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 [32P]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 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 [32P]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 nitric oxide fluid shear stress high pressure liquid chromatography protein kinase B mitogen-activated protein free intracellular calcium bovine aortic endothelial cells Dulbecco's modified Eagle's medium collision-induced dissociation tandem mass spectrometry immobilized metal affinity chromatography solid phase extraction-capillary electrophoresis polyvinylidene difluoride high voltage electrophoresis mass to charge ratio nitrogen oxides inducible nitric-oxide synthase neuronal nitric-oxide synthase polyacrylamide gel electrophoresis phosphatidylinositol 3-kinase Endothelial nitric-oxide synthase (eNOS1 or type III NOS) is one of three isoenzymes that converts l-arginine tol-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 (1Palmer R.M. Ferrige A.G. Moncada S. Nature. 1987; 327: 524-526Crossref PubMed Scopus (9366) Google Scholar), cell growth (2Garg U.C. Hassid A. J. Clin. Invest. 1989; 83: 1774-1777Crossref PubMed Scopus (1997) Google Scholar), platelet aggregation (3Ignarro L.J. Circ. Res. 1989; 65: 1-21Crossref PubMed Scopus (904) Google Scholar), and leukocyte binding to endothelium (4Radomski M.W. Vallance P. Whitley G. Foxwell N. Moncada S. Cardiovasc. Res. 1993; 27: 1380-1382Crossref PubMed Scopus (209) Google Scholar). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (5Liu J. Garcia-Cardena G. Sessa W.C. Biochemistry. 1996; 35: 13277-13281Crossref PubMed Scopus (203) Google Scholar, 6Garcia-Cardena G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (578) Google Scholar, 7Shaul P.W. Smart E.J. Robinson L.J. German Z. Yuhanna I.S. Ying Y. Anderson R.G. Michel T. J. Biol. Chem. 1996; 271: 6518-6522Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (8Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (9Garcia-Cardena G. Martasek P. Masters B.S. Skidd P.M. Couet J. Li S. Lisanti M.P. Sessa W.C. J. Biol. Chem. 1997; 272: 25437-25440Abstract Full Text Full Text PDF PubMed Scopus (696) Google Scholar, 10Feron O. Belhassen L. Kobzik L. Smith T.W. Kelly R.A. Michel T. J. Biol. Chem. 1996; 271: 22810-22814Abstract Full Text Full Text PDF PubMed Scopus (598) Google Scholar) binding motif (11Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar), located near the domain that binds Ca2+/calmodulin. Recent studies suggest that the activity of eNOS is regulated in a reciprocal manner through caveolin-1 inhibition and Ca2+/calmodulin stimulation (12Ju H. Zou R. Venema V.J. Venema R.C. J. Biol. Chem. 1997; 272: 18522-18525Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar, 13Michel J.B. Feron O. Sase K. Prabhakar P. Michel T. J. Biol. Chem. 1997; 272: 25907-25912Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 14Michel J.B. Feron O. Sacks D. Michel T. J. Biol. Chem. 1997; 272: 15583-15586Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). Increased FSS stimulates an increase in free intracellular calcium [Ca2+]i from intracellular stores (15James N.L. Harrison D.G. Nerem R.M. FASEB J. 1995; 9: 968-973Crossref PubMed Scopus (48) Google Scholar, 16Geiger R.V. Berk B.C. Alexander R.W. Nerem R.M. Am. J. Physiol. 1992; 262: C1411-C1417Crossref PubMed Google Scholar) leading to a Ca2+/calmodulin-dependent increase in eNOS activity. However, recent investigations show that increases in [Ca2+]i do not fully explain the rapid rise in NO production in response to FSS (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar). Exposure of bovine aortic endothelial cells (BAEC) to 25 dynes/cm2 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 nor any net increase in eNOS phosphorylation (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar). We therefore hypothesized that an increase in FSS could be transduced to increased NO production via activation of a protein kinase cascade resulting in phosphorylation and activation of eNOS. The objectives of the present work were to elucidate the eNOS sites phosphorylated in response to increased FSS and to identify potential protein kinase mediators of the mechanotransduction of FSS to NO production. The amino acid residues phosphorylated in small proteins may be determined by fractionation of tryptic peptides by high pressure liquid chromatography (HPLC) and mass spectrometry (18Gygi S.P. Han D.K. Gingras A.C. Sonenberg N. Aebersold R. Electrophoresis. 1999; 20: 310-319Crossref PubMed Scopus (89) Google Scholar). However, tryptic fragmentation of large proteins such as eNOS creates numerous peptides whose rapid elution from HPLC exceeds the scan rate of the triple mass spectrometer. of by collision-induced dissociation of a of the phosphopeptides from proteins from a cell as to phosphorylation with peptides and not be as In to identify phosphorylation sites in tryptic phosphopeptides were by immobilized metal affinity chromatography The peptides were separated by solid phase capillary electrophoresis which the analysis to for both increased scan of and increased of to The of two specific eNOS residues phosphorylated in response to flow was and was FSS to eNOS phosphorylation and NO production via activation of phosphatidylinositol 3-kinase and protein kinase B We present that PKB vitro at the same site phosphorylated in the cell and that the phosphorylation increases eNOS enzymatic activity. The to eNOS was from was from and and PKB were from modified and were from chromatography were from acid was from were from acid was from and were from was from difluoride was from was from and were from J. T. for HPLC was from and and were from The synthetic peptides and were from were and in in medium with as (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar). from were in and at for and shear stress were to laminar FSS in a and as O. Berk P. and the Scholar). at in were with and labeled with [32P]orthophosphate for in bovine was to the medium for to were in static or to dynes/cm2 FSS for the in a and O. Berk P. and the Scholar). were with and in of and were for at at of protein as determined by were for The were as J. Biol. Chem. Full Text PDF PubMed Google Scholar) and for at a of 1987; PubMed Scopus Google Scholar) a with of a of protein and of The were with of in and the from the were at eNOS in was a gel in a electrophoresis The gel was in of for and the proteins were to at for proteins were a gel and to for in and bovine the were with or at a the was with and was of proteins to the gel was at for in and acid analysis were as Aebersold R. PubMed Scopus Google Scholar, P. T. Electrophoresis. PubMed Scopus Google Scholar). eNOS was from and of to for The were separated a The eNOS was located by A. O. Chem. 1996; PubMed Scopus Google Scholar), from the and the incorporation was determined by The gel were in by in and in a eNOS was in the gel by in and under for at Electrophoresis. 1996; PubMed Scopus Google Scholar), was to into the and the gel were with intermittent for at 25 in the the was and the gel were with and with were in a and the and was A. O. Chem. 1996; PubMed Scopus Google Scholar). The gel and eNOS were with of as A. O. Chem. 1996; PubMed Scopus Google Scholar). of the tryptic phosphopeptides were from the gel as determined by metal affinity was and as Aebersold R. PubMed Scopus Google Scholar) with the of a of a of capillary was and in by a to the of the two in the was in a of a vessel by and the was to a of under The of capillary was in with a one of the two was in a pressure vessel and the as an The was for by at with and The in was in and by with for elution of and were after in acid and the HPLC HPLC was a with a from to which acid and acid was to peptides from the were and by from the HPLC for were to as D. G. A. Aebersold R. Chem. 1999; PubMed Scopus Google Scholar). The was a at the of the of both and the for which a small of was the D. A. Aebersold R. 1996; PubMed Scopus Google Scholar, D. A. Aebersold R. Chem. 1996; PubMed Scopus Google Scholar). were a triple mass with a D. G. A. Aebersold R. Chem. 1999; PubMed Scopus Google Scholar). was a in that and capillary and voltage were such that as an a the mass from scan to and the voltage from to the a the mass to conditions. NO by was as nitrogen oxide a as in (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar). the for the of eNOS was two the of bovine eNOS K. Harrison D.G. S.P. Nerem R.M. Alexander R.W. J. Clin. Invest. 1992; PubMed Scopus Google Scholar) was the and and was and as a to a and the with an was and and eNOS K. Harrison D.G. S.P. Nerem R.M. Alexander R.W. J. Clin. Invest. 1992; PubMed Scopus Google Scholar) was and were into and were for via was and with and Anderson 1989; PubMed Scopus Google Scholar) was and the two and as eNOS at L.J. Martasek P. Liu Masters B.S. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar) as by The was through the that was by and sequence was to be with the bovine eNOS sequence K. Harrison D.G. S.P. Nerem R.M. Alexander R.W. J. Clin. Invest. 1992; PubMed Scopus Google Scholar) in the eNOS was and from as R.C. Harrison D.G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) with was into was to phase and into of and an and were The was at for at and were and in a of of 30 and for at G. Oh P. Liu J. Schnitzer J.E. Sessa W.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6448-6453Crossref PubMed Scopus (578) Google Scholar) and were and the was at for at eNOS in the was chromatography as R.C. Harrison D.G. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The protein was by chromatography as J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). eNOS was by after and was in in at PKB to eNOS were to the of of PKB was to of eNOS with at a specific activity of the of the were in and eNOS was separated by and by The eNOS were and incorporation was determined by activation of eNOS by PKB, of PKB activity eNOS as a was with of eNOS in a protein kinase at 30 PKB were in phosphorylation, of eNOS from the kinase with and PKB were into eNOS and were as R. J.E. 1999; PubMed Scopus Google Scholar) at 30 was separated from labeled by chromatography R. J.E. 1999; PubMed Scopus Google Scholar) and were determined by and the gel in after by we and (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar, T. Li L. Proc. Natl. Acad. Sci. U. S. A. 1993; PubMed Scopus Google Scholar) that eNOS is phosphorylated in in static and that the phosphorylation of eNOS is enhanced in response to flow (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar). were labeled with [32P]orthophosphate in were in static or to laminar FSS O. Berk P. and the Scholar). The cells were eNOS was with a and the was an The proteins in the gel were to and by eNOS was phosphorylated in cells in static and to laminar FSS caused a 2-fold increase (17Corson M.A. James N.L. Latta S.E. Nerem R.M. Berk B.C. Harrison D.G. Circ. Res. 1996; 79: 984-991Crossref PubMed Scopus (411) Google Scholar) in eNOS phosphorylation and labeled endothelial cells under static with [32P]orthophosphate in the of oxide V.J. Venema R.C. Res. 1996; PubMed Scopus Google Scholar) or G. R. Liu J. Sessa W.C. J. Biol. 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The eNOS to in was to tryptic and the tryptic peptides were a The peptides were in the by and in the by of the are in static eNOS phosphopeptides two of and increased with are to dynes/cm2 FSS The of of the and phosphopeptides were by analysis in two phosphate incorporation as a of shear stress the incorporation of phosphate into eNOS a was the specific incorporation into the flow-dependent phosphorylation sites and was phosphorylation of was by to and the stimulation of phosphorylation into from to The is to the that the is the stimulation of phosphorylation the of phosphate which is by the of and increases in phosphorylation in both peptides and as early as 30 s and of the incorporation of phosphate into the peptides by These and that flow-induced eNOS phosphorylation is rapid and that flow the phosphorylation of two specific eNOS tryptic of phosphorylation of two eNOS peptides in response to dynes/cm2 was in was in The was in The was in in a eNOS for mass analysis of the eNOS was from of of the were labeled with were and were to dynes/cm2 for were eNOS was by gel and the gel was of of eNOS to of known the gel an of of eNOS from not The eNOS was from the gel and in with of the phosphopeptides were The phosphopeptides were to and from an immobilized metal affinity with of The peptides were by and the in the was determined by In of eNOS tryptic phosphopeptides by were analyzed by eNOS phosphopeptides could be by tryptic peptides of the amino acid sequence of eNOS were not and the of the phosphate was in the the is to peptides for in of the of peptides that are from in the peptides of mass and of from the of only the from a be Because the phosphorylation sites in may be modified only to phosphopeptides from proteins from cells not be The to identify eNOS phosphopeptides of was by the for analysis by the flow rate to the mass D. G. A. Aebersold R. Chem. 1999; PubMed Scopus Google Scholar), a known as “peak J. Am. 1998; 9: PubMed Scopus Google Scholar). HPLC in the was to solid phase extraction-capillary electrophoresis for and was that as the mass by the voltage for by of the peptides in is in D. G. A. Aebersold R. Chem. 1999; PubMed Scopus Google Scholar). HPLC were analyzed by The from were by for the of phosphopeptides an mass for or and 25 were to a in to These were analyzed J. S. Proc. Natl. Acad. Sci. U. S. A. 83: PubMed Scopus Google Scholar) to the The for the from is in and to bovine eNOS residues through of phosphate to is by of and the P. J. 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Full Text PDF PubMed Google Scholar) to inhibition of an upstream activator of PKB flow-dependent phosphorylation of eNOS. were for 30 with or and in static and or to laminar FSS at dynes/cm2 for and of the eNOS and a corresponding eNOS are inhibited the flow-dependent increase in eNOS phosphorylation by 97% The activation of PKB phosphorylation of and P. 1998; PubMed Scopus Google Scholar). In to FSS may the phosphorylation of PKB, were for 30 with or in static and or to laminar FSS at dynes/cm2 for and were to and with or with to increased PKB phosphorylation at by of with inhibited the flow-dependent phosphorylation of by inhibition of flow-induced eNOS phosphorylation and PKB phosphorylation at that PKB may eNOS at the site in response to flow. FSS both rapid eNOS phosphorylation and NO production in flow the production of NO by phosphorylation in is that phosphorylation eNOS enzymatic inhibited flow-dependent phosphorylation of we determined the NO production in were for 30 with or or and to FSS at dynes/cm2 for or inhibited NO production by and after and of of inhibited NO production by and after and of The inhibition by of PKB phosphorylation, eNOS phosphorylation, and NO production that laminar FSS NO production by of eNOS phosphorylation via a and Because the flow-dependent phosphorylation site is a consensus site for PKB P. 1998; PubMed Scopus Google Scholar) and flow-dependent NO production in we determined PKB could and eNOS in PKB phosphorylated eNOS to a of of of eNOS The eNOS from was separated by gel from the and in with The tryptic phosphopeptides were separated by that the phosphopeptide to a to from of the phosphopeptides in B were from the and to one was with a mass to the mass of for the the site in eNOS phosphorylated by PKB in vitro is to the site as that is phosphorylated in response to flow in to phosphorylation of eNOS with PKB, eNOS was in protein kinase with and The kinase were into eNOS and of to was phosphorylation of eNOS by PKB eNOS by the of modifications of mass spectrometry, we two flow-dependent phosphorylation sites in eNOS. We confirmed the of the tryptic phosphopeptides by co-migration of in vivo labeled tryptic phosphopeptides of eNOS with synthetic peptides the sequences by mass tryptic from the is with the PKB substrate consensus phosphorylation sequence is or P. 1998; PubMed Scopus Google Scholar). Because is an upstream activator of PKB P. 1998; PubMed Scopus Google Scholar), we the J. Biol. Chem. Full Text PDF PubMed Google Scholar), to flow-dependent PKB activity. were with to the inhibitor flow-dependent phosphorylation of PKB by eNOS phosphorylation by and NO production by 68%. 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