佛波酯促进NIH3T3细胞的铺展和聚焦粘附激酶的酪氨酸磷酸化

100nmol/L佛波酯(12-O-tetradecanoylphobol-13-acetate,TPA)能明显促进NIH3T3细胞在纤连蛋白(Fn)上的铺展,该作用能分别被酪氨酸激酶(tyrosinekinase,TK)抑制剂4′,5,7-三羟基异黄酮(genistein)和蛋白激酶C(proteinkinaseC,PKC)抑制剂calphostinC和神经鞘氨醇(sphingosine)所抑制.TPA作用于结合到Fn上的NIH3T3细胞,使其聚焦粘附激酶(focaladhe-sionkinase,FAK)的酪氨酸磷酸化程度较未处理细胞升高,于30min时达对照的204.0%,并存在浓度依赖性;该变化分别被上述抑制剂所拮抗;未经TPA处理的NIH3T3细胞和纤连蛋白结合诱导的FAK酪氨酸磷酸化亦分别被上述抑制剂所抑制.细胞松弛素D则无论TPA作用与否,都能完全阻断NIH3T3细胞的铺展和FAK的酪氨酸磷酸化.以上结果提示,TPA促进NIH3T3细胞在Fn上铺展的信号转导机制,与PKC的激活有关,进一步则可能通过影响FAK的酪氨酸磷酸化来实现,同时需要细胞骨架的参与;NIH3T3细胞和Fn结合并诱导FAK酪氨酸磷酸化的过程亦依赖于PKC和完整的细胞骨架.

TPA Enhanced Tyrosine Phosphorylation of FAK of NIH3T3 Cells and Promoted Its Spreading on Fibronectin

PKC activator TPA can regulate cell adhesion and migration. Here it was found to enhance tyrosine phosphorylation of FAK of NIH3T3 cells and promote its spreading on fibronectin. Both of the effects were inhibited by genistein, calphostin C, sphingosine and cytochalasin D respectively. These results implicated that enhancement of TPA on cell spreading and tyrosine phosphorylation of FAK required the involvement of PKC and cytoskeletal protein. In addition, the spreading of NIH3T3 cells and its tyrosine phosphorylation of FAK were observed also to be inhibited by genistein, sphingosine, calphostin C and cytochalasin D respectively, suggesting that PKC activation and the integrity of cytoskeleton were also necessary for induction of FAK tyrosine phosphorylation of NIH3T3 cells plated on Fn.