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

doi:10.1074/jbc.274.42.30101

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*

From the Departments of ^tOaMedicine and ^tOcMolecular Biotechnology, University of Washington, Seattle, Washington 98195, the ^tOgDepartment of Medicine, Emory University School of Medicine, Atlanta, Georgia 30322, and the ^tOeSchool of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332

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.

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.
↵FNb 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; E-mail: bgallis@u. washington.edu.
↵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.
↵2 B. Gallis and M. Corson, unpublished observations.
Abbreviations:

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
- Received March 16, 1999.
- Revision received July 14, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.