The cationic cluster of group IVA phospholipase A2 (Lys488/Lys541/Lys543/Lys544) is involved in translocation of the enzyme to phagosomes in human macrophages

Abstract

Group IVA cytosolic phospholipase A2 (cPLA 2) plays a role in the microbicidal machinery of immune cells by translocating to phagosomes to initiate the production of antimicrobial eicosanoids. In this work, we have studied the involvement of the cationic cluster of cPLA2 (Lys 488 / Lys 541 /Lys 543 /Lys 544) in the translocation of the enzyme to the phagosomal cup in human macrophages responding to opsonized zymosan. Phagocytosis was accompanied by an increased mobilization of free arachidonic acid, which was strongly inhibited by pyrrophenone. In transfected cells, a catalytically active enhanced green fl uorescent proteinc PLA2 translocated to the phagocytic cup, which was corroborated by frustrated phagocytosis experiments using immunoglobulin G-coated plates. However, a cPLA2 mutant in the polybasic cluster that cannot bind the anionic phospholipid phosphatidylinositol 4, 5-bisphosphate (PIP 2) did not translocate to the phagocytic cup. Moreover, an enhanced yellow fl uorescent protein (EYFP)-cPLA2 and an enhanced cyan fl uorescent protein-pleckstrin homology (PH) domain of the phospholipase C 1(PLC 1) construct that specifi cally recognizes endogenous PIP2 in the cells both localized at the same sites on the phagosome. High cellular expression of the PH domain inhibited EYFP-cPLA2 translocation. On the other hand, group V-secreted phospholipase A 2 and group VIA calcium-independent phospholipase A 2 were also studied, but the results indicated that neither of these translocated to the phagosome. Collectively, these data indicate that the polybasic cluster of cPLA2 (Lys 488 /Lys 541 /Lys 543 /Lys 544) regulates the subcellular localization of the enzyme in intact cells under physiologically relevant conditions.