The p70 S6 ribosomal protein kinase I (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires it cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6K Delta CT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (K-m) values of S6K for ATP and the Biotin-S6 Substrate peptide were determined to be 21.4 +/- 0.29 and 0.9 +/- 0.48 mu M, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide it new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors. (c) 2005 Elsevier Inc. All rights reserved.