Multiple phosphoinositide 3-kinase-dependent steps in activation of protein kinase B.
The protein kinase B (PKB)/Akt family of serine kinases is rapidly activated following agonist-induced stimulation of phosphoinositide 3-kinase (PI3K). To probe the molecular events important for the activation process, we employed two distinct models of posttranslational inducible activation and membrane recruitment. PKB induction requires phosphorylation of two critical residues, threonine 308 in the activation loop and serine 473 near the carboxyl terminus. Membrane localization of PKB was found to be a primary determinant of serine 473 phosphorylation. PI3K activity was equally important for promoting phosphorylation of serine 473, but this was separable from membrane localization. PDK1 phosphorylation of threonine 308 was primarily dependent upon prior serine 473 phosphorylation and, to a lesser extent, localization to the plasma membrane. Mutation of serine 473 to alanine or aspartic acid modulated the degree of threonine 308 phosphorylation in both models, while a point mutation in the substrate-binding region of PDK1 (L155E) rendered PDK1 incapable of phosphorylating PKB. Together, these results suggest a mechanism in which 3' phosphoinositide lipid-dependent translocation of PKB to the plasma membrane promotes serine 473 phosphorylation, which is, in turn, necessary for PDK1-mediated phosphorylation of threonine 308 and, consequentially, full PKB activation.
Scheid
Michael P
MP
Department of Experimental Therapeutics, University Health Network. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Marignani
Paola A
PA
Woodgett
James R
JR
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D019281:Dimerization; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007668:Kidney; D008565:Membrane Proteins / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011960:Receptors, Estrogen / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000494:pharmacology; D014162:Transfection
D064413:3-Phosphoinositide-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D007334:Insulin-Like Growth Factor I; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D010768:Phosphoserine; D010769:Phosphothreonine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A; D013629:Tamoxifen; C016601:afimoxifene; C546842:MTOR protein, human; C494918:AKT1 protein, human; C578562:PDPK1 protein, human