Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA₂α to Membrane

The group IVA phospholipase A₂, commonly called cytosolic phospholipase A₂α (cPLA₂α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cyclooxygenase and lipoxygenase pathways for production of prostaglandins and leukotrienes that regulate normal physiological processes and contribute to disease pathogenesis. cPLA₂α is composed of an N-terminal C2 domain and a C-terminal catalytic domain that contains the Ser-Asp catalytic dyad. The catalytic domain contains phosphorylation sites and basic residues that regulate the catalytic activity of cPLA₂α. In response to cell stimulation, cPLA₂α is rapidly activated by posttranslational mechanisms including increases in intracellular calcium and phosphorylation by mitogen-activated protein kinases. In resting cells, cPLA₂α is localized in the cytosol but translocates to membrane including the Golgi, endoplasmic reticulum, and the peri-nuclear membrane in response to increases in intracellular calcium. Calcium binds to the C2 domain, which promotes the interaction of cPLA₂α with membrane through hydrophobic interactions. In this chapter, we describe assays used to study the calcium-dependent translocation of cPLA₂α to membrane, a regulatory step necessary for access to phospholipid and release of arachidonic acid.