Phosphorylation of the ribosomal protein S6 is observed in response to treatment of target cells with growth factors, including insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor. The initial event in the regulation of the cellular response appears to be activation of a tyrosine-specific kinase which is an intrinsic activity in the plasma membrane growth factor receptor. Several S6 kinases, including an enzyme isolated from human placenta and murine lymphosarcoma, have been described. None of these enzymes appear to be substrates for the growth factor receptor kinase. The overall goal of this research is to establish the cascade of reactions which are initiated by growth factors and terminate in the activation of an S6 kinase. S6/H4PK has been purified from human placenta. The preparation contains two S6 kinases activities: S6PK (p95), which is activated by an Activating Enzyme (AE) or limited trypsin digestion and S6PK (p55) which is activated by protein kinase C. The Activating Enzyme requires MgATP for activity and apparently catalyzes phosphorylation of both S6PK (p95) and S6PK (p55). Studies with monoclonal antibodies have shown that AE contains two subunits, p116 and p72. The p72 is apparently the catalytic subunit. Experiments to continue the investigation of the regulation and reactivity of these enzymes and role of insulin receptor in those events are proposed. The specific aims of these experiments are as follows: (1) determination of the mechanism for S6 kinase activation by the homogeneous Activating Enzyme; (2) elucidation of the structure, including subunit composition, of inactive and active S6 kinases; (3) comparison of the S6 kinase regulation by Activating Enzyme and protein kinase C; (4) analyses of the phosphorylation and activation of Activating Enzyme by the insulin receptor in vivo and in vitro. Collectively, data obtained in these studies will provide a molecular basis for the action of insulin on target cells and may provide initial insights into the mechanism of cellular regulation by growth factors in general.