The 85-kDa cytosolic PLA2 (cPLA2) is present in many cells and tissues and its unusual functional properties and catalytic mechanism are being elucidated. Notably, cPLA2 becomes catalytically active in the presence of free Ca 2+ concentrations as present in stimulated cells and preferentially cleaves arachidonic acid-containing phospholipids. A variety of agonists, growth factors and cytokines, as well as stressful stimuli activate cPLA2 to hydrolyze cellular phospholipids thereby liberating fatty acids and lysophospholipids and providing the precursor substrates for the biosynthesis of eicosanoids and platelet-activating factor. These products of cPLA2 contribute to inflammatory and degenerative disease states and cPLA2 is therefore an attractive target for the development of novel therapies.
Two new cloned human cDNAs encode paralogs of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ). We propose to call these cPLA 2  (114 kDa) and cPLA 2 ␥ (61 kDa), giving the name cPLA 2 ␣ to the well known 85-kDa enzyme. cPLA 2  mRNA is expressed more highly in cerebellum and pancreas and cPLA 2 ␥ more highly in cardiac and skeletal muscle. Sequence-tagged site mapping places cPLA 2  on chromosome 15 in a region near a phosphoinositol bisphosphate phosphatase. The mRNA for cPLA 2  is spliced only at a very low level, and Northern blots in 24 tissues show exclusively the unspliced form. cPLA 2  has much lower activity on 2-arachidonoyl-phosphatidylcholine liposomes than either of the other two enzymes. Its sequence contains a histidine motif characteristic of the catalytic center of caspase proteases of the apoptotic cascade but no region characteristic of the catalytic cysteine. Sequence-tagged site mapping places cPLA 2 ␥ on chromosome 19 near calmodulin. cPLA 2 ␥ lacks the C2 domain, which gives cPLA 2 ␣ its Ca 2؉ sensitivity, and accordingly cPLA 2 ␥ has no dependence upon calcium, although cPLA 2  does. cPLA 2 ␥ contains a prenyl group-binding site motif and appears to be largely membrane-bound. cPLA 2 ␣ residues activated by phosphorylation do not appear to be well conserved in either new enzyme. In contrast, all three previously known catalytic residues, as well as one additional essential arginine, Arg-566 in cPLA 2 ␣, are conserved in both new enzyme sequences. Mutagenesis shows strong dependence on these residues for catalytic activity of all three enzymes.Enzymatic breakdown of glycerophospholipids is carried out by numerous pathways, with the production of various bioactive lipids and fatty acids (1-8). Diverse phospholipase A 2 (PLA 2 ) 1 enzymes hydrolyze the sn-2 bond of phospholipids, releasing lysophospholipids and fatty acids (9 -11). Often the released fatty acid is arachidonic acid, whose further metabolism gives rise to several types of bioactive lipids known as eicosanoids, many of which mediate inflammation. Thus PLA 2 enzymes initiate the production of inflammatory mediators (12, 13), and for that reason they have become targets for the development of anti-inflammatory therapies.The 85-kDa cytosolic phospholipase A 2 (reviewed in Refs. 12-15) has attracted special interest because it is the only one of numerous PLA 2 s that selectively releases arachidonic acid over other fatty acids (16,17). Recent results with transgenic mice ablated for this enzyme (18) have demonstrated its role in allergy and parturition. Several functional regions have been identified within its amino acid sequence, including the C2 or calcium and lipid binding region similar to the C2 regions of other proteins (19) such as the calcium-dependent protein kinase C enzymes; several serine residues capable of activation through phosphorylation (20 -22); a hydrophilic region that may have a structural role (13); and several residues essential for catalysis, identified by mutagenesis (23-25).The cDNA for the 85-kDa cP...
The Ca 2؉ -sensitive 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) is responsible for thrombin-stimulated mobilization of arachidonic acid for the synthesis of thromboxane A 2 in human platelets. We have previously shown that thrombin activates p38 kinase, a recently discovered new member of the mitogen-activated protein kinase family (Kramer, R. M., Roberts, E. F., Strifler, B. A., and Johnstone, E. M. (1995) J. Biol. Chem. 270, 27395-27398) and also induces phosphorylation of cPLA 2 , thereby increasing its intrinsic catalytic activity. In the present study we have examined the role of p38 kinase in the phosphorylation and activation of cPLA 2 in stimulated platelets. We have observed that activation of p38 kinase accompanies receptor-mediated events in platelets and coincides with cPLA 2 phosphorylation. Furthermore, in the presence of inhibitors of p38 kinase, the proline-directed phosphorylation of cPLA 2 was completely blocked in platelets stimulated with the thrombin receptor agonist peptide SFLLRN and was suppressed during the early (up to 2 min) phase of platelet stimulation caused by thrombin. Unexpectedly, we found that prevention of proline-directed phosphorylation of cPLA 2 in stimulated platelets did not attenuate its ability to release arachidonic acid from platelet phospholipids. We conclude that: 1) cPLA 2 is a physiological target of p38 kinase; 2) p38 kinase is involved in the early phosphorylation of cPLA 2 in stimulated platelets; and 3) proline-directed phosphorylation of cPLA 2 is not required for its receptor-mediated activation.On activation of platelets with physiological agonists such as thrombin, significant amounts of arachidonic acid are rapidly liberated for transformation to thromboxane A 2 via the cyclooxygenase-thromboxane synthase pathway. There is substantial evidence to indicate that this efficient receptor-mediated mobilization of arachidonic acid is mediated by a phospholipase A 2 pathway (1, 2) and that the involved phospholipase A 2 is the Ca 2ϩ -sensitive cytosolic phospholipase A 2 (cPLA 2 ) 1 (3, 4).Many studies with different cellular systems, including platelets, have documented that phosphorylation of cPLA 2 by receptor-mediated events accompanies the stimulated release of arachidonic acid from cellular phospholipids (5). Lin et al. (6) established that this phosphorylation is "activating" (i.e. it increases the catalytic activity of cPLA 2 severalfold) and occurs at Ser 505 residing within a MAP kinase consensus sequence (Pro-Leu-Ser 505 -Pro). In fact, cPLA 2 was phosphorylated and activated by the MAP kinase ERK2 in vitro and in vivo (i.e. in cultured cells overexpressing cPLA 2 and ERK2; Ref. 6), and accordingly, the ERKs were taken to be responsible for the proline-directed phosphorylation of cPLA 2 observed in various cellular systems. Surprisingly, we have noted that phosphorylation of cPLA 2 occurred in the absence of ERK activation in human platelets stimulated with the thrombin receptor agonist peptide SFLLRN (7). Furthermore, under conditions in which ER...
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.We have recently shown that thrombin stimulates the activity of the MAP 1 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 (1). We further noted that this kinase readily phosphorylates cPLA 2 but not the S505A mutant of cPLA 2 . This observation indicated that the serine residing within the MAP kinase consensus sequence (i.e. Pro-Leu-Ser 505 -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 cPLA 2 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 Incubation-Fresh human platelets were prepared from platelet-rich plasma of drug-free volunteers in the presence of prostacyclin (10 Ϫ8 M) and apyrase (0.5 units/ml) as described previously (2), suspended at 1.25 ϫ 10 9 /ml in 140 mM NaCl, 27 mM KCl, 1 mM MgCl 2 , 2.2 mM CaCl 2 , 5.5 mM glucose, 0.2 mM EGTA, 10 mM Hepes, pH 7.4, containing 30 M cyclo(S,S)-Mpr(Har)-GDWP-Pen-NH 2 (where Mpr is mercaptopropionyl, Har is homoarginine, and Pen is penicillamine) (3) (kindly provided by Dr. Robert Scarborough, COR Therapeutics), and incubated at 37°C with 5 units/ml ␣-thrombin (ϳ3500 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 Na 3 VO 4 , 100 nM microcystin (Life Technologies, Inc.), 100 M leupeptin, 0.2 mg/ml aprotinin, 10 M pepstatin A, 1 mM Pefabloc (Centerchem), and 50 mM -glycerophosphate, pH 7.5. The suspension was then briefly sonicated, centrifuged for 30 min at 100,000 ϫ g using a Sorvall RC M120EX microcentrifuge, and diluted with MonoQ buffer as indicated.Partial Purification of p38 and ERK Kinases by MonoQ Chromatography-High 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 ...
The present study identifies the phosphorylation sites of the 85-kDa cytosolic phospholipase A 2 (cPLA 2 ) in human platelets and HeLa cells. Tryptic digests of 32 Pphosphorylated and -immunoprecipitated cPLA 2 were analyzed by microbore high performance liquid chromatography and two-dimensional phosphopeptide mapping against synthetic phosphopeptide standards. Thrombin stimulated significant phosphorylation of platelet cPLA 2 at two sites, Ser-505 and Ser-727. Exclusive phosphorylation on these two sites was also seen in collagen-stimulated platelets and HeLa cells stimulated with interferon-␣ or arsenite; no tyrosine phosphorylation was detected. The inhibitor of the 38-kDa stressactivated protein kinase (p38 mapk ), SB 203580, reduced phosphorylation of both Ser-505 and Ser-727 by 50 and 60%, respectively, in thrombin-stimulated platelets. An additional p38 mapk inhibitor SB 202190 also partially (60%) inhibited the phosphorylation of cPLA 2 in arsenite-stimulated HeLa cells. These studies extend the previous work on the identification of multiple phosphorylation sites on cPLA 2 expressed in a baculovirus/insect cell system to cPLA 2 in mammalian cells stimulated with physiological agonists. They also underscore the necessity of high resolution phosphopeptide mapping combined with microbore high performance liquid chromatography for quantification of phosphorylation levels, which has lead to the conclusion that Ser-505 and Ser-727 are common phosphorylation sites on cPLA 2 in different mammalian cells stimulated with multiple agonists.Cytosolic phospholipase A 2 (cPLA 2 ) 1 catalyzes the cleavage of arachidonic acid from the sn-2 position of phospholipids (1, 2). The 85-kDa enzyme is present in many mammalian cells (3), and strong evidence is accumulating for the role of cPLA 2 in the generation of tissue mediators that are metabolites of arachidonic acid, such as prostaglandins, leukotrienes, and thromboxanes. In contrast to the small molecular weight phospholipase A 2 s that are secreted and are active on the outside of cells, cPLA 2 is regulated by intracellular signals that are propagated from surface receptors. One important regulatory mechanism appears to be a rise in the intracellular Ca 2ϩ concentration which causes translocation of cPLA 2 from the cytosol to internal membranes (4 -7) where it binds through a Ca 2ϩ -dependent lipid-binding domain (8). A second, well established mechanism of the regulation of cPLA 2 activity is by phosphorylation on Ser-505 through a mitogen-activated protein kinase (MAPK) (9) which modestly increases the intrinsic activity of the lipase measured in vitro (3, 10, 11). Phosphorylation of cPLA 2 together with release of arachidonic acid has been observed in a variety of cells (11)(12)(13)(14)(15)(16).A thorough characterization of the phosphorylation sites of human cPLA 2 heterologously expressed in Spodoptera frugiperda (Sf9) cells by high performance liquid chromatography (HPLC), mass spectrometry, and protein sequencing has revealed four sites of phosphorylation: Ser-...
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