Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress-activated protein kinase pathways

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, SE 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 SE 202190 partially (approximate to 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.