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(Circulation Research. 2005;97:1236.)
© 2005 American Heart Association, Inc.

Molecular Medicine

Gab1, SHP2, and Protein Kinase A Are Crucial for the Activation of the Endothelial NO Synthase by Fluid Shear Stress

Madhulika Dixit, Annemarieke E. Loot, Annisuddin Mohamed, Beate Fisslthaler, Chantal M. Boulanger, Bogdan Ceacareanu, Aviv Hassid, Rudi Busse, Ingrid Fleming

From the Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie (M.D., A.E.L., A.M., B.F., R.B., I.F.), Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany; Institut National de la Santé et de la Recherche Médicale (INSERM) (C.M.B.), Unit 541, Hôpital Lariboisière, Paris, France; and Department of Physiology (B.C., A.H.), University of Tennessee Health Sciences Center, Memphis, Tenn.

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

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress–induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress–induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress–induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

Key Words: Akt • blood flow • endothelial nitric oxide synthase • mechanotransduction • protein kinase A

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