Isolation of the active form of RAC-protein kinase (PKB/Akt) from transfected COS-7 cells treated with heat shock stress and effects of phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate on its enzyme activity.

RAC-protein kinase (PKB/Akt) has been shown to be activated by growth factor stimulation as a downstream target of phosphatidylinositol 3-kinase and also by heat shock through a pathway independent of phosphatidylinositol 3-kinase. RAC-protein kinase was purified by antibody affinity chromatography from COS-7 cells transfected with the epitope-tagged expression plasmid. The protein kinase activity of RAC-protein kinase purified from heat-treated cells was 9-fold higher than the enzyme isolated from untreated control cells. Phosphatidylinositol 3,4,5-trisphosphate did not enhance the activity of RAC-protein kinase purified from either heat-treated cells or control cells, whereas phosphatidylinositol 4,5-bisphosphate suppressed the enzyme isolated from heat-treated cells. These results indicate that RAC-protein kinase may interact with phosphoinositides, however, it could not be activated by simple association with the product of phosphatidylinositol 3-kinase reaction.