Characterization of insulin-like growth factor I-stimulated tyrosine kinase activity associated with the beta-subunit of type I insulin-like growth factor receptors of rat liver cells.

Abstract

We previously reported that insulin-like growth factor I (IGF-I) stimulates the phosphorylation of a Mr 98,000 protein thought to be the beta-subunit of the type I IGF receptor of BRL-3A2 rat liver cells, as well as phosphorylation of the exogenous tyrosine-containing substrate poly(Glu,Tyr), 4:1. The present study provides additional evidence that the type I IGF receptor possesses intrinsic tyrosine kinase activity and characterizes the properties of this receptor kinase. IGF-I stimulates receptor phosphorylation and phosphorylation of poly(Glu,Tyr), 4:1, by lectin-purified receptor preparations with the same concentration dependence; half-maximal stimulation was observed with approximately 3 nM IGF-I and approximately 3-fold higher concentrations of insulin. Although IGF-I-dependent receptor phosphorylation was observed within 2 min and was maximal after 10 min, phosphorylation of exogenous substrate did not begin to increase until 8 min after addition of [gamma-32P] ATP and poly(Glu,Tyr), 4:1. When IGF-I-receptor complexes were preincubated with unlabeled ATP for 10 min before addition of substrate, however, IGF-I-dependent 32P incorporation was observed within 2 min after addition of poly(Glu,Tyr), 4:1, and increased linearly for 20 min. We propose that this activation of type I IGF receptor tyrosine kinase activity results from autophosphorylation of the receptor kinase. Kinase activation is an intramolecular reaction, is specific for ATP, occurs within 3 min after addition of unlabeled ATP, and requires the presence of IGF-I before or concomitant with activation by ATP. IGF-I acts rapidly, stimulating substrate phosphorylation within 3 min. The properties of the type I-IGF receptor kinase closely resemble those of the insulin receptor kinase, suggesting that the homologies between the two receptors extend to their kinase domains.