Calcium-mediated Translocation of Cytosolic Phospholipase A to the Nuclear Envelope and Endoplasmic Reticulum (*)

Abstract

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 Ca. 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 (ΔCII) fails to translocate in response to Ca. 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 ΔCII, 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.