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

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Abstract
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 pp60c-src associated with the integrin. pp60c-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 alpha v-subunit, in the association of pp60c-src with alpha v beta 3, a cell line expressing truncated alpha v was studied. M21-L cells 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 kinase assays of anti- alpha v immunoprecipitates demonstrated that M21-L/alpha v995 cells did not exhibit pp60c-src association with alpha v, whereas the alpha v1018 complementary DNA transfected cells and FG cells had pp60c-src associated with the alpha v integrins. Immunofluorescence analysis revealed pp60c-src, alpha v beta 3 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 alpha v beta 3 demonstrated association of actin with the immune complexes. We conclude that OPN stimulates pp60c-src kinase activity associated with the alpha v beta 3 integrin and that the association requires the cytoplasmic tail of the alpha v chain.

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