Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002

Endothelial cells release nitric oxide (NO) acutely in response to increase d laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino a cid analysis of eNOS from bovine aortic endothelial cells labeled with [P-3 2]orthophosphate demonstrated that only phosphoserine was present in eNOS u nder both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s afte r initiation of flow. The flow-induced tryptic phosphopeptides were enriche d, separated by capillary electrophoresis with intermittent voltage drops, also known as 'peak parking," and analyzed by collision-induced dissociatio n in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were conf irmed as the two flow-dependent phosphopeptides by co-migration with synthe tic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consen sus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinosit ol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO prod uction by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site p hosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fo ld.