Ras Activation by Insulin and Epidermal Growth Factor through Enhanced Exchange of Guanine Nucleotides on p21^ras

Abstract

A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21^ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21^ras or by a decrease in the GTPase activity of p21^ras. To investigate whether insulin and EGF affect nucleotide exchange on p21^ras, we measured binding of [α-³²P]GTP to p21^ras in cells permeabilized with streptolysin O. For this purpose, we used a cell line which expressed elevated levels of p21 H-ras and which was highly responsive to insulin and EGF. Stimulation with insulin or EGF resulted in an increase in the rate of nucleotide binding to p21^ras. To determine whether this increased binding rate is due to the activation of a guanine nucleotide exchange factor, we made use of the inhibitory properties of a dominant negative mutant of p21^ras, p21^ras (Asn-17). Activation of p21^ras by insulin and EGF in intact cells was abolished in cells infected with a recombinant vaccinia virus expressing p21^ras (Asn-17). In addition, the enhanced nucleotide binding to p21^ras in response to insulin and EGF in permeabilized cells was blocked upon expression of p21^ras (Asn-17). From these data, we conclude that the activation of a guanine nucleotide exchange factor is involved in insulin- and EGF-induced activation of p21^ras.