粘着斑激酶在TNF-α作用下影响SMMC-7721细胞PKB的表达水平

Focal Adehision Kinase Could Affect PKB Protein Level in Response to TNF-α

探讨在肿瘤坏死因子α(TNF-α)作用下 ,粘着斑激酶 (focal adhesion kinase,FAK)对蛋白激酶 B(PKB)蛋白水平的影响 .利用构建、转染 FAK反义质粒来特异性降低 SMMC- 772 1细胞的FAK含量 ,及用 Western杂交的方法来检测 PKB的蛋白含量 .文献报道 TNF- α能够激活磷脂酰肌醇 3-激酶 (PI3K)而使 PKB发生磷酸化 .但是至于 TNF-α对 PKB蛋白水平的影响目前并无报道 .研究发现 ,当用 wortmannin特异性抑制 PI3K活性后可以显著降低 PKB的蛋白含量 .提示PI3K对维持 PKB的基础蛋白水平是必需的 .但是 TNF- α本身对 PKB的蛋白水平无明显影响 .而当用不同浓度的 TNF- α和 wortmannin处理 SMMC- 772 1细胞时 ,发现 PKB的蛋白含量随着TNF-α浓度的增加而降低 .提示 TNF-α可能除了通过 PI3K外 ,还可能通过另一条途径来下调PKB的表达 .而当用 FAK反义质粒转染 SMMC- 772 1细胞后 (FAK下降了 60 % ) ,发现在当用不同浓度的 TNF- α处理的情况下 ,FAK反义质粒转染株 AS- 772 1细胞的 PKB含量降低为对照的70 % ;而在用 TNF-α和 wortmannin处理的情况下 ,下降为对照的 40 %～ 60 % .TNF-α能够通过PI3K及另一未知途径来影响 PKB的蛋白水平 .而 FAK在 TNF- α作用下能够不通过 PI3K来影响PKB的蛋白水平 .

To explore the effect of focal adhesion kinase (FAK) on PKB protein level in response to TNF\|α.FAK antisense plasmid was constructed and transfected to SMMC\|7721 cells to study downregulation of FAK on PKB level.Western blot assay was adopted to examine PKB level.PKB was reported to be phosphorylated by TNF\|α through phosphatidyl inositol 3 kinase(PI 3K),but the effect of TNF\|α on PKB protein level has not been reported.Treatment of wortmannin alone caused dramatic reduction of PKB level in SMMC\|7721 cells,suggesting that PI 3K was essential for maintaining PKB basal level.But,in response to co\|treatment of wortmannin and TNF\|α,PKB was reduced with increase of TNF\|α concentration.And TNF\|α itself had no significant effect on PKB level.The results suggested that there existed another signal regulating PKB level.In response to TNF\|α,the FAK antisense transfectants AS\|7721 (60% reduction of FAK level) produced 70% PKB of that in controls.When exposed to wortmannin and TNF\|α,AS\|7721 produced 40%—60% PKB of that in the controls.TNF\|α could affect PKB protein level through PI 3K and another unknown pathway.And in response to TNF\|α,FAK could regulate PKB level through a signal other than PI 3K.