Regulation of cell death and neurite outgrowth by overexpressed focal adhesion kinase in rat pheochrome cytoma PC 12

我们建立了过表达局灶黏附激酶（FAK）或其突变基因Y397F、K454R或Y925F的细胞。在用神经生长因子（NGF）分化细胞后，我们估计了每个细胞对氧化应激或内质网应激诱导的细胞死亡的敏感性。我们还测定了过表达抗凋亡蛋白Bcl-2或Bcl-xL的细胞的敏感性。(1)在过表达Bcl-2或Bcl-xL的细胞中，氧化应激（鱼藤酮、多巴胺、H2O2）或内质网应激（brefeldin A、thapsigargin）诱导的细胞死亡明显受到抑制。鱼藤酮诱导的caspase-2和caspase- 3的活化也被抑制。(2) FAK过表达有抵抗氧化应激导致细胞死亡的倾向。(3)在过表达自磷酸化位点Y397F突变的细胞中，氧化应激诱导的细胞死亡比载体细胞增加。然而，在催化位点过表达K454R诱导细胞死亡的抑制。此外，鱼藤酮诱导的caspase-2或-3活性在Y397F细胞中也增强，但在K454R细胞中明显抑制。在c端过表达Y925F的细胞中，对细胞死亡的敏感性没有改变。(4)在过表达Y397F的细胞中，作为存活信号一部分的Akt的表达比载体细胞低。抗凋亡蛋白Bcl-xL的表达未见改变。在K454R细胞中，这两种蛋白的表达没有改变。上述突变Y397F和K454R能够抑制FAK依赖性信号通路，但对细胞死亡的敏感性存在差异。有必要澄清FAK激活后信号传导的变化。(5)在过表达任何基因的细胞中，ngf诱导的神经突生长与载体细胞相似。

We established cells overexpressed focal adhesion kinase (FAK) or its mutated genes named Y397F, K454R or Y925F. After cells were differentiated with nerve growth factor (NGF), we estimated the sensitivity of each cell against cell death induced by oxidative stress or ER stress. We also determined the sensitivity of cells overexpressed antiapoptotic protein Bcl-2 or Bcl-xL.(1) In the cells overexpressed Bcl-2 or Bcl-xL, cell death was clearly suppressed induced by oxidative stress (rotenone, dopamine, H2O2) or ER stress (brefeldin A, thapsigargin). Activation of caspase-2 or 3 induced by rotenone was also inhibited.(2) Overexpression of FAK tended to resist against cell death by oxidative stress.(3) In the cells overexpressed Y397F mutation which is autophosphorylation site, cell death induced by oxidative stress was increased compared with vector cells. However, overexpression of K454R at catalytic site induced suppression of cell death. Moreover, rotenone-induced caspase-2 or -3 activity was also enhanced in Y397F cells, but clearly inhibited in K454R cells. In the cells overexpressed Y925F at C-terminus, sensitivity of cell death was not altered.(4) In the cells overexpressed Y397F, expression of Akt which is a part of survival signal, was decreased than vector cells. Antiapoptotic protein Bcl-xL expression was not altered. In the K454R cells, expression of both proteins were not altered. The above mutations, Y397F and K454R, are able to suppress FAK dependent signalling pathway, however, there is a difference against the sensitivity of cell death. It is necessary to clarify the varies of signalling after FAK activation.(5) In the cells overexpressed any genes, NGF-induced neurite outgrowth was similar to vector cells.