Insulin-induced phosphorylation of the beta-4 integrin subunit expressed on murine metastatic carcinoma cells.
A tumor surface protein (TSP-180) that is highly expressed on highly malignant metastatic cells has been identified on murine lung carcinomas. On SDS-PAGE under reducing conditions, TSP-180 shows a complex banding pattern corresponding to 204, 183, 150, 135, and 116 kDa. All bands of the TSP-180 complex are glycosylated and are labeled by lactoperoxidase-catalyzed radioiodination of viable cells. The mouse TSP-180 complex described here is homologous to the human integrin alpha 6 beta 4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the beta 4 subunit of the integrin complex. It has been shown recently that monoclonal antibody to TSP-180 (MoAb 135-13C) stimulates cell growth in vitro and induces phosphorylation of the 204-kDa protein. We now report that insulin increases the phosphorylation of the 204-kDa protein 30-fold in intact carcinoma cells and epidermal growth factor (EGF) causes a threefold increase. Insulin-like growth factor (IGF-I) and platelet-derived growth factor have no effect. The effect of insulin and of IGF-I on phosphorylation of their own receptors was studied using solubilized cell membranes. Insulin and IGF-I each induced a fivefold increase in the phosphorylation of their respective receptor beta subunits. In order to test if phosphorylation of the 204-kDa protein was induced by direct binding of growth factors to TSP-180 and to identify growth factor receptors on line 1 cells, affinity cross-linking studies were performed. Affinity labeling of receptors demonstrated that insulin and IGF-I both bind to a 135-kDa protein that corresponds to the insulin and IGF-I receptor alpha subunits. Affinity labeling of EGF receptors failed to demonstrate EGF receptor molecules (175-kDa protein) on line 1 cells. Further investigations by using a different approach confirmed the very low amount of EGF receptors on line 1 cells. Direct phosphoamino acid analysis of the 204-kDa protein purified from insulin-stimulated cells demonstrated that this beta 4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that beta 4 integrin molecule is a target for phosphorylation through an indirect receptor-mediated mechanism.
Falcioni
R
R
Laboratorio di Oncogenesi Molecolare, Istituto Regina Elena per lo Studio e la Cura dei Tumori, Rome, Italy.
Perrotti
N
N
Piaggio
G
G
Kennel
S K
SK
Sacchi
A
A
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000951:Antigens, Neoplasm / Q000032:analysis / Q000378:metabolism*; D002277:Carcinoma / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000032:analysis; D005260:Female; D007328:Insulin / Q000494:pharmacology*; D039161:Integrin alpha6beta4; D016023:Integrins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D009362:Neoplasm Metastasis; D010766:Phosphorylation
D000596:Amino Acids; D000951:Antigens, Neoplasm; D007328:Insulin; D039161:Integrin alpha6beta4; D016023:Integrins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors