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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
11:23

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein

Published on: June 30, 2019

Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell

C C Kumar1, R Diao, Z Yin

  • 1Department of Tumor Biology, Schering-Plough Research Institute, Kenilworth, NJ 07033, USA. chandra.kumar@spcorp.com

Biochimica Et Biophysica Acta
|June 19, 2001
PubMed

Insights

Researchers purified active Akt1 enzyme for drug discovery, finding structural differences with PKC that suggest selective inhibitors are possible, though isoform-specific Akt inhibitors remain challenging.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Akt is a key serine/threonine kinase in cell survival signaling, implicated in tumorigenesis.
  • Up-regulation of Akt and phosphatidylinositol-3 kinase (PI3K) is common in tumors.
  • PTEN/MMAC acts as a tumor suppressor by negatively regulating this pathway.

Purpose of the Study:

  • To express and purify active Akt1 enzyme for drug discovery.
  • To characterize the enzyme's properties and develop a kinase assay.
  • To explore the potential for developing selective Akt inhibitors.

Main Methods:

  • Recombinant baculovirus expression in Sf9 cells for Akt1 purification.
  • Coexpression with PI3K or okadaic acid treatment to activate Akt1.
  • Development of a quantitative kinase assay using biotinylated peptide substrates.
  • Homology modeling to compare Akt and protein kinase C (PKC) structures.

Main Results:

  • Successfully expressed and purified active Akt1 enzyme.
  • Characterized the physical, catalytic, and kinetic properties of Akt1.
  • Identified amino acid differences in ATP binding regions between Akt and PKC, suggesting potential for selective inhibition.
  • Found high conservation in ATP binding regions across Akt isoforms, indicating challenges in developing isoform-selective inhibitors.

Conclusions:

  • Active Akt1 enzyme can be produced in an insect cell system for biochemical studies.
  • Structural comparisons suggest the feasibility of developing selective Akt inhibitors.
  • Developing isoform-specific Akt inhibitors presents significant challenges due to conserved ATP binding sites.

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