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Journal of Lipid Research, Vol. 49, 724-737, April 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

cPLA₂ phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells

Zoran Pavicevic^*, Christina C. Leslie and Kafait U. Malik^1,*

^* Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163
Department of Pediatrics, Program in Cell Biology, National Jewish Medical and Research Center, Denver, CO 80206

Published, JLR Papers in Press, January 9, 2008.

¹ To whom correspondence should be addressed. e-mail: kmalik{at}utmem.edu

Cytosolic phospholipase A₂ (cPLA₂) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA₂ at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA₂ phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA₂ antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA₂ at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA₂ at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA₂ at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA₂ wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA₂ and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA₂ at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs.

Supplementary key words cytosolic phospholipase A₂ • calcium/calmodulin-dependent kinase II • extracellular regulated kinase 1/2 • adenovirus cytosolic phospholipase A₂ wild type • mutants S505A and S515A

Abbreviations: AA, arachidonic acid; AdECFPcPLA₂ wt, adenoviral enhanced cyan fluorescent protein cytosolic phospholipase A₂ wild type; Ca²/CaMKII, calcium/calmodulin kinase II ; cPLA₂, cytosolic phospholipase A₂; ECFP, enhanced cyan fluorescent protein; ERK1/2, extracellular regulated kinase 1/2; KN-92, 2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine; KN-93, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine; MOI, multiplicity of infection; NE, norepinephrine; TCID₅₀, tissue culture infective dose 50; VSMC, vascular smooth muscle cell

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