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

Abstract

Abstract-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. (Circ Res. 2005;97:1236-1244.)Key Words: Akt Ⅲ blood flow Ⅲ endothelial nitric oxide synthase Ⅲ mechanotransduction Ⅲ protein kinase A A lthough the endothelial NO synthase (eNOS) was initially described as a Ca 2ϩ -dependent enzyme, it is now clear that the changes in the phosphorylation of several serine and threonine (and possibly also tyrosine) residues regulate NO production in response to Ca 2ϩ -elevating agonists as well as to hemodynamic stimuli, such as cyclic stretch and fluid shear stress (for reviews, see Fleming and Busse 1 and Boo and Jo 2 ). Most is known about the role played by Ser1177, which is situated in the reductase domain of the enzyme, and Thr495, which is situated in the calmodulin-binding domain, in the regulation of NO production. The phosphorylation of these sites appears to play a reciprocal role in the regulation of eNOS activity as Ser1177 becomes phosphorylated in response to endothelial cell activation, whereas Thr495 is constitutively phosphorylated but dephosphorylated on stimulation as a consequence of the activation of phosphatases. [3][4][5] The dephosphorylation of Thr495 facilitates the Ca 2ϩ -dependent association of calmodulin with eNOS, 5 whereas the phosphorylation of eNOS on Ser1177 increases NO output in an apparently Ca 2ϩ -independent manner. 6,7 As the maintained production of endothelium-derived NO in response...

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