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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...