We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A 2 (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca 2؉ leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.
The kinase inhibitors SB 203580 and PD 98059 have been reported to be specific inhibitors of the 38-and 42/44-kDa mitogen-activated protein kinase (MAPK) pathways, respectively. In this study, the two inhibitors were found to decrease platelet aggregation induced by low concentrations of arachidonic acid, suggesting that they also interfere with the metabolism of arachidonic acid to thromboxane A 2 . In support of this, SB 203580 and PD 98059 inhibited the conversion of exogenous [ 3 H]arachidonic acid to [ 3 H]thromboxane in intact platelets. Measurement of platelet cyclooxygenase-1 activity following immunoprecipitation revealed that SB 203580 and PD 98059 are direct inhibitors of this enzyme. Both compounds were shown to inhibit purified cyclooxygenase-1 and -2 by a reversible mechanism. In addition, SB 203580 (but not PD 98059) inhibited platelet aggregation induced by prostaglandin H 2 and the conversion of prostaglandin H 2 to thromboxane A 2 in intact platelets. SB 203580 also inhibited this pathway in platelet microsome preparations, suggesting a direct inhibitory effect on thromboxane synthase. These results demonstrate that direct effects of the two kinase inhibitors on active arachidonic acid metabolites have to be excluded before using these compounds for the investigation of MAPKs in signal transduction pathways. This is of particular relevance to studies on the regulation of cytosolic phospholipase A 2 as these two MAPKs are capable of phosphorylating cytosolic phospholipase A 2 , thereby increasing its intrinsic activity.Since specific inhibitors of the mitogen-activated protein kinase (MAPK) 1 and stress-activated protein kinase (SAPK) cascades were first described, they have been widely exploited to investigate the involvement of p38 mapk (also called SAPK2a) and p42/p44 mapk in intracellular signal transduction pathways. The pyridinylimidazole compound SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole) was developed from a series of bicyclic pyridin-4-ylimidazoles that exhibited potent anti-inflammatory actions mediated via inhibition of cyclooxygenase, 5-lipoxygenase, and inflammatory cytokine biosynthesis (for review, see Ref. 1). The lipopolysaccharide-stimulated production of interleukin-1 and tumor necrosis factor-␣ was decreased in human monocytes in the presence of these compounds, and they were therefore termed cytokinesuppressive anti-inflammatory drugs. Further studies revealed that the molecular target of the pyridinylimidazoles was the stress-activated p38 mapk (2, 3). SB 203580 inhibits p38 mapk and its isoform p38 (SAPK2b) with in vitro IC 50 values of 0.3-0.6 M, but has no inhibitory action on SAPK3 and SAPK4 (4 -6).The flavone compound PD 98059 (2-(2-amino-3-methoxyphenyl)oxanaphthalen-4-one) is a specific inhibitor of the mammalian MAPK kinase (MEK) (7). It acts by binding to the inactivated form of MEK, thereby preventing its phosphorylation by c-Raf or MEK kinase (8). Depending on cell type and stimulation, the IC 50 determined in intact cells r...
The Src homology (SH)2 domain-containing proteintyrosine phosphatase SHP-1 is tyrosine phosphorylated in platelets in response to the glycoprotein VI (GPVI)-selective agonist collagen-related peptide (CRP), collagen, and thrombin. Two major unidentified tyrosinephosphorylated bands of 28 and 32 kDa and a minor band of 130 kDa coprecipitate with SHP-1 in response to all three agonists. Additionally, tyrosine-phosphorylated proteins of 50 -55 and 70 kDa specifically associate with SHP-1 following stimulation by CRP and collagen. The tyrosine kinases Lyn, which exists as a 53 and 56-kDa doublet, and Syk were identified as major components of these bands, respectively. Kinase assays on SHP-1 immunoprecipitates performed in the presence of the Src family kinase inhibitor PP1 confirmed the presence of a Src kinase in CRP-but not thrombin-stimulated cells. Lyn, Syk, and SLP-76, along with tyrosinephosphorylated 28-, 32-, and 130-kDa proteins, bound selectively to a glutathione S-transferase protein encoding the SH2 domains of SHP-1, suggesting that this is the major site of interaction. Platelets isolated from motheaten viable mice (mev/mev) revealed the presence of a heavily tyrosine-phosphorylated 26-kDa protein that was not found in wild-type platelets. CRP-stimulated mev/mev platelets manifested hypophosphorylation of Syk and Lyn and reduced P-selectin expression relative to controls. These observations provide evidence of a functional role for SHP-1 in platelet activation by GPVI.In blood platelets, agonist-induced increases in protein-tyrosine phosphorylation is mediated primarily by cytosolic protein-tyrosine kinases, including members of the Src, Syk, Tec, focal adhesion kinase (FAK), and Jak tyrosine kinases (1, 2). Indeed, platelets express some of the highest levels of tyrosine kinase activity of any cell in the body, with Syk and Src each making up approximately 0.1-0.2% of cellular protein. Nevertheless, resting platelets have relatively low levels of tyrosinephosphorylated proteins, indicating that protein-tyrosine phosphatase activity must be relatively high (3,4). This is illustrated by the dramatic increase of tyrosine phosphorylation observed in platelets within 10 s of incubation with the tyrosine phosphatase inhibitor peroxovanadate (5). Several proteins undergo only transient increases in tyrosine phosphorylation upon agonist stimulation, e.g. the adapter LAT in response to GPVI 1 activation (6), further emphasizing the role of protein-tyrosine phosphatases in regulating platelet activation.The tyrosine phosphatases SHP-1 and SHP-2 both contain tandem SH2 domains within the N-terminus, enabling association with tyrosine-phosphorylated proteins. SHP-1 is restricted to hematopoietic cells, whereas SHP-2 is ubiquitous. SHP-1 has been intensively studied in the regulation of the immune system. Mutation of SHP-1 in mice is accompanied by defects in immunity and hematopoiesis (7). SHP-1 is selectively recruited to the surface membrane in hematopoietic cells through interaction with a phosphorylated immun...
Stress-activated protein kinases (SAPKs) are stimulated by cell damaging agents as well as by physiological receptor agonists. In this study we show that human platelets contain the isoforms SAPK2a, SAPK2b, SAPK3 and SAPK4 as determined by immunoblotting with specific antibodies. All four kinases were activated in thrombin-stimulated platelets whereas only SAPK2a and SAPK2b were significantly stimulated by collagen. All four isoforms were able to phosphorylate wild-type human cPLA 2 in vitro, although to different extents, but not cPLA 2 mutants that had Ser505 replaced by alanine. Phosphorylation at Ser505 was confirmed by phosphopeptide mapping using microbore HPLC. SAPK2a and 42-kDa mitogen-activated protein kinase incorporated similar levels of phosphate into cPLA 2 relative to the ability of each kinase to stimulate phosphorylation of myelin basic protein. SAPK2b and SAPK4 incorporated less phosphate, and cPLA 2 was a poor substrate for SAPK3. The inhibitor of SAPK2a and SAPK2b, SB 202190, completely blocked collagen-induced phosphorylation of cPLA 2 at its two phosphorylation sites in vivo, Ser505 and Ser727. We have also reported previously that SB 202190 partially (< 50%) blocks phosphorylation at both sites and to a similar extent in thrombin-stimulated platelets. Inhibition of phosphorylation resulted in a two-to threefold shift to the right in the concentration response curves for arachidonic acid release from thrombin-and collagen-stimulated platelets. Our data suggest that cPLA 2 is a substrate for several SAPK cascades and that phosphorylation of cPLA 2 augments arachidonic acid release.Keywords: arachidonic acid; collagen; cytosolic phospholipase A 2 ; platelet; stress-activated protein kinase; thrombin.The family of mitogen-activated protein kinases (MAPKs) is divided into two major classes, the extracellular-signal regulated protein kinases (ERKs) that are strongly activated by growth factors and tumour-promoting phorbol esters, and the stress-activated protein kinases (SAPKs) that are strongly activated by cellular stresses. The first isoform of the SAPK family, SAPK2a (also known as p38 or p38a) [1], was discovered independently by several groups [2±5]. Strong activators of this kinase include a range of stress stimuli such as hyperosmolarity, heat shock, chemical and oxidative stress and bacterial endotoxins, as well as physiological agents, notably proinflammatory cytokines [1]. Many of its substrates are transcription factors, but SAPK2a also activates a number of kinases, for example MAPK-activated protein kinases-2/3 (MAPKAP-K2/3), which in turn phosphorylates heat shock protein 27 [5,6], the MAPK-interacting kinases-1/2 (Mnk1/2) [7,8], the mitogen-and stress-activated protein kinases (e.g. MSK1) [9] and p38-regulated protein kinases (PRAK) [10]. Several other SAPKs related to SAPK2a have since been discovered. SAPK2b (p38b2) shares approximately 75% amino acid identity with SAPK2a and is similar in its activation profile and substrate specificity [11]. SAPK3 (p38g) [12±14] and SAPK4 (p38...
Collagen activates platelets through a tyrosine kinase-dependent pathway, involving phospholipase Cγ2. Functional responses such as aggregation and secretion induced by collagen are potentiated by preincubation with thrombopoietin (TPO). In this study, we show that collagen and thrombopoietin activate the phosphatidylinositol 3-kinase (PI 3-kinase) pathway and that this contributes to their respective actions. The structurally distinct inhibitors of PI 3-kinase, wortmannin, and LY294002, completely inhibit formation of phosphatidylinositol 3,4,5-trisphosphate by collagen. This leads to a substantial reduction in the formation of inositol phosphates and phosphatidic acid, 2 indices of PLC activity, and the consequent inhibition of intracellular Ca++[Ca++]i, aggregation and secretion. Potentiation of the collagen response by TPO is prevented in the presence of wortmannin and LY294002. However, when the 2 PI 3-kinase inhibitors are given after the addition of TPO but before the collagen, recovery of potentiation is observed. This suggests that potentiation is mediated through activation of PI 3-kinase. TPO stimulates aggregation of platelets from a low percentage of donors and this is also blocked by wortmannin. These results suggest that the PI 3-kinase pathway plays an important role in signaling by collagen and in the priming action of TPO.
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