C. After stimulation, platelets were briefly centrifuged (500 × g) to yield a pellet. 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, 2m thiourea, 4% (w/v) CHAPS, 15 mmdithiothreitol (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 murea, 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 Gaevertet al. (19Gevaert K. Verschelde J.-L. Puype M. Van Damme J. Goethals M. De Boeck S. Vandekerckhove J. Electrophoresis. 1996; 17: 918-924Crossref PubMed Scopus (46) Google Scholar). The concentrated gel piece was washed sequentially for 10 min in tryptic digestion buffer (10 mmNH4HCO3) 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. Washed, intact human platelets (100 μl) at a concentration of 1 × 109cells/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 mmTris (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. Mutagenesis of pAK3038-Hsp27 (20Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar) and pAK3038-Hsp27-S15D,S78D,S82D (21Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.-P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar) 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 (21Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.-P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). All mutants were verified by sequencing. 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 mmMgCl2, 1 mm EDTA, 0.2 mmdithiothreitol, and the C subunit of cAK or cGK Iα, Iβ, 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 β-glycerol phosphate, 1.25 mmEGTA, 0.25 mm sodium orthovanadate, 0.25 mmdithiothreitol, 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 [γ-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. G-actin was prepared from pig skeletal muscle according to the procedure of Pardee and Spudich (22Pardee J.D. Spudich J.A. Methods Enzymol. 1982; 85: 164-170Crossref PubMed Scopus (984) Google Scholar). 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 mmdithioerythritol 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 × 104m−1cm−1, and was found to be 70–80%. 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 mmTris (pH 7.6), 0.05 mm NaN3, 0.002 mm phenylmethylsulfonyl fluoride, 0.5 mmdithioerythritol, 10 mm MgCl2, 30 mm NH4Cl. The solutions were mixed with 1 μl of 1 m MgCl2 and 12.5 μl of 2 mKCl 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. To identify substrates of cGK in intact human platelets, cells were labeled with [32P]orthophosphate, stimulated with 500 μmof 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 (23Scheler C. Li X.-P. Salnikow J. Dunn M.J. Jungblut P.R. Electrophoresis. 1999; 20: 3623-3628Crossref PubMed Scopus (69) Google Scholar). 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.Figure 2The 27-kDa protein is immunoreactive with anti-Hsp27 antibody. Intact human platelets were labeled with [32P]orthophosphate and treated with 500 μm8-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether cGK phosphorylates Hsp27 in vitro, purified, recombinant Hsp27 was incubated with the three cGK isoforms Iα, Iβ, and II or the catalytic subunit of cAMP-dependent protein kinase in the presence of [γ-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 (15Butt E. Abel K. Krieger M. Palm D. Hoppe V. Hoppe J. Walter U. J. Biol. Chem. 1994; 269: 14509-14517Abstract Full Text PDF PubMed Google Scholar), was equally phosphorylated by all four kinases. 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 (24Mendelsohn M.E. Zhu Y. O'Neill S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11212-11216Crossref PubMed Scopus (75) Google Scholar, 25Kramer R.M. Roberts E.F. Strifer B.A. Johnstone E.M. J. Biol. Chem. 1995; 270: 27395-27398Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 26Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1038Abstract Full Text PDF PubMed Scopus (1507) Google Scholar, 27Freshney N.W. Rawlinson L. Guesdon F. Jones E. Cowley S. Hsuan J. Saklatvala J. Cell. 1994; 78: 1039-1049Abstract Full Text PDF PubMed Scopus (778) Google Scholar, 28Saklatvala J. Rawlinson L. Waller R.J. Sarsfield S. Lee J.C. Morton L.F. Barnes M.J. Farndale R.W. J. Biol. Chem. 1996; 271: 6486-6589Abstract Full Text Full Text PDF Scopus (258) Google Scholar). 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). It has been shown that Hsp27 is phosphorylated in vitro and in vivoby MAPKAP kinase 2 at Ser15, Ser78, and Ser82, with this latter residue being the most prominentin vitro phosphorylation site (29Landry J. Lambert H. Zhou M. Lavoie J.N. Hickey E. Weber L.A. Anderson C.W. J. Biol. Chem. 1992; 267: 794-803Abstract Full Text PDF PubMed Google Scholar, 30Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar). Experiments with cAMP-dependent protein kinase revealed phosphorylation of Ser15 and Ser86 of mouse Hsp25 in vitro, albeit with low efficiency (31Gaestel M. Schröder W. Benndorf R. Lippmann C. Buchner K. Hucho F. Erdmann V.A. Bielka H. J. Biol. Chem. 1991; 266: 14721-14724Abstract Full Text PDF PubMed Google Scholar). Interestingly, our sequence analysis of Hsp27 identified an additional putative phosphorylation site of Hsp27 for cAK and cGK at threonine 143 (Arg-Lys-Tyr-Thr 143-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 (21Rogalla T. Ehrnsperger M. Preville X. Kotlyarov A. Lutsch G. Ducasse C. Paul C. Wieske M. Arrigo A.-P. Buchner J. Gaestel M. J. Biol. Chem. 1999; 274: 18947-18956Abstract Full Text Full Text PDF PubMed Scopus (625) Google Scholar). 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, TableI). 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.6 A). 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 Ser15phosphorylation (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).Table IPhosphate incorporation of wild-type and mutant Hsp27KinasePhosphate incorporation (%)Wild-typeS15DS78D,S82DS15D,S78D,S82DS15D,S78D,S82D,T143EMAPKAPK-2100118 ± 950 ± 5.80.6 ± 0.40.5 ± 0.2cGK24.8 ± 318.6 ± 2.78.8 ± 0.88 ± 0.71.8 ± 0.3C-subunit40.8 ± 693 ± 7.915.1 ± 1.620.5 ± 0.50.3 ± 0.1Wild-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 [γ-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. Open table in a new tab Figure 6Phosphorylation of wild-type Hsp27 and Hsp27-T143E. A, purified recombinant wild-type Hsp27 and mutant Hsp27-T142E (each 0.5 μm) were incubated with [γ-32P]ATP in the presence of cGMP-dependent protein kinase (cGK Iβ) 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 Iβ 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 [γ-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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 [γ-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. We next examined whether the phosphorylation of Hsp27 at threonine 14
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