MAXIMAL EPIDERMAL GROWTH-FACTOR-INDUCED CYTOSOLIC PHOSPHOLIPASE A(2) ACTIVATION IN-VIVO REQUIRES PHOSPHORYLATION FOLLOWED BY AN INCREASED INTRACELLULAR CALCIUM-CONCENTRATION

The 85 kDa cytosolic phospholipase A(2) (cPLA(2)) preferentially catal yses the hydrolysis of arachidonic acid from the sn-2 position of phos pholipids. cPLA(2) can be activated by extracellular stimuli such as t hrombin, platelet-derived growth factor and epidermal growth factor (E GF). A full activation of cPLA(2) requires an increase of intracellula r Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activat ed protein (MAP) kinase. Because EGF can provoke an increase in intrac ellular [Ca2+] ([Ca2+](i)) and activation of MAP kinase, we investigat ed the role of these pathways in EGF-induced activation of cPLA(2). Ch aracterization of two cell lines expressing different numbers of EGF r eceptors (HERc13 and HER14) revealed that both were activating MAP kin ase in response to EGF, but only HER14 responded with an increase in [ Ca2+](i). In this study we used both cell lines as a tool to clarify t he role of each pathway in cPLA(2) activation. We show that EGF stimul ates cPLA(2) activity in both cell lines in vitro as measured in cytos olic fractions, but only in HER14 in vivo as measured by H-3 release f rom cells prelabelled with [H-3]arachidonic acid. This latter activati on can be restored in HERc13 cells by the addition of the ionophore A2 3187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA(2) activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+](i) concentration.