Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells.
Fluid shear stress generated by blood flow modulates endothelial cell function via specific intracellular signaling events. We showed previously that flow activated the phosphatidylinositol 3-kinase (PI3K), Akt, and endothelial nitric-oxide synthase (eNOS) via Src kinase-dependent transactivation of vascular endothelial growth factor receptor 2 (VEGFR2). The scaffold protein Gab1 plays an important role in receptor tyrosine kinase-mediated signal transduction. We found here that laminar flow (shear stress = 12 dynes/cm2) rapidly stimulated Gab1 tyrosine phosphorylation in both bovine aortic endothelial cells and human umbilical vein endothelial cells, which correlated with activation of Akt and eNOS. Gab1 phosphorylation as well as activation of Akt and eNOS by flow was inhibited by the Src kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and VEGFR2 kinase inhibitors SU1498 and VTI, suggesting that flow-mediated Gab1 phosphorylation is Src kinase-dependent and VEGFR2-dependent. Tyrosine phosphorylation of Gab1 by flow was functionally important, because flow stimulated the association of Gab1 with the PI3K subunit p85 in a time-dependent manner. Furthermore, transfection of a Gab1 mutant lacking p85 binding sites inhibited flow-induced activation of Akt and eNOS. Finally, knockdown of endogenous Gab1 by small interference RNA abrogated flow activation of Akt and eNOS. These data demonstrate a critical role of Gab1 in flow-stimulated PI3K/Akt/eNOS signal pathway in endothelial cells.
Jin
Zheng-Gen
ZG
Center for Cardiovascular Research and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA. zheng-gen_jin@urmc.rochester.edu
Wong
Chelsea
C
Wu
Jie
J
Berk
Bradford C
BC
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001011:Aorta / Q000166:cytology; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D002934:Cinnamates / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011743:Pyrimidines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D033081:Shear Strength; D015398:Signal Transduction; D013314:Stress, Mechanical; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002934:Cinnamates; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011743:Pyrimidines; D034741:RNA, Small Interfering; D014443:Tyrosine; D040301:Vascular Endothelial Growth Factor Receptor-2; C412373:AG 1879; C098587:GAB1 protein, human; C483044:SU 1498; C496314:NOS3 protein, human; C494918:AKT1 protein, human