Effect of Phosphoinositide-Dependent Kinase 1 on Protein Kinase B Translocation and Its Subsequent Activation

Abstract

In this report we investigated the function of phosphoinositide-dependent protein kinase 1 (PDK1) in protein kinase B (PKB) activation and translocation to the cell surface. Wild-type and PDK1 mutants were transfected into HeLa cells, and their subcellular localization was analyzed. PDK1 was found to translocate to the plasma membrane in response to insulin, and this process did not require a functional catalytic activity, since a catalytically inactive kinase mutant (Kd) of PDK1 was capable of translocating. The PDK1 presence at the cell surface was shown to be linked to phospholipids and therefore to serum-dependent phosphatidylinositol 3-kinase activity. Using confocal microscopy in HeLa cells we found that PDK1 colocalizes with PKB at the 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 insulin, a PKB mutant with its PH domain deleted (ΔPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that ΔPH-PKB was highly active independent of insulin stimulation when cotransfected with PDK1 mutants defective in their PH domain. 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 negative regulator of its enzyme activity.

ACKNOWLEDGMENTS

Our research was supported by the Institut National de la Santé et de la Recherche Médicale, the Association pour la Recherche sur le Cancer, the Université de Nice-Sophia Antipolis, the Ligue contre le Cancer, Groupe LIPHA-Merck (Lyon, France), and the European Community (grant QLG1-CT-1999-00674). C.S. was a recipient of a Poste Vert from INSERM.

We thank P. T. Hawkins for the generous gift of Myc-tagged PDK1 constructs and proteins purified from baculovirus-expressing Sf9 cells.