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

The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A(2) (cPLA(2)alpha). This work dissects the pathway leading to cPLA(2)alpha activation and LD biogenesis. Both processes were Ca2+-independent, as they took place after pharmacological blockade of Ca2+ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA(2)alpha, which abrogates its Ca2+ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca2+ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA(2)alpha at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of cPLA(2)alpha at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA(2)alpha and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase.