Regulation of Cytosolic Phospholipase A₂ in a New Perspective:  Recruitment of Active Monomers from an Inactive Clustered Pool

Abstract

cPLA 2 plays a key role in many signal transduction cascades by hydrolyzing arachidonic acid from membrane phospholipids. Tight control of cPLA 2 activity by a number of regulatory mechanisms is essential to its cellular function. We recently described the localization of cPLA 2 in clusters in fibroblasts and now propose that these clusters reflect a localized inactive pool from which active monomers can be recruited to keep cPLA 2 activity under control on the subcellular level. Using an electron microscopic in vitro approach, we show that cPLA 2 monomers, but not the clusters, bind to membranes in a Ca 2+dependent manner. This binding is accompanied by hydrolytic activity. The present data combined with our previous observation of a relative abundance of clusters over monomers in fixed fibroblasts [Bunt,

Niemann-Pick Type C2 Deficiency in Human Fibroblasts Confers Robust and Selective Activation of Prostaglandin E2 Biosynthesis

“…6A), whereas in NPC2-null cells cPLA2 displayed a reticular/punctate cytoplasmic staining pattern (Fig. 6B) that was reported earlier for both unstimulated and stimulated fibroblasts (43,44). These fluorescence microscopy data correlated well with those of Western blot analysis (Fig.…”

“…cPLA2 is now considered to be a central enzyme in mediating AA release for eicosanoid production, including PGE2 (48,49). The most likely mechanism for constitutive cPLA2 activation and subsequent PGE2 production could include, in addition to its Ser-505 phosphorylation, the release of active cPLA2 monomers from inactive intracellular clusters (43,44). Indeed, it has been previously reported that in fibroblasts, cPLA2 is present in small clusters that were identified as punctuate structures by immunofluorescence and immunogold electron microscopy (43).…”

“…These cPLA2 clusters were randomly distributed throughout the cytosol and localized in the vicinity of major intracellular membranes, except for the Golgi apparatus. More importantly, upon cell stimulation with EGF or calcium ionophore A23187 that are known to activate cPLA2 and stimulate AA metabolism, the cPLA2 punctate localization pattern did not change (43), but the release of activated cPLA2 monomeric forms from inactive clusters and their binding to the nearing intracellular membranes were observed (44). The latter was accompanied by an induction of cPLA2 hydrolytic activity, indicating that the release of active cPLA2 monomers from the existent inactive cPLA2 clusters could be an important mechanism that controls cPLA2 activity in fibroblasts and quite possibly in other nonimmune cells (44).…”

“…More importantly, upon cell stimulation with EGF or calcium ionophore A23187 that are known to activate cPLA2 and stimulate AA metabolism, the cPLA2 punctate localization pattern did not change (43), but the release of activated cPLA2 monomeric forms from inactive clusters and their binding to the nearing intracellular membranes were observed (44). The latter was accompanied by an induction of cPLA2 hydrolytic activity, indicating that the release of active cPLA2 monomers from the existent inactive cPLA2 clusters could be an important mechanism that controls cPLA2 activity in fibroblasts and quite possibly in other nonimmune cells (44). Given that very similar punctate cPLA2 structures were observed in NPC2-null fibroblasts by immunofluorescence in the current study and that cPLA2 phosphorylation by ERK1/2 was proposed to be a prerequisite for cPLA2 release from the inactive clusters (44), one may suggest that RTK/GFR 3 ERK1/2 3 cPLA2 phosphorylation axis could be a primary regulatory target for NPC2 within the AA metabolic pathway.…”

Phosphorylation of p42/44MAPK by Various Signal Transduction Pathways Activates Cytosolic Phospholipase A2to Variable Degrees

“…cPLA 2 in Her14 fibroblasts was reported to be localized in clusters near the vicinity of all organellar membranes except for the Golgi apparatus and the nucleus (63). Subsequently, it was shown that these clusters represent the inactive form of cPLA 2 from which active monomers can be recruited that might occur via phosphorylation by p42/44 MAPK (64). Also, p42/44 MAPK phosphorylation has been reported to occur at different locations induced by different agonists.…”