Subcellular targeting and agonist-induced site-specific phosphorylation of endothelial nitric-oxide synthase.

The endothelial isoform of nitric-oxide synthase (eNOS) undergoes a complex pattern of covalent modifications, including acylation with the fatty acids myristate and palmitate as well as phosphorylation on multiple sites. eNOS acylation is a key determinant for the reversible subcellular targeting of the enzyme to plasmalemmal caveolae. We transfected a series of hemagglutinin epitope-tagged eNOS mutant cDNAs deficient in palmitoylation (palm(-)) and/or myristoylation (myr(-)) into bovine aortic endothelial cells; after treatment with the eNOS agonists sphingosine 1-phosphate or vascular endothelial growth factor, the recombinant eNOS was immunoprecipitated using an antibody directed against the epitope tag, and patterns of eNOS phosphorylation were analyzed in immunoblots probed with phosphorylation state-specific eNOS antibodies. The wild-type eNOS underwent agonist-induced phosphorylation at serine 1179 (a putative site for phosphorylation by kinase Akt), but phosphorylation of the myr(-) eNOS at this residue was nearly abrogated; the palm(-) eNOS exhibited an intermediate phenotype. The addition of the CD8 transmembrane domain to the amino terminus of eNOS acylation-deficient mutants rescued the wild-type phenotype of robust agonist-induced serine 1179 phosphorylation. Thus, membrane targeting, but not necessarily acylation, is the critical determinant for agonist-promoted eNOS phosphorylation at serine 1179. In striking contrast to serine 1179, phosphorylation of eNOS at serine 116 was enhanced in the myr(-) eNOS mutant and was markedly attenuated in the CD8-eNOS membrane-targeted fusion protein. We conclude that eNOS targeting differentially affects eNOS phosphorylation at distinct sites in the protein and suggest that the inter-relationships of eNOS acylation and phosphorylation may modulate eNOS localization and activity and thereby influence NO signaling pathways in the vessel wall.

Lysophosphatidic acid and receptor-mediated activation of endothelial nitric-oxide synthase.

Both lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are platelet-derived phospholipids that elicit diverse biological responses. In endothelial cells, S1P stimulates the EDG-1 receptor-mediated activation of the endothelial isoform of nitric oxide synthase (eNOS), but the role of LPA in eNOS regulation is less well understood. We now report that LPA treatment of bovine aortic endothelial cells (BAEC) activates eNOS enzyme activity in a pathway that involves phosphorylation of eNOS on serine 1179 by protein kinase Akt. In contrast to the cellular responses elicited by S1P in COS-7 cells, LPA can stimulate the activation of eNOS and Akt independently of EDG-1 receptor transfection. LPA-stimulated enzyme activation was significantly attenuated in an eNOS mutant lacking the site that is phosphorylated by kinase Akt (eNOS S1179A). In BAEC, activation of eNOS by LPA is completely blocked by pertussis toxin, by the intracellular calcium chelator BAPTA (1,2-bis(aminophenoxy) ethane-N,N,N',N'-tetraacetic acid), and by the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, but is unaffected by U0126, an inhibitor of mitogen-activated protein (MAP) kinase pathways. Analysis of the LPA dose response for eNOS activation reveals an EC(50) of approximately 40 nM, a concentration well below the potency of LPA at the EDG-1 receptor. Taken together, these results indicate that LPA potently activates eNOS in BAEC in a pathway distinct from the EDG-1 receptor, but mediated by a similar receptor-mediated pathway dependent on pertussis toxin-sensitive G proteins and involving activation of the PI3-K/Akt pathway. These studies have identified a role for the phospholipid LPA in eNOS activation, and point out the complementary role of distinct platelet-derived lipids in endothelial signaling pathways.