Osteopontin activation of c-src in human melanoma cells requires the cytoplasmic domain of the integrin alpha(v)-subunit

In human melanoma cells, expression of the alpha(v) beta(3) integrin is correlated with the metastatic potential. The expression of osteopontin (OPN or OP), a protein ligand for the integrin alpha(v) beta(3), also correlates with metastatic potential of some tumors. Analysis of signal transduction, stimulated by OPN/alpha(v) beta(3) in human melanoma cells (M21), revealed activation of pp(60c-src) associated with the integrin. pp(60c-src) stimulation by OPN was dose dependent, and it was inhibition in vitro by a tyrosine kinase inhibitor, herbimycin-A. To determine the need for the cytoplasmic domain of the alpha(v)-subunit, in the association of pp(60c-src) with alpha(v) beta(3), a cell line expressing truncated alpha(v) was studied. M21-L cell lacked alpha(v) expression but stably transfected with complementary DNAs encoding alpha(v) full length protein alpha(v)1018 or alpha(v)995 (lacking 23 carboxyl-terminal amino acids), and a fibroblast cell line (FG) expressing alpha(v) beta(5) but not alpha(v) beta(3), were used. Western analysis and immune complex kinases assays of anti-alpha(v) immunoprecipitates demonstrated that M21-L/alpha(v)995 cells did not exhibit pp(60c-src) association with alpha(v), whereas the alpha(v)1018 complementary DNA transfected cells and FG cells had pp(60c-src) associated with the alpha v integrins. Immunofluorescence analysis revealed pp(60c-src), alpha(v) beta(3) integrin, and actin distribution along the plasma membrane of M21 cells. S-35-labeling of cells and analysis of complexes immunoprecipitated by a monoclonal antibody against alpha(v) beta(3) demonstrated association of actin with the immune complexes. We conclude that OPN stimulates pp(60c-src) kinase activity associated with the alpha(v) beta(3) integrin and that the association requires the cytoplasmic tail of the alpha(v) chain.