Shi Huaiping, Zhang Tianying, Yi Yongqing, Ma Yue
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, P.R. China.
Oncol Rep. 2016 Jun;35(6):3593-9. doi: 10.3892/or.2016.4696. Epub 2016 Mar 21.
Although previous studies have shown that Ras-ERK signaling in mitosis is closed due to the inhibition of signal transduction, the events involved in the molecular mechanisms are still unclear. In the present study, we investigated the Ras-ERK signaling pathway in mitotic COS7 cells. The results demonstrated that treatment with epidermal growth factor (EGF) failed to increase the endocytosis of EGF-EGFR (EGF receptor) complexes in mitotic COS7 cells, although a large amount of endosomes were found in asynchronous COS7 cells. Clathrin expression levels in mitotic COS7 cells were inhibited whereas caveolin expression levels in mitotic COS7 cells were almost unaffected. Y1068 and Y1086 residues of EGFR in the mitotic COS7 cells were activated. However, Grb2 and Shc in the mitotic COS7 cells did not bind to activated EGFR. Ras activity was inhibited in the mitotic COS7 cells whereas its downstream protein, Raf, was obviously phosphorylated by EGF in mitosis. Treatment with phorbol 12-myristate 13-acetate (PMA) also increased the phosphorylation levels of Raf in the mitotic COS7 cells. Nevertheless, Raf phosphorylation in mitosis was significantly inhibited by AG1478. Lastly, activation of EGF-mediated MEK and ERK in the mitotic COS7 cells was obviously inhibited. In summary, our results suggest that the Ras-ERK pathway is inhibited in mitotic COS7 cells which may be the dual result of the difficulty in the transduction of EGF signaling by EGFR or Raf to downstream proteins.
尽管先前的研究表明，有丝分裂过程中的Ras-ERK信号由于信号转导的抑制而关闭，但分子机制所涉及的事件仍不清楚。在本研究中，我们研究了有丝分裂期COS7细胞中的Ras-ERK信号通路。结果表明，用表皮生长因子（EGF）处理未能增加有丝分裂期COS7细胞中EGF-EGFR（EGF受体）复合物的内吞作用，尽管在非同步化的COS7细胞中发现了大量的内体。有丝分裂期COS7细胞中的网格蛋白表达水平受到抑制，而有丝分裂期COS7细胞中的小窝蛋白表达水平几乎未受影响。有丝分裂期COS7细胞中EGFR的Y1068和Y1086残基被激活。然而，有丝分裂期COS7细胞中的Grb2和Shc不与激活的EGFR结合。有丝分裂期COS7细胞中的Ras活性受到抑制，而其下游蛋白Raf在有丝分裂过程中被EGF明显磷酸化。用佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）处理也增加了有丝分裂期COS7细胞中Raf的磷酸化水平。然而，AG1478显著抑制了有丝分裂过程中的Raf磷酸化。最后，有丝分裂期COS7细胞中EGF介导的MEK和ERK的激活明显受到抑制。总之，我们的结果表明，有丝分裂期COS7细胞中的Ras-ERK通路受到抑制，这可能是EGFR或Raf难以将EGF信号转导至下游蛋白的双重结果。
