Thrombin Induces Activation of p38 MAP Kinase in Human Platelets

Abstract

In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca2+-dependent cytosolic phospholipase A2 (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J. A.(1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation. In human platelets a proline-directed kinase distinct from the ERK MAP kinases is stimulated by both thrombin and the thrombin receptor agonist peptide SFLLRN and may be involved in the activation of Ca2+-dependent cytosolic phospholipase A2 (Kramer, R. M., Roberts, E. F., Hyslop, P. A., Utterback, B. G., Hui, K. Y., and Jakubowski, J. A.(1995) J. Biol. Chem. 270, 14816-14823). Here we show that this kinase is identical with or closely related to p38 (the mammalian homolog of HOG1 from yeast), a recently discovered protein kinase typically activated by inflammatory cytokines and environmental stress. Further, we demonstrate that activation of this kinase by thrombin is transient (with maximal stimulation at 1 min), is accompanied by tyrosine phosphorylation, and precedes the activation of the ERK kinases. This is the first report to show that p38 kinase is activated by thrombin and to suggest a role for this MAP kinase in the thrombin-mediated signaling events during platelet activation. INTRODUCTIONWe have recently shown that thrombin stimulates the activity of the MAP 1The abbreviations used are: MAPmitogen-activated proteinERKextracellular signal-regulated kinasecPLA2cytosolic phospholipase A2JNKJun nuclear kinaseCAPS3-(cyclohexylamino)-1-propanesulfonic acidDTTdithiothreitolIEPisoelectric pointPAGEpolyacrylamide gel electrophoresis. kinases ERK1 and ERK2 but also activates another proline-directed kinase that is distinguishable from ERK1/2 based on its strong binding to anion exchange resin and the lack of reactivity with anti-ERK1/2 antibodies (1Kramer R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar). We further noted that this kinase readily phosphorylates cPLA2 but not the S505A mutant of cPLA2. This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser505-Pro) is the target phosphorylation site for the kinase. Significantly, the thrombin receptor agonist peptide SFLLRN also activated this proline-directed kinase but completely failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated activation of cPLA2 by phosphorylation, and we reasoned that this unidentified kinase could play a role in the signal transduction pathways activated through the thrombin receptor. We therefore further characterized the kinase with the goal to determine its identity and define its role in the thrombin-induced signaling events during platelet activation.EXPERIMENTAL PROCEDURESPlatelet Isolation and IncubationFresh human platelets were prepared from platelet-rich plasma of drug-free volunteers in the presence of prostacyclin (10-8M) and apyrase (0.5 units/ml) as described previously(2Kramer R.M. Roberts E.F. Manetta J.V. Hyslop P.A. Jakubowski J.A. J. Biol. Chem. 1993; 268: 26796-26804Google Scholar), suspended at 1.25 × 109/ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl2, 2.2 mM CaCl2, 5.5 mM glucose, 0.2 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 μM cyclo(S,S)-Mpr(Har)-GDWP-Pen-NH2 (where Mpr is mercaptopropionyl, Har is homoarginine, and Pen is penicillamine) (3Scarborough R.M. Naughton M.A. Teng W. Rose J.W. Phillips D.R. Nannizzi L. Arfsten A. Campbell A.M. Charo I.F. J. Biol. Chem. 1993; 268: 1066-1073Google Scholar) (kindly provided by Dr. Robert Scarborough, COR Therapeutics), and incubated at 37°C with 5 units/ml α-thrombin (~3500 NIH units/mg, Enzyme Research Laboratories). Reactions were terminated by adding (final concentrations) 1% Triton X-100, 5 mM EGTA, 1 mM DTT, 0.2 mM Na3VO4, 100 nM microcystin (Life Technologies, Inc.), 100 μM leupeptin, 0.2 mg/ml aprotinin, 10 μM pepstatin A, 1 mM Pefabloc (Centerchem), and 50 mM β-glycerophosphate, pH 7.5. The suspension was then briefly sonicated, centrifuged for 30 min at 100,000 × g using a Sorvall RC M120EX microcentrifuge, and diluted with MonoQ buffer as indicated.Partial Purification of p38 and ERK Kinases by MonoQ ChromatographyHigh speed supernatants were subjected to chromatography on a MonoQ HR 5/5 column (Pharmacia Biotech Inc.) at a flow rate of 1.5 ml/min collecting 0.5-ml fractions using two different procedures. In order to partially purify the p38 kinase, the column was first equilibrated in buffer A (1 mM EGTA, 1 mM DTT, 100 μM Na3VO4, 50 mM β-glycerophosphate, pH 7.5) containing 150 mM NaCl and subjected to a 30-ml linear salt gradient from 150 to 530 mM NaCl. For rapid partial purification of the p38 and the ERK kinases, the MonoQ column was equilibrated in buffer A containing 50 mM NaCl and then subjected to a step gradient from 50 to 250 mM NaCl (for elution of ERK1/2), followed by a step gradient from 250 to 450 mM NaCl (for elution of p38). MonoQ fractions (0.5 ml) were collected into 10 μl of a mixture providing (final concentrations) 100 nM microcystin, 100 μM leupeptin, 0.1 mg/ml aprotinin, and 1 mM Pefabloc.Assay for Proline-directed KinasesKinase assays were performed as described before (1Kramer R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar) using the Thr669 peptide substrate (KRELVEPLTPSGEAPNQALLR from Macromolecular Resources, Colorado State University) and the SpinZyme system (Pierce).SDS-PAGE and ImmunoblottingSamples were electrophoresed in 7.5% Tris/HCl gels (1 mm thick; Daiichi) or 10% Tris/glycine gels (1.5 mm thick; Novex) according to instructions of the manufacturer using Rainbow markers (range, 14.3-200 kDa; Amersham Corp.) as standards. Proteins were electroblotted onto polyvinyldifluoride membranes (Bio-Rad) using as blotting buffer 10 mM CAPS, pH 11.0, containing 0.01% SDS and 1% methanol. The membranes were incubated as follows: 1 h at room temperature in TBST (137 mM NaCl, 2.6 mM KCl, 0.1% Tween 20, 25 mM Tris/HCl, pH 7.5, containing 0.2% I-Block (Tropix)), followed by 1 h with primary antibody as indicated (see figure legends) in TBST and 30 min with secondary antibody (either goat anti-rabbit or goat anti-mouse IgG(H+L)/horseradish peroxidase conjugate (from Jackson ImmunoResearch and Bio-Rad, respectively)) in TBST. For immunoblotting with anti-phosphotyrosine antibodies we used bovine serum albumin (0.75%) instead of I-Block. The blots were developed using the ECL detection system (Amersham Corp.).RESULTS AND DISCUSSIONIn human platelets thrombin activates several kinases that readily phosphorylate the Thr669 peptide derived from the epidermal growth factor receptor(1Kramer R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar). Based upon the distinct chromatographic and immunological characteristics, these kinases could be distinguished and found to consist of the MAP kinases ERK1/2, as well as another unidentified proline-directed kinase. When extracts from control and thrombin-stimulated platelets were applied to MonoQ in buffer containing 150 mM NaCl, ERK1/2 flowed through the column. The unknown thrombin-activated Thr669 kinase, on the other hand, bound tightly to the column and eluted at ≥350 mM NaCl (Fig. 1A). Consequently, the isoelectric point (IEP) of this kinase had to be significantly lower than that of ERK1/2 (~6.8). For example, the IEP of cPLA2 that binds similarly to MonoQ eluting with ~400 mM NaCl is 5.1(4Kramer R.M. Roberts E.F. Manetta J. Putnam J.E. J. Biol. Chem. 1991; 266: 5268-5272Google Scholar). Only three recently identified proline-directed kinases exhibit calculated IEPs in agreement with the observed chromatographic behavior of the platelet Thr669 kinase. These include JNK2 (p54α1)(5Kyriakis J.M. Banerjee P. Nikolakaki E. Dal T. Ruble E.A. Ahmad M.F. Avruch J. Woodgett J.R. Nature. 1994; 369: 156-160Google Scholar), p38 kinase(6Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Google Scholar), and an ERK3 homolog (referred to as p97MAPK) (7Zhu A.X. Zhao Y. Moller D.E. Flier J.S. Mol. Cell. Biol. 1994; 14: 8202-8211Google Scholar) with IEPs of 5.7, 5.6, and 4.8, respectively.We therefore subjected extracts from thrombin-stimulated platelets and the most active Thr669 kinase MonoQ fractions 34 and 35 (see Fig. 1A) to SDS-PAGE/immunoblotting, probing with antibodies against JNK2, p38, and ERK3. As shown in Fig. 2A anti-p38 antibodies specifically recognized a protein of ~40 kDa that was enriched in the MonoQ fractions (lane 2) compared with the loaded platelet extract (lane 1). The same ~40-kDa protein strongly reacted with anti-phosphotyrosine antibodies (lane 4). Although ERK1 and ERK2 could be readily detected in extracts (lane 5), they were absent in the MonoQ fractions containing the Thr669 kinase activity (lane 6). As demonstrated in Fig. 2B, no immunoreactivity could be detected in extracts or active MonoQ fractions when probing with anti-JNK antibodies (lanes 8 and 9) or anti-ERK3 antibodies (lanes 11 and 12). By comparison both kinases could be readily seen when standard proteins were tested (lane 7 and lane 10). We confirmed the specificity of two anti-p38 antibodies (anti-p38N and anti-p38C) for the ~40-kDa protein by competition experiments with the respective C- and N-terminal peptides of p38 used for immunization. As demonstrated in Fig. 2C, the reactivity with the ~40-kDa protein was significantly decreased when the immunizing peptides were present during immunoblotting, including the anti-p38N (lane 14 versus lane 13) and the anti-p38C (lane 16 versus lane 15) antibodies. We further examined whether the kinase activity eluting from the MonoQ column (Fig. 1A) paralleled the immunoreactivity of the eluting kinase protein probing with both anti-p38 and anti-phosphotyrosine antibodies. As shown in Fig. 1B (upper right panel), the MonoQ elution of the ~40-kDa protein recognized by the anti-p38 antibodies correlated with the thrombin-induced Thr669 kinase activity. Likewise, coincident with the peak of kinase activity we detected thrombin-induced tyrosine phosphorylation of the same ~40-kDa protein (Fig. 1B, lower panel). Taken together, these data indicate that the proline-directed kinase activated by thrombin is identical with, or closely related to, the p38 MAP kinase.Figure 2:Immunological identification of thrombin-stimulated Thr669 kinase. Soluble extracts (C) from thrombin-stimulated platelets (10 μl), MonoQ fractions containing the Thr669 kinase (K) (10 μl) (as in Fig. 1A), and standard proteins (St) were subjected to SDS-PAGE/immunoblotting probing with different antibodies as detailed under “Experimental Procedures.” A, anti-p38 (C-20) polyclonal antibody at 0.1 μg/ml (Santa-Cruz Biotechnology) (lanes 1 and 2), anti-phosphotyrosine (αP-Y) monoclonal antibodies 4G10 (Upstate Biotechnology) plus PY20 (ICN) at 1 μg/ml each (lanes 3 and 4), and anti-ERK1/2 polyclonal antibody erk1-CT (Upstate Biotechnology) at 1 μg/ml (lanes 5 and 6). B, anti-JNK2 polyclonal antibody (Santa-Cruz Biotechnology) at 0.1 μg/ml (lanes 7-9) and anti-ERK3 antibody (Transduction Laboratories) at 1 μg/ml (lanes 10-12). The ability of anti-JNK2 and anti-ERK3 antibodies to recognize JNK2 and ERK3, respectively, was verified with purified human JNK2 (Santa-Cruz Biotechnology) (lane 7) and human fibroblast ERK3 (Transduction Laboratories) (lane 10). C, anti-p38(N-20) antibodies (Santa-Cruz Biotechnology) in the absence (lane 13) and presence of immunizing peptide N-20 (lane 14), and anti-p38 (C-20) antibody in the absence (lane 15) and presence of immunizing peptide C-20 (lane 16). Migration position of molecular mass marker is indicated on the right.View Large Image Figure ViewerDownload (PPT)We determined the kinetics of thrombin-mediated activation of the p38 kinase, resolving it from ERK1/2 by MonoQ chromatography. As shown in Fig. 3A, thrombin induced a transient stimulation of p38 kinase activity that reached a maximum at 1 min and was still detected at the latest time point measured (5 min). The amount of p38 protein purified by MonoQ chromatography was the same for all time points examined, as verified by SDS-PAGE immunoblotting (Fig. 3B). The appearance and disappearance of p38 kinase activity in thrombin-stimulated platelets temporally coincided with the tyrosine phosphorylation of p38 (Fig. 3C). By comparison, activation of the ERKs was delayed with maximal stimulation at 2 min following thrombin stimulation, as shown by the ability of ERK1/2 to phosphorylate the Thr669 peptide substrate (Fig. 4A) and the decreased electrophoretic mobility of the ERK proteins (Fig. 3C and Fig. 4B), indicative of activation(8Leevers S.J. Marshall C.J. EMBO J. 1992; 11: 569-574Google Scholar). The data in Fig. 4 also reveal that activation by thrombin of p38 is more prominent than that of ERK1/2. A similar robust activation of p38 and delayed stimulation of ERK1/2 were observed in aspirinized platelets (where the synthesis of endogenous thromboxane A2 is inhibited), demonstrating that p38 kinase is the target of thrombin and not of the secondary agonist thromboxane A2 that is released from activated platelets.Figure 3:Time course of thrombin-mediated activation of p38 kinase. Platelets (0.6 ml at 1.25 × 109/ml) were incubated at 37°C in the absence (-THR) and presence of 5 units/ml of thrombin (+THR) as described under “Experimental Procedures.” After addition of 150 μl of Triton X-100 stopping mixture and ultracentrifugation, the extracts were diluted with 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 100 μM Na3VO4, and 50 mM β-glycerophosphate, pH 7.5 and subjected to MonoQ chromatography as described under “Experimental Procedures.” A, determination of kinase activity in pooled MonoQ fractions containing the p38 kinase using 2 mM Thr669 peptide substrate as detailed under “Experimental Procedures.” B, SDS-PAGE/immunoblotting verifying that equal amounts of p38 kinase protein were present in the pooled active MonoQ fractions derived from platelets incubated without thrombin (-THR) for 30 s to 5 min (lanes 1-4) and with thrombin (+THR) for 0-5 min (lanes 5-10). C, SDS-PAGE/immunoblotting of solubilized lysates (10 μl) from platelets incubated with thrombin (5 units/ml) for 0-5 min as indicated, probing for the presence of tyrosine-phosphorylated proteins (αP-Y, lanes 1-5), p38 kinase (lanes 6-10), and ERK1/2 (lanes 11-14) using the antibodies as described in Fig. 2. The data shown are representative of two independent experiments yielding similar results, and the values shown in A are means ± range of duplicate incubations.View Large Image Figure ViewerDownload (PPT)Figure 4:Differential activation of p38 and ERK kinases by thrombin. Platelets (0.6 ml at 1.25 × 109/ml) were incubated at 37°C with 5 units/ml thrombin, and the reaction was quenched as described under “Experimental Procedures.” The solubilized lysates were cleared by ultracentrifugation, diluted with 1 mM EGTA, 1 mM DTT, 100 μM Na3VO4, and 50 mM β-glycerophosphate, pH 7.5 (buffer A), and passed over a MonoQ column equilibrated in buffer A containing 50 mM NaCl. ERK1/2 and p38 were eluted with a step salt gradient and recovered in 1.5 ml of buffer A containing 250 mM NaCl and 1.5 ml of buffer A containing 450 mM NaCl, respectively, as described under “Experimental Procedures.” A, determination of kinase activity in 8.3 μl of the 1.5-ml MonoQ pools using 2 mM Thr669 peptide substrate as detailed under “Experimental Procedures.” B, SDS-PAGE/immunoblotting demonstrating the presence of ERK1/2 (lanes 5-8) and p38 (lanes 1-4) in the respective 1.5-ml MonoQ pools probing with the antibodies described in Fig. 2. The data shown are representative of two independent experiments yielding similar results, and the values shown in A are means ± range of duplicate incubations, each assayed in duplicate.View Large Image Figure ViewerDownload (PPT)The p38 kinase belongs to a new subfamily of stress-activated MAP kinases related to the HOG1 gene product, a kinase required for adaptation to osmotic stress in Saccharomyces cerevisiae(9Brewster J.L. de Valoir T. Dwyer N.D. Winter E. Gustin M.C. Science. 1993; 259: 1760-1763Google Scholar), and has only recently been identified in mammalian cells. Thus, Han et al.(10Han J. Lee J.-D. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 1993; 268: 25009-25014Google Scholar) first described this novel kinase of apparent molecular mass of 38 kDa (therefore referred to as p38) in cells of monocytic lineage, observing that it is rapidly phosphorylated on tyrosine residues in response to endotoxin. Cloning of the p38 kinase revealed that its predicted sequence is 52% identical to the yeast kinase HOG1 (6Han J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Google Scholar) and shares with HOG1 the unique sequence TGY comprising the dual phosphorylation site typical of MAP kinases. Lee et al.(11Lee J.C. Layton J.T. McDonnell P.C. Gallagher T.F. Kumar J.R. J.E. J.R. J.L. Nature. 1994; Scholar) identified the new kinase a target of synthesis that was found to be identical with p38 kinase. et J. P. A. T. Cell. 1994; Scholar) discovered a stress-activated kinase recognized by antibodies against the kinase a kinase closely related to HOG1 from and et L. E. J. J. Cell. 1994; Scholar) purified an kinase from human epidermal cells closely of the p38 kinase. by et J. J.S. Han J. Ulevitch R.J. R.J. J. Biol. Chem. 1995; 270: Scholar) that p38 kinase is activated not only by osmotic stress and but is also stimulated by inflammatory and to In the p38 kinase was only activated by growth and J. Lee J.-D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Google J. Lee J.-D. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 1993; 268: 25009-25014Google J. J.S. Han J. Ulevitch R.J. R.J. J. Biol. Chem. 1995; 270: Scholar). we report that the serine thrombin to a receptor a activation of the p38 kinase. The stimulation of p38 kinase by thrombin not only precedes that of the ERKs but is also more than that of the This that p38, than the may be involved in proline-directed phosphorylation events during thrombin-mediated platelet activation. The in the of activation is with the that the p38 and ERK MAP kinases are by distinct signaling pathways J. Biol. Chem. 1995; 270: kinase to the activation of the ERK MAP kinases of and of two protein kinases and MAP kinase to the J. Biol. Chem. 1995; 270: Scholar). In the and protein kinases involved in the activation of p38 are not of p38 kinase dual phosphorylation on and J. J.S. Han J. Ulevitch R.J. R.J. J. Biol. Chem. 1995; 270: Scholar). a MAP kinase kinase referred to as A. A. Science. 1995; 268: Scholar) or B. J. T. Han J. Ulevitch R.J. R.J. Science. 1995; Scholar) was identified that activates the p38 and also the kinases. kinase, as the human homolog of the yeast MAP kinase kinase activated only the p38 kinase and not the ERK and MAP B. J. T. Han J. Ulevitch R.J. R.J. Science. 1995; Scholar). Thus, may be a of an independent signaling that specifically activates the p38 kinase. the proteins and were shown to be of the signaling the p38 kinase A. A. A. Cell. 1995; show that the p38 kinase is present in human it is and stimulated by thrombin. Although the of the signaling pathways that in stimulation of platelets upon activation of the thrombin receptor has not been completely it is that the activated thrombin receptor to and of the proteins and 1994; Scholar). to proteins are to the ERK MAP kinase activated and protein 1994; Scholar). The thrombin receptor agonist peptide SFLLRN also stimulates the proline-directed kinase, identified as p38, thrombin, not R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar). These suggest the of pathways to the activation of p38 or the both pathways be stimulated by thrombin, SFLLRN activates the signaling p38 at in the p38 kinase is for of cPLA2. other of the p38 kinase to be p38 is to be an of a signaling by the thrombin receptor in and it be of to further the role of p38 in platelet rapidly and stimulates the p38 kinase in human demonstrating that other than events and cytokines this proline-directed kinase. Taken with (1Kramer R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar) these suggest that in platelets thrombin activates two distinct signaling pathways that in the activation of the p38 or the ERK MAP kinases, the thrombin receptor agonist peptide SFLLRN through the p38 and cPLA2 to be of the of p38 kinase. INTRODUCTIONWe have recently shown that thrombin stimulates the activity of the MAP 1The abbreviations used are: MAPmitogen-activated proteinERKextracellular signal-regulated kinasecPLA2cytosolic phospholipase A2JNKJun nuclear kinaseCAPS3-(cyclohexylamino)-1-propanesulfonic acidDTTdithiothreitolIEPisoelectric pointPAGEpolyacrylamide gel electrophoresis. kinases ERK1 and ERK2 but also activates another proline-directed kinase that is distinguishable from ERK1/2 based on its strong binding to anion exchange resin and the lack of reactivity with anti-ERK1/2 antibodies (1Kramer R.M. Roberts E.F. Hyslop P.A. Utterback B.G. Hui K.Y. Jakubowski J.A. J. Biol. Chem. 1995; 270: 14816-14823Google Scholar). We further noted that this kinase readily phosphorylates cPLA2 but not the S505A mutant of cPLA2. This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser505-Pro) is the target phosphorylation site for the kinase. Significantly, the thrombin receptor agonist peptide SFLLRN also activated this proline-directed kinase but completely failed to stimulate ERK1/2. Nonetheless SFLLRN, like thrombin, mediated activation of cPLA2 by phosphorylation, and we reasoned that this unidentified kinase could play a role in the signal transduction pathways activated through the thrombin receptor. We therefore further characterized the kinase with the goal to determine its identity and define its role in the thrombin-induced signaling events during platelet activation.