title: Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain construct of the 70 kDa 40S ribosomal protein S6 kinase-1 alphaII isoform (S6K1alphaII).
abstract: S6K1alphaII 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 [S6K1alphaII(DeltaAID); 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)-S6K1alphaII(DeltaAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His(6)-S6K1alphaII(DeltaAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 ( approximately 75 nmol/min/mg). Most significantly, we report that the His(6)-S6K1alphaII(DeltaAID)-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(DeltaPH)]. Approximately equal amounts of fully activated His(6)-S6K1alphaII(DeltaAID)-T389E (5+/-1 mg) and His(6)-PDK1(DeltaPH) (8+/-2 mg) were His(6) affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2x10(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(DeltaPH) to phosphorylate T229 to approximately 100% after co-expression in Sf9 insect cells as compared to approximately 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.
authors.affiliation: Department of Chemistry, University of Miami, P. O. Box 249118, Coral Gables, FL 33124-0431, USA.
authors.forename: Malik M
authors.initials: MM
authors.lastname: Keshwani
authors.forename: Duncan B
authors.initials: DB
authors.lastname: Ross
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Ragan
authors.forename: Thomas K
authors.initials: TK
authors.lastname: Harris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D020134:Catalytic Domain; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D038762:Ribosomal Protein S6 Kinases, 70-kDa; C448901:ribosomal protein S6 kinase, 70kD, polypeptide 1