Study Of Egf-mediated Important Signal Protein Activity Of Mapk/pi3k/plc-γ1 Pathways

EGF-mediated signal pathways are included in various cell processes, such as cellular proliferation, cellular differentiation and cell migration,and also promote the growth of tumors. In present experiments, the expressions of some important signal proteins of EGF-mediated signal pathways in cell interphase and mitosis are investigated in response to EGF. The results were shown following:1. To investigate the protein phosphorylation levels in different signal pathways, interphase cells and mitotic cells were respectively collected and lysated after they were treated with or without nocodazole (200 ng/mL) and EGF (50 ng/mL). The results showed that the phosphorylation level of EGFR tyrosine kinase stimulated by EGF in cellular mitosis was probably higher than that in cellular interphase. EGF-activated pathways (EGF-mediated MAPK, PI3K, PLC-γ1 cascades) were inhibited at the same time when the cells were blocked in the mitosis (G2/M time); EGF-mediated ERK and AKT phosphorylation in cell mitosis were significantly blocked (P<0.05), which showed that MAPK and PI3K pathways by EGF were significantly inhibited. In addition, FAK and Cortactin phosphorylation in cell mitosis were inhibited and Cbl phosphorylation level in cell interphase was almost quiet compared with that in mitosis in response to 50 ng/mL EGF (P>0.05).2. The expressions of EGFR and its downstream signal proteins were affected by AG1478, U0126, U73122 and Wortmannin; the phosphorylations of EGFR and related singal proteins were inhibited, the roles of which also were possibly related with the concentration of AG1478. After EGFR phosphorylation in response to EGF (50 ng/mL) were blocked by AG1478 (0.5μmol/L), EGF-mediated Raf and MEK phosphorylation levels were greatly decreased; 0.5μmol/L AG1478 made ERK phosphorylation level of Hela-H2B cells down but 2.5μmol/L AG1478 decreased ERK phosphorylation level of COS7 cells. AKT, PLC-γ1, Cbl and Cortactin phosphorylations in response to EGF (50 ng/mL) also were inhibited by 0.5μmol/L AG1478. MEK phosphorylation in response to EGF (50 ng/mL) was blocked by 10μmol/L U0126; PLC-γ1 phosphorylation level in response to EGF (50 ng/mL) was decreased by U73122, especially very significant when 100μmol/L U73122; AKT phosphorylation level in response to EGF (50 ng/mL) was significantly downregulated by 100 nmol/L Wortmannin.3. ERK phosphorylation was affected by EGF. ERK phosphorylation level in nocodazole-untreated cells was down with the time of serum starvation extending, especially very significant after 12 h (P<0.05); and ERK phosphorylation level in nocodazole-treated cells was possibly decreased with nocodazole-treated time lengthening. After nocodazole-untreated or–treated cells were stimulated by EGF (50 ng/mL), ERK phosphorylation level was increased in nocodazole-untreated cells (P<0.05) and decreased in nocodazole-treated cells. MEK phosphorylation level of nocodazole-treated cells was lower than that of nocodazole-untreated cells when cells were treated with or without nocodazole (250 ng/mL) for 8 h; and EGF-mediated ERK phosphorylation level of nocodazole-treated cells was lower than that of nocodazole-untreated cells when cells were treated with or without nocodazole (250 ng/mL) for 12 h, especially very significant for 24 h (P<0.05). Next the results showed that the cell migration by EGF stimulation was blocked in response to nocodazole.4. The quantity of EGF-EGFR complexes in nocodazole-untreated cells was more than that in nocodazole-treated cells, which suggested that the endocytosis of EGF-EGFR complex was inhibited in cell mitosis. Through the investigation of Clathrin and Caveolin related with EGFR endocytosis pathway, it is found that Clathrin expression in cell mitosis was greatly less than that in cell interphase in response to EGF (P<0.05) but Caveolin expression in cell interphase was quiet compared with that in mitosis, which suggested that EGFR endocytosis by Clathrin in cell mitosis was blocked. Then EGFR tyrosine kinase phosphorylation levels were analyzed. It is certified that the phosphorylation levels of Y992, Y1045, Y1068, Y1086 and Y1173 sites were dependent on cell lines. In COS7 cells, the phosphorylation levels of 5 sites had almost no different in cell interphase and mitosis (P>0.05). In Hela-H2B cells, the phosphorylation level from Y992 site in mitosis was higher than that in cell interphase. In Cho-EGFR cells, the phosphorylation levels of 5 sites in mitosis were higher than that in cell interphase. Additionally, the results showed that the interaction of EGFR with Grb2 or Shc in cell mitosis was blocked and Ras phosphorylation also was inhibited in cell mitosis. Finally,it was centified that PMA-mediated phosphorylation levels of MEK and ERK were decreased in the mitosis. According to the above results, it is concluded that MAPK pathway is blocked in cell mitosis.On the whole, present experiments showed that the phosphoryaltion levels of signal proteins of EGF-mediated signal pathways in cell interphase are more than that in cell mitosis; EGF-mediated protein activities are blocked by protein kinase inhibitors such as AG1478, U0126, U73122 and Wortmannin; when cells enter into the mitosis, MAPK cascade is significantly inhibited.