Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum.

Cytosolic phospholipase A2 (cPLA2) is activated by a wide variety of stimuli to release arachidonic acid, the precursor of the potent inflammatory mediators prostaglandin and leukotriene. Specifically, cPLA2 releases arachidonic acid in response to agents that increase intracellular Ca2+. In vitro data have suggested that these agents induce a translocation of cPLA2 from the cytosol to the cell membrane, where its substrate is localized. Here, we use immunofluorescence to visualize the translocation of cPLA2 to distinct cellular membranes. In Chinese hamster ovary cells that stably overexpress cPLA2, this enzyme translocates to the nuclear envelope upon stimulation with the calcium ionophore A23187. The pattern of staining observed in the cytoplasm suggests that cPLA2 also translocates to the endoplasmic reticulum. We find no evidence for cPLA2 localization to the plasma membrane. Translocation of cPLA2 is dependent on the calcium-dependent phospholipid binding domain, as a calcium-dependent phospholipid binding deletion mutant of cPLA2 (delta CII) fails to translocate in response to Ca2+. In contrast, cPLA2 mutated at Ser-505, the site of mitogen-activated protein kinase phosphorylation, translocates normally. This observation, combined with the observed phosphorylation of delta CII, establishes that translocation and phosphorylation function independently to regulate cPLA2. The effect of these mutations on cPLA2 translocation was confirmed by subcellular fractionation. Each of these mutations abolished the ability of cPLA2 to release arachidonic acid, establishing that cPLA2-mediated arachidonic acid release is strongly dependent on both phosphorylation and translocation. These data help to clarify the mechanisms by which cPLA2 is regulated in intact cells and establish the nuclear envelope and endoplasmic reticulum as primary sites for the liberation of arachidonic acid in the cell.