Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases

Abstract

THE critical pathways through which protein-tyrosine kinases induce cellular proliferation and malignant transformation are not well defined. As microinjection of antibodies against p21^ras can block the biological effects of both normal and oncogenic tyrosine kinases, it is likely that they require functional p21^ras to transmit their mitogenic signals^1,2. No biochemical link has been established, however, between tyrosine kinases and p21^ras. We have identified a non-catalytic domain of cytoplasmic tyrosine kinases, SH2, that regulates the activity and specificity of the kinase domain^3–9. The presence of two adjacent SH2 domains in the p21^ras GTPase-activating protein (GAP)^6,10,11indicates that GAP might interact directly with tyrosine kinases. Here we show that GAP, and two co-precipitating proteins of relative molecular masses 62,000 and 190,000 (p62 and p190) are phosphorylated on tyrosine in cells that have been transformed by cytoplasmic and receptor-like tyrosine kinases. The phosphorylation of these polypeptides correlates with transformation in cells expressing inducible forms of thev-src or v-fps encoded tyrosine kinases. Furthermore, GAP, p62 and p190 are also rapidly phosphorylated on tyrosine in fibroblasts stimulated with epidermal growth factor. Our results suggest a mechanism by which tyrosine kinases might modify p21^ras function, and implicate GAP and its associated proteins as targets of both oncoproteins and normal growth factor receptors with tyrosine kinase activity. These data support the idea^4–6 that SH2 sequences direct the interactions of cytoplasmic proteins involved in signal transduction.