S6K1 alpha II is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (T229) and hydrophobic motif (HM; T389) regions of its catalytic kinase domain [S61 alpha II(Delta AID); deletion of C-terminal autoinhibitory domain residues 399-502]. With regard to mimicking the synergistic effect of full dual site phosphorylation, baculovirus-mediated expression and affinity purification of the His(6)-S6K1 alpha II(Delta AID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His(6)-S6K1 alpha II(Delta AID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 (similar to 75 nmol/min/mg). Most significantly, we report that the His(6)-S6K1 alpha II(Delta AID)-T389E construct was generated in its most highly active form (250 nmol/min/mg) by baculovirus-mediated expression and purification from Sf9 insect cells that were coinfected with recombinant baculovirus expressing the catalytic kinase domain of PDK1 [His(6)-PDK1(Delta PH)]. Approximately equal amounts of fully activated His(6)-S6K1 alpha II(Delta AID)-T389E (5 +/- 1 mg) and His(6)-PDK1(Delta PH) (8 2 mg) were His(6) affinity co-purified 60 It after initial coinfection of 200 mL of Sf9 insect cells (2 x 10(6) cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His(6)-PDK1(Delta PH) to phosphorylate T229 to similar to 100% after coexpression in Sf9 insect cells as compared to similar to 50% under in vitro conditions, raising interest to mechanistic components not fully achieved in the in vitro reaction. Generation of fully activated S6K1 will facilitate more rigorous analysis of its structure and mechanism. (c) 2007 Elsevier Inc. All rights reserved.