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

Akt is a serine/threonine kinase that plays a critical role in cell surviva l signaling and its activation has been linked to tumorigenesis. Upregulati on of Akt as well as its upstream regulator phosphatidylinositol-3 kinase ( PI3K) has been found in many tumors and the negative regulator of this path way PTEN/MMAC is a tumor suppressor. As a target for drug discovery, we hav e expressed and purified an active Akt1 enzyme from a recombinant baculovir us-infected Sf9 cell culture. Coexpression of Akt1 with the catalytic subun it of PI3K or treatment with okadaic acid during expression was found to ge nerate an active enzyme in the insect cell culture system. We have optimize d the kinase activity and developed a simple quantitative kinase assay usin g biotinylated peptide substrates. Using the purified active enzyme, we hav e characterized its physical, catalytic and kinetic properties. Since Akt i s closely related to protein kinase C (PKC) and protein kinase A, the issue of obtaining selective inhibitors of this enzyme was addressed by comparis on of the structures of catalytic domains of Akt and PKC, derived by homolo gy modeling methods. A number of amino acid differences in the ATP binding regions of these kinases were identified, suggesting that selective inhibit ors of Akt can be discovered. However, the ATP binding regions are highly c onserved in the three isoforms of Akt implying that the discovery of isofor m-selective inhibitors would be very challenging. (C) 2001 Elsevier Science B.V. All rights reserved.