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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*

  • Byron Gallis
    Correspondence
    To whom all correspondence should be addressed: Division of Cardiology, Box 359748, University of Washington, Seattle, WA 98195. Tel.: 206-685-6960; Fax: 206-616-1580
    Affiliations
    From the Departments of Medicine and Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Garry L. Corthals
    Footnotes
    Affiliations
    Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • David R. Goodlett
    Affiliations
    Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Hiroto Ueba
    Affiliations
    From the Departments of Medicine and Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Francis Kim
    Affiliations
    From the Departments of Medicine and Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Steven R. Presnell
    Affiliations
    School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332
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  • Daniel Figeys
    Footnotes
    Affiliations
    Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • David G. Harrison
    Affiliations
    Department of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322
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  • Bradford C. Berk
    Footnotes
    Affiliations
    From the Departments of Medicine and Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Ruedi Aebersold
    Affiliations
    Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Marshall A. Corson
    Affiliations
    From the Departments of Medicine and Molecular Biotechnology, University of Washington, Seattle, Washington 98195
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  • Author Footnotes
    * This work was supported by the Washington American Heart Association Grant-in-aid WA97GB31 and National Institutes of Health Grant (NIH) 1RO1HL30946 (B. G. and M. A. C.), by the Georgia Tech/Emory Seed Grant Program (to S. R. P.), by NIH Grants 1R01HL58000 and 1R01HL39006 (to D. G. H.), by NIH Grant 5P01HL18645 (to B. C. B.), by the NSF Science and Technology Center for Molecular Biotechnology, NIH Grant 1RO1AI41109, and the NIH Resource Technology Center for Comprehensive Biology (to R. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    FNd Present address: The Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia.
    FNf Present address: Institute for Marine Biosciences, Halifax, Nova Scotia B3H 3Z1, Canada.
    FNh Present address: the University of Rochester School of Medicine and Dentistry, Rochester, NY 14642.
Open AccessPublished:October 15, 1999DOI:https://doi.org/10.1074/jbc.274.42.30101
      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.
      eNOS
      endothelial nitric-oxide synthase
      NO
      nitric oxide
      FSS
      fluid shear stress
      HPLC
      high pressure liquid chromatography
      PKB
      protein kinase B
      MAP
      mitogen-activated protein
      [Ca2+]i
      free intracellular calcium
      BAEC
      bovine aortic endothelial cells
      DMEM
      Dulbecco's modified Eagle's medium
      CID
      collision-induced dissociation
      MS/MS
      tandem mass spectrometry
      IMAC
      immobilized metal affinity chromatography
      SPE-CE
      solid phase extraction-capillary electrophoresis
      PVDF
      polyvinylidene difluoride
      HVE
      high voltage electrophoresis
      m/z
      mass to charge ratio
      NOx
      nitrogen oxides
      iNOS
      inducible nitric-oxide synthase
      nNOS
      neuronal nitric-oxide synthase
      PAGE
      polyacrylamide gel electrophoresis
      PI3-kinase
      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 (
      • Palmer R.M.
      • Ferrige A.G.
      • Moncada S.
      ), cell growth (
      • Garg U.C.
      • Hassid A.
      ), platelet aggregation (
      • Ignarro L.J.
      ), and leukocyte binding to endothelium (
      • Radomski M.W.
      • Vallance P.
      • Whitley G.
      • Foxwell N.
      • Moncada S.
      ). In vivo eNOS is both myristoylated and palmitoylated. These modifications increase eNOS compartmentalization to plasmalemmal caveolae and facilitate release of NO from cells (
      • Liu J.
      • Garcia-Cardena G.
      • Sessa W.C.
      ,
      • Garcia-Cardena G.
      • Oh P.
      • Liu J.
      • Schnitzer J.E.
      • Sessa W.C.
      ,
      • Shaul P.W.
      • Smart E.J.
      • Robinson L.J.
      • German Z.
      • Yuhanna I.S.
      • Ying Y.
      • Anderson R.G.
      • Michel T.
      ). In caveolae, which are small plasmalemmal invaginations that sequester signaling proteins (
      • Okamoto T.
      • Schlegel A.
      • Scherer P.E.
      • Lisanti M.P.
      ), eNOS specifically interacts with the scaffolding protein caveolin-1 through a caveolin (
      • Garcia-Cardena G.
      • Martasek P.
      • Masters B.S.
      • Skidd P.M.
      • Couet J.
      • Li S.
      • Lisanti M.P.
      • Sessa W.C.
      ,
      • Feron O.
      • Belhassen L.
      • Kobzik L.
      • Smith T.W.
      • Kelly R.A.
      • Michel T.
      ) binding motif (
      • Couet J.
      • Li S.
      • Okamoto T.
      • Ikezu T.
      • Lisanti M.P.
      ), 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 (
      • Ju H.
      • Zou R.
      • Venema V.J.
      • Venema R.C.
      ,
      • Michel J.B.
      • Feron O.
      • Sase K.
      • Prabhakar P.
      • Michel T.
      ,
      • Michel J.B.
      • Feron O.
      • Sacks D.
      • Michel T.
      ).
      Increased FSS stimulates an increase in free intracellular calcium [Ca2+]i from intracellular stores (
      • James N.L.
      • Harrison D.G.
      • Nerem R.M.
      ,
      • Geiger R.V.
      • Berk B.C.
      • Alexander R.W.
      • Nerem R.M.
      ) 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 (
      • Corson M.A.
      • James N.L.
      • Latta S.E.
      • Nerem R.M.
      • Berk B.C.
      • Harrison D.G.
      ). 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 (
      • Corson M.A.
      • James N.L.
      • Latta S.E.
      • Nerem R.M.
      • Berk B.C.
      • Harrison D.G.
      ). 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 (
      • Gygi S.P.
      • Han D.K.
      • Gingras A.C.
      • Sonenberg N.
      • Aebersold R.
      ). However, tryptic fragmentation of large proteins such as eNOS creates numerous peptides whose rapid elution from HPLC exceeds the scan rate of the triple quadrupole mass spectrometer. This prevents acquisition of sequences, by collision-induced dissociation (CID), of a substantial fraction of the peptides. Additionally, phosphopeptides from proteins isolated from a cell will nearly always exist as minor ions due to sub-stoichiometric phosphorylation compared with nonphosphorylated peptides and will not be scanned as major parent ions. In order to identify phosphorylation sites in eNOS, tryptic phosphopeptides were first enriched by immobilized metal affinity chromatography (IMAC). The peptides were then separated by peak parking solid phase extraction capillary electrophoresis (SPE-CE), which extends the peak analysis time to provide for both increased scan times of parent ions and increased MS/MS analyses of each selected peptide according to optimized CID parameters. The identity of two specific eNOS residues phosphorylated in response to flow was determined, and evidence was obtained linking FSS to eNOS phosphorylation and NO production via activation of phosphatidylinositol 3-kinase (PI3-kinase) and protein kinase B (PKB). We present evidence that PKB phosphorylates eNOSin vitro at the same site phosphorylated in the cell and that the phosphorylation increases eNOS enzymatic activity.

      DISCUSSION

      Through the use of innovative modifications of mass spectrometry, we have identified two flow-dependent phosphorylation sites in eNOS. We have confirmed the identities of the tryptic phosphopeptides by co-migration of in vivo labeled tryptic phosphopeptides of eNOS with synthetic peptides based on the sequences discovered by mass spectrometry. One tryptic phosphopeptide, designated F1, TQpSFSLQER, cleaved from the sequence, RIRTQpSFSLQER, is consistent with the PKB substrate consensus phosphorylation sequence RXRXXSX (where X is F, I, or V) (
      • Alessi D.R.
      • Cohen P.
      ). Because PI3-kinase is an upstream activator of PKB (
      • Alessi D.R.
      • Cohen P.
      ), we used the PI3-kinase inhibitor, LY294002 (
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ), to partially inhibit flow-dependent PKB activity. When BAEC were pretreated with LY294002 prior to flow, the inhibitor decreased flow-dependent phosphorylation of PKB by 80%, eNOS phosphorylation by 97%, and NO production by 68%. These data suggest that flow-dependent regulation of NO production in BAEC occurs in part by regulation of the level of phosphorylation of Ser-1179 in eNOS. Indeed, in vitro phosphorylation of eNOS by PKB occurred at Ser-1179 and activated the enzyme by 15–20-fold.
      Flow-dependent phosphorylation of eNOS occurs near the amino terminus, at Ser-116, and near the carboxyl terminus, at Ser-1179. The amino acid residues surrounding Ser-1179 suggest that eNOS may be phosphorylated by PKB as well as possibly other protein kinases (
      • Pearson R.B.
      • Kemp B.E.
      ). Unlike Ser-1179, the sequence surrounding Ser-116 contains a proline residue at the n + 1 position, suggesting that a proline-directed protein kinase may phosphorylate this site. Thus FSS appears to activate at least two different protein kinases that phosphorylate eNOS. The significance of these modifications for regulation of eNOS function will be better understood when a crystal structure of the enzyme or site-directed eNOS mutants become available. Recently the crystal structure of an eNOS fragment (amino acid residues 39–482) revealed that pairs of cysteine residues (96 and 101) at the dimer interface tetrahedrally coordinate a zinc ion that stabilizes the tetrahydrobiopterin-binding site that interacts with Ser-104 and Val-106 (
      • Raman C.S.
      • Li H.
      • Martasek P.
      • Kral V.
      • Masters B.S.
      • Poulos T.L.
      ). We speculate that incorporation of phosphate at Ser-116 may alter the structure of the tetrahydrobiopterin-binding site to enhance eNOS catalytic activity.
      The two phosphorylation sites identified in this study and a third site described elsewhere (
      • Figeys D.
      • Corthals G.
      • Gallis B.
      • Ducret A.
      • Corson M.
      • Aebersold R.
      ) are the first in vivophosphorylation sites identified for any isoenzyme of nitric-oxide synthase. The other two isoenzymes of nitric-oxide synthase, inducible nitric-oxide synthase (iNOS) and neuronal nitric-oxide synthase (nNOS), share 50–60% amino acid identity (
      • Lamas S.
      • Marsden P.A.
      • Li G.K.
      • Tempst P.
      • Michel T.
      ,
      • Marsden P.A.
      • Schappert K.T.
      • Chen H.S.
      • Flowers M.
      • Sundell C.L.
      • Wilcox J.N.
      • Lamas S.
      • Michel T.
      ,
      • Xie Q.W.
      • Cho H.J.
      • Calaycay J.
      • Mumford R.A.
      • Swiderek K.M.
      • Lee T.D.
      • Ding A.
      • Troso T.
      • Nathan C.
      ) with eNOS. The mechanism of regulation of nNOS remains unclear, but this enzyme contains the conserved PKB consensus sequence (
      • Lamas S.
      • Marsden P.A.
      • Li G.K.
      • Tempst P.
      • Michel T.
      ) we have identified as being phosphorylated in eNOS. nNOS lacks the F2 site and iNOS contains neither the F1 nor the F2 sites we have identified as being phosphorylated in eNOS in response to flow. Indeed, levels of NO produced by iNOS are believed to be regulated not by phosphorylation of iNOS but by the levels of mRNA for this protein (
      • Xie Q.W.
      • Cho H.J.
      • Calaycay J.
      • Mumford R.A.
      • Swiderek K.M.
      • Lee T.D.
      • Ding A.
      • Troso T.
      • Nathan C.
      ). In contrast to the sequence divergence between eNOS and the other isoforms of NOS, the primary amino acid sequences of mammalian eNOS are more than 90% conserved (
      • Marsden P.A.
      • Schappert K.T.
      • Chen H.S.
      • Flowers M.
      • Sundell C.L.
      • Wilcox J.N.
      • Lamas S.
      • Michel T.
      ) and contain the phosphorylation sites we have described.
      Previous investigators have shown that eNOS contains predominantly phosphoserine with phosphotyrosine as a minor modification (
      • Venema V.J.
      • Marrero M.B.
      • Venema R.C.
      ,
      • Garcia-Cardena G.
      • Fan R.
      • Stern D.F.
      • Liu J.
      • Sessa W.C.
      ). In these studies, either phenylarsine oxide or pervanadate was used. Both of these protein tyrosine phosphatase inhibitors greatly increase the overall tyrosine phosphate content of many cellular proteins (
      • Zick Y.
      • Sagi-Eisenberg R.
      ,
      • Fletcher M.C.
      • Samelson L.E.
      • June C.H.
      ). We have found only phosphoserine in eNOS under both static and flow conditions in BAEC. Tandem MS/MS analysis of eNOS tryptic peptides derived from cells under static or flow conditions yielded CID spectra that covered 40% of the amino acid sequence of eNOS (data not shown). These spectra were screened and matched against the known sequence of eNOS using the SEQUEST (
      • Yates J.R.D.
      • Eng J.K.
      • McCormack A.L.
      • Schieltz D.
      ) program, for addition of phosphate to serine, threonine, and tyrosine. No phosphothreonine or phosphotyrosine residues were found. Whereas such modifications may exist and may not be detected by the mass spectrometer because of low stoichiometry, the SEQUEST search did confirm the phosphoamino acid analysis in this study by identifying three phosphoserine-containing peptides (
      • Figeys D.
      • Corthals G.
      • Gallis B.
      • Ducret A.
      • Corson M.
      • Aebersold R.
      ).
      Repeated attempts to identify tryptic phosphopeptides of eNOS by MS/MS analysis after conventional means of enrichment (IMAC) and separation (HPLC) were not successful, despite a 40% coverage of the eNOS primary amino acid sequence. In this study, the data-dependent modulation of the rate at which peptide ions entered the MS/MS, produced by a peptide peak-generated drop in the electric field of the CE, expanded the analytical window for each peptide to be analyzed. This peak parking procedure (
      • Figeys D.
      • Corthals G.
      • Gallis B.
      • Ducret A.
      • Corson M.
      • Aebersold R.
      ,
      • Davis M.T.
      • Lee T.D.
      ) should be applicable to identification of in vivo phosphorylation sites in other large phosphoproteins or in cases in which the phosphoprotein cannot be purified to homogeneity.
      It has recently been shown (
      • Chen Z.P.
      • Mitchelhill K.I.
      • Michell B.J.
      • Stapleton D.
      • Rodriguez-Crespo I.
      • Witters L.A.
      • Power D.A.
      • Ortiz de Montellano P.R.
      • Kemp B.E.
      ) that the AMP-activated kinase phosphorylates Ser-1179 on eNOS in vitro and increases eNOS activity. While this manuscript was under review, two groups (
      • Fulton D.
      • Gratton J.-P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ,
      • Dimmeler S.
      • Fleming I.
      • Fisslthaler B.
      • Hermann C.
      • Busse R.
      • Zeiher A.M.
      ) showed that Ser-1179 is phosphorylated in response to VEGF and shear stress, that PI3-kinase inhibitors decrease agonist-induced phosphorylation of eNOS and NO production, and that PKB phosphorylates and activates eNOS in vitro.

      Acknowledgments

      B. G. thanks Dr. Steve Gygi for support, encouragement, and explanations of mass spectra.

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