Sanders Matthew A, Basson Marc D
Department of Surgery, Wayne State University, Detroit, MI 48201, USA.
Biol Chem. 2008 Jan;389(1):47-55. doi: 10.1515/BC.2008.008.
We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130(Cas) phosphorylation and stimulates focal adhesion kinase (FAK). However, the role of FAK and the related kinase, Pyk2, in Caco-2 spreading and migration is unclear. FAK- or Pyk2-specific siRNAs reduced protein levels by 90%. However, when detached cells were replated on collagen IV neither individual nor combined FAK and Pyk2 siRNAs affected the cell spreading rate. As combined FAK and Pyk2 siRNAs increased p130(Cas) protein levels, we cotransfected cells with 1 nm p130(Cas) siRNA to partially reduce p130(Cas) protein to control levels. Although p130(Cas) Tyr(P)(249) phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130(Cas) increased cell spreading by 20% compared to p130(Cas) siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130(Cas). FAK-binding mutant SH3 domain-deleted rat p130(Cas) was not phosphorylated after adhesion and, unlike full-length p130(Cas), did not restore spreading after human-specific p130(Cas) siRNA knockdown of endogenous p130(Cas). Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130(Cas) phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130(Cas).
我们之前观察到，IV型胶原蛋白通过Src依赖的p130（Cas）磷酸化调节Caco-2肠上皮细胞的铺展和迁移，并刺激粘着斑激酶（FAK）。然而，FAK和相关激酶Pyk2在Caco-2铺展和迁移中的作用尚不清楚。FAK或Pyk2特异性siRNA使蛋白水平降低了90%。然而，当将脱离的细胞重新接种到IV型胶原蛋白上时，单独的或联合的FAK和Pyk2 siRNA均不影响细胞铺展速率。由于联合的FAK和Pyk2 siRNA增加了p130（Cas）蛋白水平，我们用1 nM的p130（Cas）siRNA共转染细胞，以将p130（Cas）蛋白部分降低至对照水平。尽管p130（Cas）Tyr（P）（249）磷酸化降低了60%，但细胞铺展未受影响。与单独使用p130（Cas）siRNA相比，联合使用siRNA降低FAK、Pyk2和p130（Cas）可使细胞铺展增加20%，这表明FAK和Pyk2除了通过p130（Cas）刺激铺展外，还对铺展起负调节作用。FAK结合突变体SH3结构域缺失的大鼠p130（Cas）在粘附后未被磷酸化，并且与全长p130（Cas）不同，在内源性p130（Cas）被人特异性p130（Cas）siRNA敲低后不能恢复铺展。总之，这些数据表明FAK通过p130（Cas）磷酸化对IV型胶原蛋白上的Caco-2铺展起正调节作用，但也表明FAK可能通过其他机制对铺展起负调节作用，并且存在额外的不依赖FAK的调节p130（Cas）的途径。
