Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release1
- *Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe-Universität, D-60590, Frankfurt am Main, Germany; and
- †Department of Botany, UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway
- ↵Correspondence: 2Correspondence: Institute of Pharmacology and Toxicology, Klinikum der J.W. Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail: Pfeilschifter{at}em.uni-frankfurt.de
SPECIFIC AIM
Our interest focuses on the sphingolipid-ceramide signaling cascade and on the identification of molecular targets of ceramide action, which has proved to be difficult and so far mainly indirect. We show that ceramide, which is an early messenger of inflammatory cytokine action exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation.
PRINCIPAL FINDINGS
1. Ceramide induces arachidonic acid release from mesangial cells
Stimulation of [3H]arachidonic acid-labeled mesangial cells with short-chain (C6) or long-chain (C16) ceramides reveals a dose-dependent increase in [3H]arachidonic acid release after 60 min of stimulation. The long-chain C16-ceramide was found to be more potent than the short-chain C6-ceramide to stimulate arachidonic acid release.
Preincubation of mesangial cells with either an inhibitor of the classical MAPK cascade, PD 98059 (upto 20 μM), or an inhibitor of protein kinase C (PKC), Ro 31–8220 (at 1 μM), revealed no significant reduction of ceramide-induced arachidonic acid release, thus suggesting that the classical MAPK cascade and the conventional and novel PKCs are not involved in the ceramide-triggered response.
2. Direct binding of TID-ceramide to cPLA2
To evaluate whether cPLA2 is a direct target of ceramide due to its amino-terminal CaLB domain, we used a photoaffinity labeling analog of ceramide, [125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramide (TID-ceramide), of high [125I]-iodine-specific radioactivity (2000 Ci/mmol) and incubated recombinant cPLA2 with this compound in a cell-free system for 5 min, followed by photolysis at 364 nm. As seen in Fig. 1A⇓ , there is a strong labeling of cPLA2 by TID-ceramide. There was no difference in labeling between the D- and L-erythro isomers of TID-ceramide, suggesting that the absolute configuration is not critical for binding to cPLA2.
Binding of [125I]TID-ceramide to wild-type cPLA2, the catalytic domain, a hinge region-deficient mutant, and the CaLB domain …
