cPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells.
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.
Pavicevic
Zoran
Z
Department of Pharmacology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Leslie
Christina C
CC
Malik
Kafait U
KU
D000818:Animals; D000918:Antibody Specificity / Q000276:immunology; D016718:Arachidonic Acid / Q000378:metabolism*; D001158:Arteries / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002478:Cells, Cultured; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D054510:Phospholipases A2, Cytosolic / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D021381:Protein Transport; D011817:Rabbits
D010768:Phosphoserine; D016718:Arachidonic Acid; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D054510:Phospholipases A2, Cytosolic; D009638:Norepinephrine