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Journal of Lipid Research, Vol. 46, 2122-2133, October 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Group IVC cytosolic phospholipase A₂ is farnesylated and palmitoylated in mammalian cells

Dawn E. Tucker^*, Allison Stewart^*, Laxman Nallan, Pravine Bendale, Farideh Ghomashchi, Michael H. Gelb^1, and Christina C. Leslie^1,*,

^* Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206
Departments of Chemistry and Biochemistry, University of Washington, Seattle, WA 98195
Departments of Pathology and Pharmacology, University of Colorado School of Medicine, Denver, CO 80206

Published, JLR Papers in Press, August 1, 2005. DOI 10.1194/jlr.M500230-JLR200

¹ To whom correspondence should be addressed. e-mail: gelb{at}chem.washington.edu (M.H.G.); lesliec{at}njc.org (C.C.L.)

Cytosolic phospholipase A₂ (cPLA₂) is a member of the group IV family of intracellular phospholipase A₂ enzymes, but unlike the well-studied cPLA₂, it is constitutively bound to membrane and is calcium independent. cPLA₂ contains a C-terminal CaaX sequence and is radiolabeled by mevalonic acid when expressed in cPLA₂-deficient immortalized lung fibroblasts (IMLF^–/–). The radiolabel associated with cPLA₂ was identified as the farnesyl group. The protein farnesyltransferase inhibitor BMS-214662 prevented the incorporation of [³H]mevalonic acid into cPLA₂ and partially suppressed serum-stimulated arachidonic acid release from IMLF^–/– and undifferentiated human skeletal muscle (SkMc) cells overexpressing cPLA₂, but not from cells overexpressing cPLA₂. However, BMS-214662 did not alter the amount of cPLA₂ associated with membrane.

These results were consistent in COS cells expressing the C538S cPLA₂ prenylation mutant. cPLA₂ also contains a classic myristoylation site and several potential palmitoylation sites and was found to be acylated with oleic and palmitic acids but not myristoylated. Immunofluorescence microscopy revealed that cPLA₂ is associated with mitochondria in IMLF^–/–, SkMc cells, and COS cells.

Abbreviations: cPLA₂, cytosolic phospholipase A₂; GFP, green fluorescent protein; GST, glutathione S-transferase; His, 6x histidine tag; IMLF, immortalized mouse lung fibroblasts; PLA₂, phospholipase A₂; Sf 9, Spodoptera frugiperda; SH, sulfhydryl; SkMc, skeletal muscle

Supplementary key words prenylation • fatty acylation • mitochondria

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