Human Caco-2 intestinal epithelial motility is associated with tyrosine kinase and cytoskeletal focal adhesion kinase signals

Intestinal epithelial cells assume a specialized phenotype adapted to motility and mucosal healing during mucosal restitution. Since cell-matrix interactions initiate tyrosine kinase (TK) signals, we hypothesized that the regulation of the intestinal epithelial migratory phenotype may also involve TK signals, particularly via focal adhesion kinase (FAK), Caco-2 cells were seeded simultaneously at 26,000 and 6000 cells/cm(2). After 4 days, the first cells were confluent, while cells in the second population were not contact-inhibited and expressed migrating lamellipodia. Cells were fractionated into Triton X-100-soluble (membrane/cytoskeletal) and -insoluble (cytosolic) fractions, TK activity in each fraction was assayed by ELISA using a synthetic substrate. FAR protein was assessed by immunoprecipitation with monoclonal anti-FAR and Western blotting. Because active FAK autophosphorylates, we also measured FAK tyrosine phosphorylation, immunoprecipitating with anti-FAR and then Western blotting for phosphotyrosine. TK activity was increased in both cytosolic and membrane/cytoskeletal fractions of migrating cells by 17.6 +/- 3.6 and 28.9 +/- 4.1%, respectively, compared to static cells (n = 11, P < 0.01). FAK protein increased in the cytosolic fraction by 90.4 +/- 20.0% (n = 5, P = 0.01), but did not change in the membrane/cytoskeletal fraction. Tyrosine phosphorylated FAR increased by 62.8 +/- 21.4% in the cytosolic fraction of migrating cells but also by 46.6 +/- 38.4% in the membrane/cytoskeletal fraction (n = 5, P < 0.05), These data suggest that intestinal epithelial cell migration is associated with increases in both cytosolic and cytoskeletal TB:activity and upregulation of cytosolic FAK protein. The increase in cytoskeletal FAR phosphorylation without increased FAK protein suggests autophosphorylation and increased cytoskeletal FAK activity. The migrating intestinal epithelial phenotype may therefore be modulated by TK signals including cytoskeletal FAK activity. (C) 1998 Academic Press.