Requirement of JNK-Mediated Phosphorylation for Translocation of Group IVA Phospholipase A2 to Phagosomes in Human Macrophages

Abstract

Eicosanoids are a broad family of lipids that play a critical role in host defense against bacterial and fungal infections. The first enzyme in the metabolic pathway for the generation of eicosanoids is group IVA phospholipase A2, also known as cytosolic phospholipase A2α (cPLA2α). During phagocytosis, cPLA2α has been found to translocate to the phagosome, although the molecular mechanism involved in such a translocation has not been elucidated. By using enhanced GFP-tagged proteins we show in this work that a nonphosphorylatable cPLA2α mutant (S505A) does not translocate to the phagosomes, but a mutant that mimics phosphorylation on Ser505 (S505E) does it so readily. During phagocytosis, endogenous cPLA2α is phosphorylated at Ser505, and inhibitors of JNK, but not of other related kinases such as p38 or the extracellular-regulated kinases 1 and 2, completely block such a phosphorylation. Inhibition of JNK activity also inhibits the translocation of cPLA2α to phagosomal membranes, as well as arachidonic acid release to the extracellular medium. Moreover, the S505E mutant makes the enzyme refractory to JNK inhibition, translocating normally to phagosomal membranes. Collectively, these data support a key role for JNK-mediated cPLA2α phosphorylation at Ser505 in the sequence of events leading to translocation and activation of the enzyme to phagosomal membranes in human macrophages.

Group V Secreted Phospholipase A2 Is Upregulated by IL-4 in Human Macrophages and Mediates Phagocytosis via Hydrolysis of Ethanolamine Phospholipids

“…In this regard, it is striking that sPLA 2 -V translocates to the forming phagosome in zymosan-stimulated murine macrophages (50, 66) but not in zymosan-stimulated human macrophages (33,34). These data suggest that, at least in humans, the regulatory actions of the enzyme on the phagocytosis process itself occur at a level distinct from that of the phagosome, probably at the plasma membrane level.…”

“…It should be noted that human macrophages are relatively large cells (diameter . 30 mm) (33,34); hence, their membrane phospholipid content is quite high (35). Thus, if the hydrolytic action of IL-4-induced sPLA 2 -V on membrane phospholipids is limited, the small changes involved easily could be obscured by the high phospholipid amount present in the cells.…”

“…Pools of cells corresponding to five donors were used in all experiments, and replicates were performed with different pools of cells. Macrophage differentiation (95% monocytes) was achieved by incubating the adhered monocytes in RPMI 1640 supplemented with 2 mM L-glutamine, 40 mg/ml gentamicin, and 5% human serum (which contains M-CSF) in a humidified atmosphere with 5% CO 2 , for 2 wk in the absence of exogenous cytokine mixtures, in accordance with previously published procedures (26,(33)(34)(35)(36). To generate classically (M1) or alternatively (M2) activated macrophages, the cells were treated with IFN-g (500 U/ml) plus LPS (10 ng/ml) or IL-4 (1000 U/ml), respectively, in serum-free medium for the indicated periods of time (37,38).…”

JNK and Ceramide Kinase Govern the Biogenesis of Lipid Droplets through Activation of Group IVA Phospholipase A2

“…Lee et al (35) showed that secretion of monocyte chemoattractant protein-1 induced by stimulation of macrophages with synthetic oligodeoxynucleotides containing CpG motifs depends on cPLA 2 ␣ activity, which can be blocked by pharmacological inhibition or down-regulation of JNK. Casas et al (36), on the other hand, showed that translocation of cPLA 2 ␣ to phagosomal membranes of macrophages, an event required for eicosanoid generation and killing of the ingested microbe, requires enzyme phosphorylation at Ser-505 and is blocked after pharmacological inhibition of JNK. Therefore, this study describes for the first time the crucial role of the JNK cascade in the activation of cPLA 2 ␣ for LD biogenesis.…”

Phospholipase A2IVα Regulates Phagocytosis Independent of Its Enzymatic Activity

“…The role of the phosphorylation of PLA 2 IV␣ in induction of membrane translocation is not fully defined, although a requirement for PLA 2 IV␣ phosphorylation for its full enzymatic activation has been reported by different groups (15,21,22). Serine phosphorylation on PLA 2 IV␣ (Ser-505 or Ser-727) is mediated mainly by the mitogen-activated protein kinases (MAPKs) ERK1/2, and by the stress kinases p38 and JNK, and this has been shown to increase the intrinsic enzymatic activity of PLA 2 IV␣ (23,24).…”