Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells
Flow shear stress stimulates Gab1 tyrosine phosphorylation to mediate protein kinase B and endothelial nitric-oxide synthase activation in endothelial cells
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DOI：
10.1074/jbc.m500294200
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发表时间：
2005-04-01
影响因子：
4.8
通讯作者：
Berk, BC
中科院分区：
文献类型：
--
作者：
Jin, ZG;Wong, C;Berk, BC
血流产生的流体剪切应力通过特定的细胞内信号传导事件调节内皮细胞功能。我们之前表明，流通过血管内皮生长因子受体 2 (VEGFR2) 的 Src 激酶依赖性反式激活来激活磷脂酰肌醇 3 激酶 (PI3K)、Akt 和内皮一氧化氮合酶 (eNOS)。支架蛋白Gab1在受体酪氨酸激酶介导的信号转导中发挥重要作用。我们在这里发现层流（剪切应力= 12 dynes/cm(2)）快速刺激牛主动脉内皮细胞和人脐静脉内皮细胞中的Gab1酪氨酸磷酸化，这与Akt和eNOS的激活相关。 Gab1 磷酸化以及流对 Akt 和 eNOS 的激活受到 Src 激酶抑制剂 PP2（4-氨基-5-(4-氯苯基)-7-(叔丁基)吡唑并[3,4-d]嘧啶）和 VEGFR2 激酶抑制剂 SU1498 和 VTI 的抑制，表明流介导的 Gab1 磷酸化是 Src 激酶依赖性和 VEGFR2 依赖性的。流对 Gab1 的酪氨酸磷酸化在功能上很重要，因为流以时间依赖性方式刺激 Gab1 与 PI3K 亚基 p85 的结合。此外，转染缺乏 p85 结合位点的 Gab1 突变体会抑制流动诱导的 Akt 和 eNOS 激活。最后，通过小干扰 RNA 敲低内源性 Gab1 消除了 Akt 和 eNOS 的流动激活。这些数据证明了 Gab1 在内皮细胞中流动刺激的 PI3K/Akt/eNOS 信号通路中的关键作用。
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/cm(2)) 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.