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

Abstract

Endothelial cells release nitric oxide (NO) acutely in response to increased laminar fluid shear stress, and the increase is correlated with enhanced phosphorylation of endothelial nitric-oxide synthase (eNOS). Phosphoamino acid analysis of eNOS from bovine aortic endothelial cells labeled with [ 32 P]orthophosphate demonstrated that only phosphoserine was present in eNOS under both static and flow conditions. Fluid shear stress induced phosphate incorporation into two specific eNOS tryptic peptides as early as 30 s after initiation of flow. The flow-induced tryptic phosphopeptides were enriched, separated by capillary electrophoresis with intermittent voltage drops, also known as "peak parking," and analyzed by collision-induced dissociation in a tandem mass spectrometer. Two phosphopeptide sequences determined by tandem mass spectrometry, TQpSFSLQER and KLQTRPpSPGPPPAEQLLSQAR, were confirmed as the two flow-dependent phosphopeptides by co-migration with synthetic phosphopeptides. Because the sequence (RIR)TQpSFSLQER contains a consensus substrate site for protein kinase B (PKB or Akt), we demonstrated that LY294002, an inhibitor of the upstream activator of PKB, phosphatidylinositol 3-kinase, inhibited flow-induced eNOS phosphorylation by 97% and NO production by 68%. Finally, PKB phosphorylated eNOS in vitro at the same site phosphorylated in the cell and increased eNOS enzymatic activity by 15-20-fold.Endothelial nitric-oxide synthase (eNOS 1 or type III NOS) is one of three isoenzymes that converts L-arginine to L-citrulline and nitric oxide (NO). Endothelial cells synthesize NO tonically and increase NO production in response to agonists and increased fluid shear stress (FSS). Endothelial NO contributes to blood vessel homeostasis by regulating vessel tone (1), cell growth (2), platelet aggregation (3), and leukocyte binding to endothelium (4). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (5-7). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (8), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (9, 10) binding motif (11), located near the domain that binds Ca 2ϩ /calmodulin. Recent studies suggest that the activity of eNOS is regulated in a reciprocal manner through caveolin-1 inhibition and Ca 2ϩ /calmodulin stimulation (12-14).Increased FSS stimulates an increase in free intracellular calcium [Ca 2ϩ ] i from intracellular stores (15, 16) leading to a Ca 2ϩ /calmodulin-dependent increase in eNOS activity. However, recent investigations show that increases in [Ca 2ϩ ] i do not fully explain the rapid rise in NO production in response to FSS (17). Exposure of bovine aortic endothelial cells (BAEC) to 25 dynes/cm 2 FSS for 30 s caused a 7-fold rise in NO production and a corresponding 2-fold increase in eNOS phosphorylation, whereas the calcium ionophore A23187 neither caused rapid NO production...

Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS

“…Finally, we have confirmed the importance of employing ammonium dihydrogen phosphate as buffer to achieve effective liberation of mono-and all poly-phosphopeptide species from I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].In addition to the usual conditions documented above for solubilization of peptides bound in their immobilized, chelated form attached to the resin, evidence has been presented which suggests that monophosphopeptides may be released directly from the resin during the MALDI process [7,9]. It was recognized that the ferric IMAC resin must bind multiple phosphorylated components as well, but that laser irradiation does not easily dissociate these components from the agarose IMAC beads [7].…”

“…The use of sodium salt solutions was therefore somewhat undesirable, although widely used in IMAC [2, 20 -22]. The method reported by Gallis et al [3] involves the use of monobasic ammonium phosphate (NH 4 H 2 PO 4 ) in a 0.1% aqueous solution; a method analogous to that used by other groups with sodium phosphate. Ammonium phosphate solution not only proved to be MALDI-TOFMS compatible, but also enabled detection of multiply phosphorylated peptides, and can be pH-buffered.…”

“…I mmobilized metal ion affinity chromatography (IMAC) has been used for a number of years for the selective enrichment of phosphopeptides from proteolytic digest mixtures containing both phosphorylated and non-phosphorylated components [1][2][3][4][5][6][7][8]. The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].…”

“…The established methods have largely relied upon the use of high-pH elution buffers to disrupt the interaction of phosphopeptides remaining bound to the metal ioncontaining IMAC media after separation of all other peptides [2][3][4][5].…”

PIKE-A is a proto-oncogene promoting cell growth, transformation and invasion

“…Furthermore, Akt stimulates tumor progression by promoting cell invasiveness and angiogenesis (Gallis et al, 1999;Lee et al, 2001). These observations establish Akt as an attractive target for cancer therapy (Hill and Hemmings, 2002).…”