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Abstract
Cytosolic phospholipase A(2) (cPLA(2)) 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 cPLA(2) at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA(2) 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 cPLA(2) antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA(2) at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA(2) at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA(2) 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 cPLA(2) 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 cPLA(2) and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA(2) 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.
Highlights
Cytosolic phospholipase A2 is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2)
When the blots were probed with anti-phospho-S505 antibody, a single phosphorylated band was observed on Western blots from the samples of HEK293 cells transfected with pECFPcPLA2 wt and pECFPcPLA2S515A mutant but not pECFPcPLA2S505A (Fig. 1B, middle panel)
In HEK293 cells expressing pECFPcPLA2 wt, stimulation with ionomycin (5 mM) for 10 min increased its phosphorylation by 5-fold at S515, by 2-fold at S505, and by 7-fold at ERK1/2 above basal (Fig. 1D). These results indicate that pECFPcPLA2 wt overexpressed in HEK293 cells is constitutively phosphorylated on both S515 and S505 and that its phosphorylation is enhanced by ionomycin, an agent known to increase the influx of extracellular Ca21
Summary
Cytosolic phospholipase A2 (cPLA2) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). ECFPcPLA2 wt and its mutants S505A, S515A, and S505A/S515A inserted in pacAd5CMV were cotransfected with cPLA2 phosphorylation sites by NE in VSMCs 725 adenoviral DE1 backbone vector into low-passage HEK293 cells (American Type Culture Collection) using the FuGENE 6 transfection reagent (Roche, Palo Alto, CA).
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