Collagen IV regulates Caco-2 cell spreading and p130Cas phosphorylation by FAK-dependent and FAK-independent pathways.
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).
Sanders
Matthew A
MA
Department of Surgery, Wayne State University, Detroit, MI 48201, USA. aj2198@wayne.edu
Basson
Marc D
MD
D018938:Caco-2 Cells; D002448:Cell Adhesion / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D024141:Collagen Type IV / Q000494:pharmacology*; D050719:Crk-Associated Substrate Protein / Q000378:metabolism*; D003470:Culture Media; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D006801:Humans; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
C493841:BCAR1 protein, human; D024141:Collagen Type IV; D050719:Crk-Associated Substrate Protein; D003470:Culture Media; D034741:RNA, Small Interfering; D051418:Focal Adhesion Kinase 2