Maximal epidermal growth-factor-induced cytosolic phospholipase A₂ activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration

The 85 kDa cytosolic phospholipase A₂ (cPLA₂) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA₂ can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF). A full activation of cPLA₂ requires an increase of intracellular Ca²⁺ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca²⁺] ([Ca²⁺](i)) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA₂. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase 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 the role of each pathway in cPLA₂ activation. We show that EGF stimulates cPLA₂ activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by ³H release from cells prelabelled with [³H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. 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 cPLA₂ activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca²⁺](i) concentration.