The phosphoinositide-3 kinase (PI-3K)/AKT cell survival pathway is an important pathway activated by EGFR signaling. Here we show, that in addition to previously described critical components of this pathway, i.e., the docking protein Gab1, the PI-3K/AKT pathway in epithelial cells is regulated by the exocyst complex, which is a vesicle tether that is essential for exocytosis. Using live-cell imaging, we demonstrate that PI(3,4,5)P 3 levels fluctuate at the membrane on a minutes time scale and that these fluctuations are associated with local PI(3,4,5)P 3 increases at sites where recycling vesicles undergo exocytic fusion. Supporting a role for exocytosis in PI(3,4,5)P 3 generation, acute promotion of exocytosis by optogenetically driving exocyst-mediated vesicle tethering up-regulates PI(3,4,5)P 3 production and AKT activation. Conversely, acute inhibition of exocytosis using Endosidin2, a small-molecule inhibitor of the exocyst subunit Exo70 (also designated EXOC7), or inhibition of exocyst function by siRNA-mediated knockdown of the exocyst subunit Sec15 (EXOC6), impairs PI(3,4,5)P 3 production and AKT activation induced by EGF stimulation of epithelial cells. Moreover, prolonged inhibition of EGF signaling by EGFR tyrosine kinase inhibitors results in spontaneous reactivation of AKT without a concomitant relief of EGFR inhibition. However, this reactivation can be negated by acutely inhibiting the exocyst. These experiments demonstrate that exocyst-mediated exocytosis—by regulating PI(3,4,5)P 3 levels at the plasma membrane—subserves activation of the PI-3K/AKT pathway by EGFR in epithelial cells.

中文翻译：

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通过胞吐介导的胞吐作用调节 EGF 刺激的 PI-3K/AKT 通路激活

磷酸肌醇 3 激酶 (PI-3K)/AKT 细胞存活通路是 EGFR 信号激活的重要通路。在这里，我们表明，除了先前描述的该通路的关键成分（即对接蛋白 Gab1）外，上皮细胞中的 PI-3K/AKT 通路还受胞吐复合体调节，胞吐复合体是胞吐作用所必需的囊泡系链. 使用活细胞成像，我们证明了 PI(3,4,5)P3个水平在膜上以分钟的时间尺度波动，并且这些波动与局部 PI(3,4,5)P 有关3个在回收囊泡进行胞吐融合的位点增加。支持 PI(3,4,5)P 中的胞吐作用3个通过光遗传学驱动胞吐介导的囊泡束缚上调 PI(3,4,5)P 来产生、急性促进胞吐作用3个生产和 AKT 激活。相反，使用胞吐亚基 Exo70（也称为 EXOC7）的小分子抑制剂 Endosidin2 急性抑制胞吐作用，或通过 siRNA 介导的胞吐亚基 Sec15 (EXOC6) 敲低抑制胞吐功能，会损害 PI(3,4) ,5)P3个EGF 刺激上皮细胞诱导的 AKT 产生和激活。此外，EGFR 酪氨酸激酶抑制剂对 EGF 信号传导的长期抑制会导致 AKT 自发重新激活，而不会同时缓解 EGFR 抑制。然而，这种重新激活可以通过急剧抑制胞囊来抵消。这些实验表明，exocyst 介导的胞吐作用——通过调节 PI(3,4,5)P3个质膜水平——促进上皮细胞中 EGFR 对 PI-3K/AKT 通路的激活。