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目的研究上皮生长因子受体和FAK的相互作用以及对下游信号的影响。方法建立聚集粘连激酶(FAK)缺失突变和绿色荧光蛋白(GFP)融合基因del1-693FAK-GFP、del1-100FAK-GFP和FAK-GFP稳定表达细胞系。结果同野生型FAK-GFP相比,N-端1-100氨基酸残基的缺失突变体,缺失1-693氨基酸残基的突变体结合在黏附点的能力被完全抑制。应用等电聚焦和SDS-PAGE双向电泳证明,EGF和纤维连接蛋白诱导FAK磷酸化的位点不同,进一步证实del1-693FAK-GFP、del1-100FAK-GFP,抑制MAPK的磷酸化,增强Akt的磷酸化;而FAK-GFP增强MAPK磷酸化,抑制Akt磷酸化。结论FAK通过和EGFR的相互作用调节MAPK和Akt之间的相对平衡。
Aim To investigate the interaction between epithelial growth factor receptor and FAK and its effect on downstream signaling. Methods FAK deletion mutation and green fluorescent protein (GFP) fusion gene del1-693FAK-GFP, del1-100FAK-GFP and FAK-GFP stable expression cell lines were established. As a result, compared with the wild-type FAK-GFP, the mutant having the N-terminal 1-100 amino acid residue and the mutant lacking the amino acid residue 1-693 were completely inhibited in binding ability at the adhesion site. Application of isoelectric focusing and SDS-PAGE two-dimensional electrophoresis demonstrated that EGF and fibronectin induced different sites of FAK phosphorylation, further confirming that del1-693FAK-GFP, del1-100FAK-GFP, inhibits MAPK phosphorylation and enhances Akt phosphorylation While FAK-GFP enhanced MAPK phosphorylation and inhibited Akt phosphorylation. Conclusions FAK regulates the relative balance of MAPK and Akt through its interaction with EGFR.