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Collagen IV Regulates Caco-2 Cell Spreading And P130Cas Phosphorylation By FAK-dependent And FAK-independent Pathways

M. Sanders, M. Basson
Published 2008 · Biology, Medicine

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Abstract We previously observed that collagen IV regulates Caco-2 intestinal epithelial cell spreading and migration via Src-dependent p130Cas 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 p130Cas protein levels, we cotransfected cells with 1 nm p130Cas siRNA to partially reduce p130Cas protein to control levels. Although p130Cas Tyr(P)249 phosphorylation was reduced by 60%, cell spreading was unaffected. Combined siRNA reduction of FAK, Pyk2 and p130Cas increased cell spreading by 20% compared to p130Cas siRNA alone, suggesting that FAK and Pyk2 negatively regulate spreading in addition to stimulating spreading via p130Cas. FAK-binding mutant SH3 domain-deleted rat p130Cas was not phosphorylated after adhesion and, unlike full-length p130Cas, did not restore spreading after human-specific p130Cas siRNA knockdown of endogenous p130Cas. Together, these data suggest that FAK positively regulates Caco-2 spreading on collagen IV via p130Cas phosphorylation, but also suggests that FAK may negatively regulate spreading through other mechanisms and the presence of additional FAK-independent pathways regulating p130Cas.
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