Mechanism of group IVA cytosolic phospholipase A2 activation by phosphorylation

Group IVA cytosolic phospholipase A(2) (cPLA(2)) has been shown to play a critical role in the agonist-induced release of arachidonic acid. To understand the mechanism by which phosphorylation of Ser(505) and Ser(727) activates cPLA(2), we systematically analyzed the effects of S505A, S505E, S727A, S727E, S505A/S727A, S505A/S727E, and S505E/S727E mutations on its enzyme activity and membrane affinity. In vitro membrane binding measurements showed that S505A has lower affinity than the wild type or S505E for phosphatidylcholine membranes, which is exclusively due to faster desorption of the membrane-bound S505A. In contrast, neither S727A nor S727E mutation had a significant effect on the phosphatidylcholine vesicle binding affinity of cPLA(2). The difference in in vitro membrane affinity between wild type ( or S505E) and S505A increased with the decrease in Ca2+ concentration, reaching > 60-fold at 2.5 muM Ca2+. When HEK293 cells transfected with cPLA(2) and mutants were stimulated with ionomycin, the wild type and S505E translocated to the perinuclear region and caused the arachidonic acid release at 0.4 muM Ca2+, whereas S505A showed no membrane translocation and little activity to release arachidonic acid. Further mutational analysis of hydrophobic residues in the active site rim (Ile(399), Leu(400), and Leu(552)) indicate that a main role of the Ser(505) phosphorylation is to promote membrane penetration of these residues, presumably by inducing a conformational change of the protein. These enhanced hydrophobic interactions allow the sustained membrane interaction of cPLA(2) in response to transient calcium increases. On the basis of these results, we propose a mechanism for cPLA(2) activation by calcium and phosphorylation.