Effect of phosphoinositide-dependent kinase 1 on protein kinase B translocation and its subsequent activation

In this report we investigated the function of phosphoinositide-dependent p rotein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocati on to the cell surface. Wild-type and PDK1 mutants were transfected into He La cells, and their subcellular localization was analyzed. PDK1 was found t o translocate to the plasma membrane in response to insulin, and this proce ss did not require a functional catalytic activity, since a catalytically i nactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 p resence at the cell surface was shown to be linked to phospholipids and the refore to serum-dependent phosphatidylinositol 3-kinase activity. Using con focal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at t he plasma membrane. Further, after cotransfection of PKB and a PDK1 mutant (Mut) unable to translocate to the plasma membrane, PKB was prevented from moving to the cell periphery after insulin stimulation. In response to insu lin, a PKB mutant with its PH domain deleted (Delta PH-PKB) retained the ab ility to translocate to the plasma membrane when coexpressed with PDK1. Fin ally, we found that Delta PH-PKB was highly active independent of insulin s timulation when cotransfected with PDK1 mutants defective in their PH domai n. These findings suggest that PDK1 brings PKB to the plasma membrane upon exposure of cells to insulin and that the PH domain of PDK1 acts as a negat ive regulator of its enzyme activity.