Differential Activation of Mitogen-activated Protein Kinase and S6 Kinase Signaling Pathways by 12-O-Tetradecanoylphorbol-13-acetate (TPA) and Insulin

AG-18, an inhibitor of protein-tyrosine kinases, was employed to study the role of tyrosine-phosphorylated proteins in insulin- and phorbol ester-induced signaling cascades. When incubated with Chinese hamster ovary cells overexpressing the insulin receptor, AG-18 reversibly inhibited insulin-induced tyrosine phosphorylation of insulin receptor substate-1, with minimal effects either on receptor autophosphorylation or on phosphorylation of Shc64. Under these conditions, AG-18 inhibited insulin-stimulated phosphorylation of the ribosomal protein S6, while no inhibition of insulin-induced activation of mitogen-activated protein kinase (MAPK) kinase or MAPK was detected. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of MAPK kinase and MAPK and phosphorylation of S6 were inhibited by AG-18. This correlated with inhibition of TPA-stimulated tyrosine phosphorylation of several proteins, the most prominent ones being pp114 and pp120. We conclude that Tyr-phosphorylated insulin receptor substrate-1 is the main upstream regulator of insulin-induced S6 phosphorylation by p70s6k, whereas MAPK signaling seems to be activated in these cells primarily through the adaptor molecule Shc. In contrast, TPA-induced S6 phosphorylation is mediated by the MAPK/p90rsk cascade. A key element of this TPA-stimulated signaling pathway is an AG-18-sensitive protein-tyrosine kinase.