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

Albert Gubern,Miquel Barceló-Torns,David Barneda,José M López,Roser Masgrau,Fernando Picatoste,Charles E Chalfant,Jesús Balsinde,María A Balboa,Enrique Claro

doi:10.1074/jbc.m109.061515

Abstract

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 Ca(2+)-independent, as they took place after pharmacological blockade of Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.

Highlights

NOVEMBER 20, 2009 VOLUME 284 NUMBER 47 glycerols (TAG) and cholesteryl esters, surrounded by a phospholipid monolayer and by specific proteins, among which the best characterized belong to the perilipin family [1,2,3]
Still promoted ADRP expression (Fig. 1C) and lipid droplets (LD) biogenesis that was sensitive to the cPLA2␣ inhibitor methylarachidonyl fluorophosphonate (MAFP) (Fig. 1E)
This study addresses the mechanism of cPLA2␣ activation needed for the biogenesis of LD induced by FBS

Summary

JNK and CERK Govern LD Biogenesis

505 depends on MAPK, among which most reports implicate ERK and p38 [11, 12]. We have shown that phosphorylation of cPLA2␣ at Ser-505 is key for LD biogenesis, as evidenced by the inability of the S505A mutant to recapitulate the effect of the endogenous enzyme [10]. This work was undertaken to delineate the mechanism of cPLA2␣ activation that generates LD in the cell after serum addition. We show that the activation of cPLA2␣ is independent of increases of intracellular Ca2ϩ; we identify JNK as the MAPK implicated in cPLA2␣ activation. We show that ceramide kinase (CERK), the enzyme responsible for the synthesis of Cer-1-P, mediates LD biogenesis by inducing the phosphorylation of cPLA2␣ by JNK

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION