Osteopontin Activation of c-src in Human Melanoma Cells Requires the Cytoplasmic Domain of the Integrin α_v-Subunit

In human melanoma cells, expression of the α_vβ₃ integrin is correlated with the metastatic potential. The expression of osteopontin (OPN or OP), a protein ligand for the integrin α_vβ₃, also correlates with metastatic potential of some tumors. Analysis of signal transduction, stimulated by OPN/α_vβ₃ 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 inhibited in vitro by a tyrosine kinase inhibitor, herbimycin-A. To determine the need for the cytoplasmic domain of the α_v-subunit, in the association of pp^60c-src with α_vβ₃, a cell line expressing truncated α_v was studied. M21-L cells lacked α_v expression but stably transfected with complementary DNAs encoding α_v full length protein α_v1018 or α_v995 (lacking 23 carboxyl-terminal amino acids), and a fibroblast cell line (FG) expressing α_vβ₅ but not α_vβ₃, were used. Western analysis and immune complex kinase assays of anti-α_v immunoprecipitates demonstrated that M21-L/α_v995 cells did not exhibit pp^60c-src association with α_v, whereas the α_v1018 complementary DNA transfected cells and FG cells had pp^60c-src associated with the av integrins. Immunofluorescence analysis revealed pp^60c-src, α_vβ₃ integrin, and actin distribution along the plasma membrane of M21 cells. ³⁵S-labeling of cells and analysis of complexes immunoprecipitated by a monoclonal antibody against α_vβ₃ demonstrated association of actin with the immune complexes. We conclude that OPN stimulates pp^60c-src kinase activity associated with the α_vβ₃ integrin and that the association requires the cytoplasmic tail of the α_v chain.