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, 58 (1), 32-41

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)

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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)

Malik M Keshwani et al. Protein Expr Purif.

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.

Figures

Fig. 1
Fig. 1
S6K1 expression construct. (A) Amino acid and DNA coding sequence of the N-terminal catalytic kinase domain of the 70 kDa 40S ribosomal protein S6 kinase-1 [S6K1(ΔAID); residues 1-398 in the S6K1αII isoform). The regulatory sites of T-loop (T229) and hydrophobic motif phosphorylation (T389) are shaded. (B) S6K1(ΔAID) was engineered in the pFastbac™1 vector to contain and N-terminal His6 affinity tag, the PreScission protease recognition sequence, and the T389E phosphomimicking mutation.
Fig. 2
Fig. 2
Expression, purification, and activation of His6-S6K1αII(ΔAID)-T389E by PDK1. (A) SDS-PAGE analysis with Coomassie staining of His6-S6K1αII(ΔAID)-T389E purified after baculovirus-mediated expression and His6 affinity purification from Sf9 insect cells. Lane 1 shows the total soluble lysate; lane 2 shows the proteins from the soluble lysate that were not retained after passage over the His6 affinity column; and lane 3 shows purified His6-S6K1αII(ΔAID)-T389E subsequently eluted from the His6 affinity column. (B) SDS-PAGE analysis with Coomassie staining of His6-S6K1αII(ΔAID)-T389E purified from Sf9 insect cells that were coinfected with His6-PDK1(ΔPH). Lane 1 shows the total soluble lysate; lane 2 shows the proteins from the soluble lysate that were not retained after passage over the His6 affinity column; lane 3 shows both His6-S6K1αII(ΔAID)-T389E and His6-PDK1(ΔPH), which co-eluted from the His6 affinity column; lane 4 shows purified His6-PDK1(ΔPH) that was not retained by the MonoQ anion exchange column and shown below is Western detection of S241 phosphorylated His6-PDK1(ΔPH) using the phospho-PDK1 (S241) polyclonal antibody; and lane 5 shows His6-S6K1αII(ΔAID)-T389E that eluted from the MonoQ anion exchange column. (C) Comparison of His6-S6K1αII(ΔAID)-T389E activation by (i) S6K1 activity towards the model S6K1-Tide and (ii) SDS-PAGE-Western analysis using the phospho-S6K1 (T229) polyclonal antibody. Lane 1 shows His6-S6K1αII(ΔAID)-T389E purified from cells solely infected; lane 2 shows His6-S6K1αII(ΔAID)-T389E purified from cells coinfected with His6-PDK1(ΔPH); and lane 3 shows His6-S6K1αII(ΔAID)-T389E purified from cells solely infected after 30 min in vitro treatment with His6-PDK1(ΔPH). (D) Progress curves for PDK1-catalyzed phosphorylation of His6-S6K1αII(ΔAID)-T389E. Either 2 μM (●) or 15 μM (○) His6-S6K1αII(ΔAID)-T389E was incubated at 25 °C with 10 nM active His6-PDK1(ΔPH) and 100 μM [γ-32P]ATP in kinase reaction buffer. At different times, aliquots were removed from the reaction mixture, and the mole fraction of phosphorylated His6-S6K1αII(ΔAID)-T389E was determined. (E) After 30 min treatment of His6-S6K1αII(ΔAID)-T389E with His6-PDK1(ΔPH) and [γ-32P]ATP-Mg2+, the reaction mixture was subjected to proteolytic digestion with trypsin and the resulting peptides were resolved by reversed-phase HPLC. Scintillation counting of the individual fractions detected the 32P-radiolabeled peptide that eluted near 31% acetonitrile, which has been identified by Edmond degradation to be the monophosphorylated tryptic peptide containing 32P-T229 [8].
Fig. 3
Fig. 3
ESI-TOF and MonoQ analysis of His6-S6K1αII(ΔAID)-T389E phospho-isoform species. (A, C, E) ESI-TOF reconstructed mass spectra and (B, D, F) MonoQ elution profiles and the corresponding S6K1 activities measured for fractionated phospho-isoform species are shown for (A, B) His6-S6K1αII(ΔAID)-T389E purified from cells solely infected; (C, D) His6-S6K1αII(ΔAID)-T389E purified from cells solely infected after 30 min in vitro treatment with His6-PDK1(ΔPH); and (E, F) His6-S6K1αII(ΔAID)-T389E purified from cells coinfected with His6-PDK1(ΔPH). For each reconstructed mass spectrum, species a (46,558 ± 10 Da), b (46,638 ± 10 Da), and c (46,714 ± 10 Da) correspond to the calculated molecular masses of the unmodified (46,568 Da), monophosphorylated (46,648 Da), and diphosphorylated forms of His6-S6K1αII(ΔAID)-T389E (46,728 Da). For His6-S6K1αII(ΔAID)-T389E purified from cells coinfected with His6-PDK1(ΔPH), ESI-TOF detected a small amount of triphosphorylated enzyme and very small amounts of even higher order phosphorylated species. The MonoQ elution peaks of species a, b, and c were maximum at 42.4%, 45.5%, and 48.5% of Buffer B; and the fractions corresponding to the peak maximums were assayed for S6K1 activity towards the model S6K1-Tide. In cases where no significant amount of enzyme eluted as a designated species, activities could not be determined and are so denoted (×).
Fig. 4
Fig. 4
Steady-state kinetic titrations of His6-S6K1αII(ΔAID)-T389E with nucleotide and peptide substrates. (A) Titration of in vivo (●) and in vitro (○) PDK1-activated His6-S6K1αII(ΔAID)-T389E with varying S6K1-Tide concentrations. (B) Titration of in vivo (▲) and in vitro (△) PDK1-activated His6-S6K1αII(ΔAID)-T389E with varying S6K1-Tide concentrations. Initial velocities normalized to enzyme concentration (k = ν/[Et]) were measured (A) using 50 μM ATP for varying S6K1-Tide concentrations (0.5, 1, 2, 3, 5, 10, 15, 30, and 50 μM) and (B) using 50 μM S6K1-Tide for varying ATP concentrations (0.5, 1, 2, 3, 5, 10, 15, 30, and 50 μM). The assays were initiated by addition of 20 nM kinase.

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