P96	Grb2-associated binder protein-1 and the tyrosine phosphatase SHP2 regulate activation of endothelial nitric oxide synthase in response to fluid shear stress by a PKA-dependent but Akt-independent mechanism.
1M.Dixit, 1A.E.Loot, 1A.Mohamed, 1B.Fisslthaler, 3C.M.Boulanger, 2B.Ceacareanu, 2A.Hassid, 1R.Busse, 1I.Fleming
1Johann Wolfgang Goethe-Universität, Frankfurt/Main, DE; 2University of Tennessee Health Science Center, Memphis, US; 3Institue National de la Santé et de Recherche Médicale, Paris, FR.

Fluid shear stress, the frictional force on the luminal endothelial surface caused by the flowing blood induces nitric oxide (NO) production by a mechanism involving activation of phosphatidylinositol-3-kinase (PI 3-K) and the phosphorylation of endothelial NO synthase (eNOS). Grb2-associated binder protein-1 (Gab1) through its interaction with the p85 subunit of PI 3-K and the protein tyrosine phosphatase, SHP2, is implicated in multiple signaling pathways initiated by growth factors and cytokines. Although Gab1 and SHP2 are reported to translocate to plasma membrane in a PECAM-1 dependent manner in endothelial cells in response to fluid shear stress, their role in endothelial cell physiology is largely undefined. Here we assessed the involvement of Gab1 and SHP2 in shear stress-induced signaling for activation of eNOS by over-expressing mutants of the two proteins through recombinant adenoviral vectors in porcine aortic endothelial cells (PAECs). Fluid shear stress elicited the phosphorylation of Akt and eNOS (Ser1177) as well as tyrosine phosphorylation of Gab1 and its association with SHP2 and the p85 subunit of PI 3-K. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain (ΔPHGab1) abrogated the shear stress-induced phosphorylation of Akt but failed to block the shear stress-mediated phosphorylation of eNOS. The latter response was sensitive to a protein kinase A (PKA) inhibitor. On the other hand overexpression of a Gab1 mutant unable to bind SHP2 (YFGab1) completely prevented the shear stress-induced phosphorylation and activation of eNOS without affecting the phosphorylation of Akt. Additionally, a dominant negative SHP2 mutant (DSH2) also prevented the shear stress-induced activation of PKA and subsequent phosphorylation of eNOS without affecting Akt. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor, as well as by the overexpression of either the YFGab1 mutant or the dominant negative SHP2 mutant. In conclusion, these data indicate that the shear stress-induced activation of eNOS depends upon Gab1 and the tyrosine phosphatase SHP2 which in turn regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.

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