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

M. Chellaiah, C. Fitzgerald, E. J. Filardo, D. A. Cheresh, K. A. Hruska

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Abstract

In human melanoma cells, expression of the αvβ3 integrin is correlated with the metastatic potential. The expression of osteopontin (OPN or OP), a protein ligand for the integrin αvβ3, also correlates with metastatic potential of some tumors. Analysis of signal transduction, stimulated by OPN/αvβ3 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 αv-subunit, in the association of pp60c-src with αvβ3, a cell line expressing truncated αv was studied. M21-L cells lacked αv expression but stably transfected with complementary DNAs encoding αv full length protein αv1018 or αv995 (lacking 23 carboxyl-terminal amino acids), and a fibroblast cell line (FG) expressing αvβ5 but not αvβ3, were used. Western analysis and immune complex kinase assays of anti-αv immunoprecipitates demonstrated that M21-L/αv995 cells did not exhibit pp60c-src association with αv, whereas the αv1018 complementary DNA transfected cells and FG cells had pp60c-src associated with the av integrins. Immunofluorescence analysis revealed pp60c-src, αvβ3 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 αvβ3 demonstrated association of actin with the immune complexes. We conclude that OPN stimulates pp60c-src kinase activity associated with the αvβ3 integrin and that the association requires the cytoplasmic tail of the αv chain.

Original languageEnglish
Pages (from-to)2432-2440
Number of pages9
JournalEndocrinology
Volume137
Issue number6
DOIs
StatePublished - 1996

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