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

Biochim Biophys Acta. 2001 Jun 15;1526(3):257-68. doi: 10.1016/s0304-4165(01)00143-x.


Akt is a serine/threonine kinase that plays a critical role in cell survival signaling and its activation has been linked to tumorigenesis. Up-regulation of Akt as well as its upstream regulator phosphatidylinositol-3 kinase (PI3K) has been found in many tumors and the negative regulator of this pathway PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we have expressed and purified an active Akt1 enzyme from a recombinant baculovirus-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subunit of PI3K or treatment with okadaic acid during expression was found to generate an active enzyme in the insect cell culture system. We have optimized the kinase activity and developed a simple quantitative kinase assay using biotinylated peptide substrates. Using the purified active enzyme, we have characterized its physical, catalytic and kinetic properties. Since Akt is closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparison of the structures of catalytic domains of Akt and PKC, derived by homology modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibitors of Akt can be discovered. However, the ATP binding regions are highly conserved in the three isoforms of Akt implying that the discovery of isoform-selective inhibitors would be very challenging.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Baculoviridae / metabolism
  • Cell Line
  • Cell Survival
  • Class Ib Phosphatidylinositol 3-Kinase
  • Cloning, Molecular
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Drug Design
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation / drug effects
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / biosynthesis
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Weight
  • Okadaic Acid / pharmacology
  • Phosphatidylinositol 3-Kinases / biosynthesis
  • Protein Kinase C / chemistry
  • Protein-Serine-Threonine Kinases / biosynthesis*
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Proto-Oncogene Proteins c-akt
  • Proto-Oncogene Proteins*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Signal Transduction


  • Isoenzymes
  • Proto-Oncogene Proteins
  • Okadaic Acid
  • Phosphatidylinositol 3-Kinases
  • Class Ib Phosphatidylinositol 3-Kinase
  • PIK3CG protein, human
  • AKT1 protein, human
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt
  • Cyclic AMP-Dependent Protein Kinases
  • Protein Kinase C