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

Abstract

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.

Keywords

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