Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain construct of the 70 kDa 40S ribosomal protein S6 kinase-1 αII isoform (S6K1αII)

Malik M. Keshwani, Duncan B. Ross, Timothy J. Ragan, Thomas K Harris

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

S6K1αII 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 [S6K1αII(ΔAID); 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 His6-S6K1αII(ΔAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His6-S6K1αII(ΔAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 (∼75 nmol/min/mg). Most significantly, we report that the His6-S6K1αII(ΔAID)-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 [His6-PDK1(ΔPH)]. Approximately equal amounts of fully activated His6-S6K1αII(ΔAID)-T389E (5 ± 1 mg) and His6-PDK1(ΔPH) (8 ± 2 mg) were His6 affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2 × 106 cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His6-PDK1(ΔPH) to phosphorylate T229 to ∼100% after co-expression in Sf9 insect cells as compared 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.

Original languageEnglish
Pages (from-to)32-41
Number of pages10
JournalProtein Expression and Purification
Volume58
Issue number1
DOIs
StatePublished - Mar 1 2008

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Ribosomal Protein S6 Kinases
70-kDa Ribosomal Protein S6 Kinases
Baculoviridae
Purification
Catalytic Domain
Protein Isoforms
Phosphotransferases
Sf9 Cells
Insects
Phosphorylation
Chromatography
Anions
Electrospray Ionization Mass Spectrometry
Protein-Serine-Threonine Kinases
ribosomal protein S6 kinase, 90kDa, polypeptide 3
Coinfection
Mass spectrometry
Assays
Chemical activation
Kinetics

Keywords

  • Baculoviral coinfection
  • ESI-TOF
  • MonoQ anion exchange
  • PDK1
  • Phosphoinositide-dependent protein kinase-1
  • Protein phosphorylation
  • S6K1-T389E
  • Sf9 insect cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{a1a28d97d4034a298047b51fc5d6d8a5,
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 αII isoform (S6K1αII)",
abstract = "S6K1αII 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 [S6K1αII(ΔAID); 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 His6-S6K1αII(ΔAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His6-S6K1αII(ΔAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 (∼75 nmol/min/mg). Most significantly, we report that the His6-S6K1αII(ΔAID)-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 [His6-PDK1(ΔPH)]. Approximately equal amounts of fully activated His6-S6K1αII(ΔAID)-T389E (5 ± 1 mg) and His6-PDK1(ΔPH) (8 ± 2 mg) were His6 affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2 × 106 cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His6-PDK1(ΔPH) to phosphorylate T229 to ∼100{\%} after co-expression in Sf9 insect cells as compared 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.",
keywords = "Baculoviral coinfection, ESI-TOF, MonoQ anion exchange, PDK1, Phosphoinositide-dependent protein kinase-1, Protein phosphorylation, S6K1-T389E, Sf9 insect cells",
author = "Keshwani, {Malik M.} and Ross, {Duncan B.} and Ragan, {Timothy J.} and Harris, {Thomas K}",
year = "2008",
month = "3",
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doi = "10.1016/j.pep.2007.11.003",
language = "English",
volume = "58",
pages = "32--41",
journal = "Protein Expression and Purification",
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publisher = "Academic Press Inc.",
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TY - JOUR

T1 - Baculovirus-mediated expression, purification, and characterization of a fully activated catalytic kinase domain construct of the 70 kDa 40S ribosomal protein S6 kinase-1 αII isoform (S6K1αII)

AU - Keshwani, Malik M.

AU - Ross, Duncan B.

AU - Ragan, Timothy J.

AU - Harris, Thomas K

PY - 2008/3/1

Y1 - 2008/3/1

N2 - S6K1αII 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 [S6K1αII(ΔAID); 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 His6-S6K1αII(ΔAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His6-S6K1αII(ΔAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 (∼75 nmol/min/mg). Most significantly, we report that the His6-S6K1αII(ΔAID)-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 [His6-PDK1(ΔPH)]. Approximately equal amounts of fully activated His6-S6K1αII(ΔAID)-T389E (5 ± 1 mg) and His6-PDK1(ΔPH) (8 ± 2 mg) were His6 affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2 × 106 cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His6-PDK1(ΔPH) to phosphorylate T229 to ∼100% after co-expression in Sf9 insect cells as compared 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.

AB - S6K1αII 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 [S6K1αII(ΔAID); 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 His6-S6K1αII(ΔAID)-T229E,T389E double mutant from Sf9 insect cells yielded enzyme with compromised activity. Higher activity preparations were generated using the Sf9 purified His6-S6K1αII(ΔAID)-T389E single mutant isoform, which was in vitro phosphorylated by the upstream T229 kinase, PDK1 (∼75 nmol/min/mg). Most significantly, we report that the His6-S6K1αII(ΔAID)-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 [His6-PDK1(ΔPH)]. Approximately equal amounts of fully activated His6-S6K1αII(ΔAID)-T389E (5 ± 1 mg) and His6-PDK1(ΔPH) (8 ± 2 mg) were His6 affinity co-purified 60 h after initial coinfection of 200 mL of Sf9 insect cells (2 × 106 cells/mL), which were resolved by MonoQ anion exchange chromatography. ESI-TOF mass spectrometry, MonoQ anion exchange chromatography, and kinetic assays showed His6-PDK1(ΔPH) to phosphorylate T229 to ∼100% after co-expression in Sf9 insect cells as compared 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.

KW - Baculoviral coinfection

KW - ESI-TOF

KW - MonoQ anion exchange

KW - PDK1

KW - Phosphoinositide-dependent protein kinase-1

KW - Protein phosphorylation

KW - S6K1-T389E

KW - Sf9 insect cells

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U2 - 10.1016/j.pep.2007.11.003

DO - 10.1016/j.pep.2007.11.003

M3 - Article

VL - 58

SP - 32

EP - 41

JO - Protein Expression and Purification

JF - Protein Expression and Purification

SN - 1046-5928

IS - 1

ER -