title: 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 (DeltaPH-PKB) retained the ability to translocate to the plasma membrane when coexpressed with PDK1. Finally, we found that DeltaPH-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.
authors.affiliation: INSERM U145, IFR 50, Faculté de Médecine, 06107 Nice Cedex 2, France.
authors.forename: N
authors.initials: N
authors.lastname: Filippa
authors.forename: C L
authors.initials: CL
authors.lastname: Sable
authors.forename: B A
authors.initials: BA
authors.lastname: Hemmings
authors.forename: E
authors.initials: E
authors.lastname: Van Obberghen
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D018613:Microscopy, Confocal; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; C526292:IRS1 protein, human; C578562:PDPK1 protein, human