Collagen IV regulates Caco-2 cell spreading and p130^Cas phosphorylation by FAK-dependent and FAK-independent pathways

Abstract

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.