正常受体酪氨酸激酶(RTK)需要到达质膜(PM)以进行配体诱导的激活,而其致癌突变体可以在到达细胞器的PM之前被激活,例如高尔基/跨高尔基网络(TGN)。来自内质网(ER)的蛋白质输出抑制剂,如brefeldinA(BFA)和2-甲基亲原酰胺(M-COPA),可以抑制癌细胞中突变RTK的激活,表明RTK突变体不能在ER中启动信号传导。BFA和M-COPA阻断在ER-高尔基体蛋白运输中起关键作用的ADP-核糖基化因子(ARFs)的功能。然而,在ARF家族蛋白中,BFA或M-COPA抑制的特定ARF,也就是说,从ER传输RTK所涉及的ARF,仍然不清楚。在这项研究中,我们表明,M-COPA不仅阻断了试剂盒的输出,而且还阻断了PDGFRA/EGFR/METRTK从ER的输出。ER保留的RTK不能完全转导抗凋亡信号,从而导致癌细胞凋亡。此外,ARF1、ARF3、ARF4、ARF5或ARF6的单次击倒不能阻止RTK的ER输出,表明BFA/M-COPA治疗不能通过仅一个ARF成员的击倒来模仿。有趣的是,同时转染ARF1、ARF4和ARF5siRNA反映了BFA/M-COPA处理的效果。与这些结果一致,体外下拉实验显示BFA/M-COPA阻断ARF1、ARF4和ARF5的功能。一起来看,这些结果表明,BFA/M-COPA至少靶向ARF1,ARF4和ARF5;换句话说,RTK需要同时激活ARF1、ARF4和ARF5来导出它们的ER。
Normal receptor tyrosine kinases (RTKs) need to reach the plasma membrane (PM) for ligand-induced activation, whereas its cancer-causing mutants can be activated before reaching the PM in organelles, such as the Golgi/trans-Golgi network (TGN). Inhibitors of protein export from the endoplasmic reticulum (ER), such as brefeldin A (BFA) and 2-methylcoprophilinamide (M-COPA), can suppress the activation of mutant RTKs in cancer cells, suggesting that RTK mutants cannot initiate signaling in the ER. BFA and M-COPA block the function of ADP-ribosylation factors (ARFs) that play a crucial role in ER-Golgi protein trafficking. However, among
ARF family proteins, the specific ARFs inhibited by BFA or M-COPA, that is, the ARFs involved in RTKs transport from the ER, remain unclear. In this study, we showed that M-COPA blocked the export of not only KIT but also PDGFRA/EGFR/MET RTKs from the ER. ER-retained RTKs could not fully transduce anti-apoptotic signals, thereby leading to cancer cell apoptosis. Moreover, a single knockdown of ARF1, ARF3, ARF4, ARF5, or ARF6 could not block ER export of RTKs, indicating that BFA/M-COPA treatment cannot be mimicked by the knockdown of only one
ARF member. Interestingly, simultaneous transfection of ARF1, ARF4, and ARF5 siRNAs mirrored the effect of BFA/M-COPA treatment. Consistent with these results, in vitro pulldown assays showed that BFA/M-COPA blocked the function of ARF1, ARF4, and ARF5. Taken together, these results suggest that BFA/M-COPA targets at least ARF1, ARF4, and ARF5; in other words, RTKs require the simultaneous activation of ARF1, ARF4, and ARF5 for their ER export.