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 humanspecific 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 .

N-WASP-directed actin polymerization activates p130Cas phosphorylation and lamellipodium spreading

“…In addition to the present findings, others have reported that both FAK and SFKs are crucial for CasSD phosphorylation (Fonseca et al, 2004;Hamamura et al, 2008;Sanders and Basson, 2008). Results from an earlier study of FAK mutants indicate that the ability of overexpressed FAK to enhance CasSD phosphorylation relies on the autophosphorylation of FAK residue Y397 (Ruest et al, 2001).…”

“…Cas and FAK complex formation is important for CasSD phosphorylation FAK is a crucial binding partner of Cas and is responsible for phosphorylation of CasSD (Fonseca et al, 2004;Sanders and Basson, 2008). FAK and Cas coimmunoprecipitated with each other in adherent fibroblasts and fibroblasts in suspension (Fig.…”

“…The C-terminus of Cas harbors binding sites for SFKs (Nakamoto et al, 1996). Binding of FAK and Src family kinases to the N-and C-terminus of Cas is important for the proper localization and phosphorylation of Cas (Fonseca et al, 2004;Hamamura et al, 2008;Nakamoto et al, 1997;Sanders and Basson, 2008). At the same time, FAK and SFKs are directly involved in rigidity sensing (Jiang et al, 2006;Kostic and Sheetz, 2006).…”

Extracellular matrix-mediated cellular communication in the heart

“…It has been reported that collagen can promote cell survival in vitro by inhibition of apoptosis, via a β1 integrin-dependent mechanism [72]. In addition, collagen participates in cell spreading through p130 Cas phosphorylation via FAK-dependent and FAK-independent integrin receptor pathways [73]. Collagen is also implicated in the induction of proliferation via FAK activation and downstream signaling pathways (Src, MEK, PI3-kinase, and p38 MAPK) [70, 74] (Figure 1).…”

Heart Remodelation: Role of MMPs

“…In addition, collagen participates in cell spreading through p130Cas phosphorylation via FAK-dependent and FAK-independent integrin receptor pathways [9,10]. Finally, collagen plays a key role in cell migration through the activation of FAK and PI3-K, leading to elevated Rac1 activity as a downstream consequence in activated cell migration [11].…”