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 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.
Chellaiah
M
M
Renal Division, Jewish Hospital of St. Louis, Missouri 63110, USA.
Fitzgerald
C
C
Filardo
E J
EJ
Cheresh
D A
DA
Hruska
K A
KA
D000199:Actins / Q000032:analysis; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry* / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007163:Immunosorbent Techniques; D039482:Integrin alpha5; D008545:Melanoma / Q000378:metabolism*; D008969:Molecular Sequence Data; D053495:Osteopontin; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000032:analysis / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000032:analysis / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012795:Sialoglycoproteins / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000199:Actins; D015703:Antigens, CD; D039482:Integrin alpha5; D019030:Receptors, Vitronectin; D011993:Recombinant Fusion Proteins; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D016392:Proto-Oncogene Proteins pp60(c-src)