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 α6 β4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the β4 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. Insulinlike 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 β 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 α 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 insulinstimulated cells demonstrated that this β4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that β4 integrin molecule is a target for phosphorylation through an indirect receptor‐mediated mechanism. Copyright © 1989 Wiley‐Liss, Inc., A Wiley Company
Insulin‐Induced Phosphorylation of the Beta‐4 Integrin Subunit Expressed on Murine Metastatic Carcinoma Cells
Perrotti N.;
1989-01-01
Abstract
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 α6 β4 complex, and in particular it has been demonstrated that protein corresponding to 204 kDa is homologous to the β4 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. Insulinlike 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 β 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 α 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 insulinstimulated cells demonstrated that this β4 integrin subunit is phosphorylated on serine and tyrosine. We conclude that β4 integrin molecule is a target for phosphorylation through an indirect receptor‐mediated mechanism. Copyright © 1989 Wiley‐Liss, Inc., A Wiley CompanyI documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
