VISUALIZING LOCALIZATION & TRANSLOCATION OF THREE TYPES OF PHOSPHOLIPASE A2

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phospholipase A2 (PLA2) plays important roles in diverse cellular responses including fundamental metabolism and signal transduction by generating lysophospholipids and fatty acids such as arachidonic acid (AA), which is the precursor of eicosanoids. To date, many subtypes of mammalian PLA2s have been identified and classified into subgroups. In this study, we focus on three subtypes of them; the cytosolic group IV PLA2 (cPLA2), the secretory group V (sPLA2), and the cytosolic Ca2+independent group VI PLA2 (iPLA2). In the previous year (2003), we examined the secreted form of PLA2 and conducted a series of studies on its activation in macrophages following chronic exposure to lipopolysaccharide. Because sPLA2 is a secreted enzyme, it has been suggested that after cellular stimulation, it must be released to the extra-cellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. Using confocal laser scanning microscopy and GFP-tagged versions of sPLA2, we found that chronic exposure to lipopolysaccharide results in sPLA2 being associated with caveolin-2- containing granules close to the perinuclear region. This association is blocked by heparin (a cell-impermeable compound with high affinity for sPLA2), suggesting that the granules are formed by the internalization of sPLA2 previously associated with the outer cell surface. Perinuclear localization is not observed if the cells are treated with the group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, further indicating the important role played by cPLA2 in the activation process. These studies provided evidence that the encapsulation of sPLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to sPLA2 and COX-2 (cyclo-oxygenase-2) for efficient prostaglandin synthesis. Results from this project have been published on the Journal of Biological Chemistry in 2003 [Balboa et al., ¿Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells.¿(2003). Journal of Biological Chemistry, 278 (48), 48059-48065].