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/cm2. 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. FAK protein was assessed by immunoprecipitation with monoclonal anti-FAK and Western blotting. Because active FAK autophosphorylates, we also measured FAK tyrosine phosphorylation, immunoprecipitating with anti-FAK 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 FAK 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 TK activity and upregulation of cytosolic FAK protein. The increase in cytoskeletal FAK 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.
Liu
Y W
YW
Department of Surgery, Yale University, New Haven, Connecticut, 06520, USA.
Sanders
M A
MA
Basson
M D
MD
D000595:Amino Acid Sequence; D000906:Antibodies; D015153:Blotting, Western; D018938:Caco-2 Cells / Q000166:cytology; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology*; D003599:Cytoskeleton / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology* / Q000201:enzymology*; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D013379:Substrate Specificity
D000906:Antibodies; D015815:Cell Adhesion Molecules; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human