This Article Full Text Full Text (PDF) Data Supplement All Versions of this Article: 102/12/1520    most recent CIRCRESAHA.108.172072v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Fisslthaler, B. Articles by Fleming, I. Search for Related Content PubMed PubMed Citation Articles by Fisslthaler, B. Articles by Fleming, I. Related Collections Signal transduction Endothelium/vascular type/nitric oxide

(Circulation Research. 2008;102:1520.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Inhibition of Endothelial Nitric Oxide Synthase Activity by Proline-Rich Tyrosine Kinase 2 in Response to Fluid Shear Stress and Insulin

Beate Fisslthaler, Annemarieke E. Loot, Annisuddin Mohamed, Rudi Busse, Ingrid Fleming

From the Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany.

Correspondence to Ingrid Fleming PhD, Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail fleming{at}em.uni-frankfurt.de

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS^–/– mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.

Key Words: blood flow • insulin • mechanotransduction • nitric oxide • nitric oxide synthases • phosphorylation

This article has been cited by other articles:

				A. E. Loot, J. G. Schreiber, B. Fisslthaler, and I. Fleming Angiotensin II impairs endothelial function via tyrosine phosphorylation of the endothelial nitric oxide synthase J. Exp. Med., November 23, 2009; (2009) jem.20090449v1. [Abstract] [Full Text] [PDF]

				F. Finetti, S. Donnini, A. Giachetti, L. Morbidelli, and M. Ziche Prostaglandin E2 Primes the Angiogenic Switch via a Synergic Interaction With the Fibroblast Growth Factor-2 Pathway Circ. Res., September 25, 2009; 105(7): 657 - 666. [Abstract] [Full Text] [PDF]

				D. J.R. Fulton Mechanisms of Vascular Insulin Resistance: A Substitute Akt? Circ. Res., May 8, 2009; 104(9): 1035 - 1037. [Full Text] [PDF]

				H. Korhonen, B. Fisslthaler, A. Moers, A. Wirth, D. Habermehl, T. Wieland, G. Schutz, N. Wettschureck, I. Fleming, and S. Offermanns Anaphylactic shock depends on endothelial Gq/G11 J. Exp. Med., February 16, 2009; 206(2): 411 - 420. [Abstract] [Full Text] [PDF]

				R. Martinelli, M. Gegg, R. Longbottom, P. Adamson, P. Turowski, and J. Greenwood ICAM-1-mediated Endothelial Nitric Oxide Synthase Activation via Calcium and AMP-activated Protein Kinase Is Required for Transendothelial Lymphocyte Migration Mol. Biol. Cell, February 1, 2009; 20(3): 995 - 1005. [Abstract] [Full Text] [PDF]

				W.-Z. Ying, K. Aaron, and P. W. Sanders Dietary Salt Activates an Endothelial Proline-Rich Tyrosine Kinase 2/c-Src/Phosphatidylinositol 3-Kinase Complex to Promote Endothelial Nitric Oxide Synthase Phosphorylation Hypertension, December 1, 2008; 52(6): 1134 - 1141. [Abstract] [Full Text] [PDF]