Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress.
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.
Dixit
Madhulika
M
Vascular Signalling Group, Institut für Kardiovaskuläre Physiologie, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Loot
Annemarieke E
AE
Mohamed
Annisuddin
A
Fisslthaler
Beate
B
Boulanger
Chantal M
CM
Ceacareanu
Bogdan
B
Hassid
Aviv
A
Busse
Rudi
R
Fleming
Ingrid
I
D000109:Acetylcholine / Q000494:pharmacology; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012039:Regional Blood Flow; D015398:Signal Transduction; D013314:Stress, Mechanical; D013552:Swine; D014443:Tyrosine / Q000378:metabolism; D014664:Vasodilation / Q000187:drug effects
D048868:Adaptor Proteins, Signal Transducing; C490897:Gab1 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D014443:Tyrosine; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; D000109:Acetylcholine