SELECTIVE-INHIBITION OF P70 S6 KINASE ACTIVATION BY PHOSPHATIDYLINOSITOL 3-KINASE INHIBITORS

Treatment of fibroblasts with wortmannin or demethoxyviridin, two pote nt inhibitors of phosphatidylinositol 3-kinase, prevents the activatio n of ribosomal protein S6 kinase, which is induced by a variety of ext ernal stimuli. Concentrations giving 50% inhibition of 45 nM (wortmann in) and 400 nM (demethoxyviridin) were obtained when epidermal growth factor was used as an S6 kinase activator; with platelet-derived growt h factor, the concentrations giving 50% inhibition were about three-ti mes higher. Western-blot analysis and immunocomplex kinase assays show ed that wortmannin and demethoxyviridin specifically block the phospho rylation and activation of p70 S6 kinase without affecting the M(r) 90 000 ribosomal S6 kinase (p90(rsk)) or mitogen-activated protein kinase s. Consistent with the irreversible nature of the inhibition of phosph atidylinositol 3-kinase by these compounds, treatment of cells with wo rtmannin, followed by washing out of the inhibitor, still led to inhib ition of p70 S6 kinase activation. Several S6 kinase agonists not prev iously known to activate phosphatidylinositol 3-kinase (A23187, bombes in and phorbol 12-myristate 13-acetate) were found to increase the pro duction of phosphatidylinositol 3,4,5-trisphosphate in a wortmannin-se nsitive manner. These results support a model in which phosphatidylino sitol 3-kinase acts upstream of p70 S6 kinase in a mitogenic signallin g pathway; the existence of a phosphatidylinositol 3-kinase-independen t pathway is also evident.