Changes in
integrin expression during malignant transformation have been observed in many
tumors. Colon-
carcinoma cells show reduced expression or even loss of the
alpha5beta1 integrin compared to normal or
adenoma cells. To determine the significance of absent
alpha5beta1 integrin signaling, we transfected the
cDNA coding for the
alpha5 integrin sub-unit into the human colon-
carcinoma cell line HT29, which constitutively lacks this subunit but does express the beta1 subunit. We show here that the newly expressed
fibronectin receptor alpha5beta1 generates multiple signals, causing marked changes in cytoskeletal arrangements within a few minutes of adhesion to
fibronectin. Cells expressing the
alpha5beta1 integrin exhibit the formation of actin stress fibers and focal adhesions, as well as the induction of
tyrosine phosphorylation of several
proteins, within 10 min. We identified the
focal adhesion kinase pp125FAK and the cytoskeletal
protein paxillin as major phosphorylation substrates in these cells. These
proteins remained hypophosphorylated when alpha5-negative control cells were plated on
fibronectin. The
tyrosine kinase pp60c-src, regarded as central in the regulation of cellular proliferation and constitutively over-expressed in HT29 and in colon-
carcinoma cells, showed reduced intrinsic
kinase activity in unstimulated HT29alpha5 cells. In contrast,
fibronectin-induced signaling through alpha5beta1 increased pp60c-src activity. Moreover, immunoprecipitation of pp60c-src from extracts of HT29alpha5 cells cultivated on
fibronectin for 20 min revealed complex formation of pp60c-src and
tyrosine-phosphorylated pp125FAK. Our data suggest that de novo expression of the
alpha5beta1 integrin in HT29
colon-cancer cells restores signaling via pp125FAK and pp60c-src. Thus, loss of this receptor during malignant transformation may contribute to
tumor-cell autonomy, while reduced activity of pp60c-src in HT29alpha5-cells may participate directly in growth control.