title: The phosphoinositide-dependent kinase, PDK-1, phosphorylates conventional protein kinase C isozymes by a mechanism that is independent of phosphoinositide 3-kinase.
abstract: Phosphorylation by the phosphoinositide-dependent kinase, PDK-1, is required for the activation of diverse members of the AGC family of protein kinases, including the protein kinase C (PKC) isozymes. Here we explore the subcellular location of the PDK-1-mediated phosphorylation of conventional PKCs, and we address whether this phosphorylation is regulated by phosphoinositide 3-kinase. Pulse-chase experiments reveal that newly synthesized endogenous PKC alpha is primarily phosphorylated in the membrane fraction of COS-7 cells, where it is processed to a species that is phosphorylated at the activation loop and at two carboxyl-terminal positions. This "mature" species is then released into the cytosol. Deletion of the plekstrin homology domain of PDK-1 results in a 4-fold increase in the rate of processing of PKC indicating an autoinhibitory role for this domain. Autoinhibition by the plekstrin homology domain is not relieved by binding 3'-phosphoinositides; PKC is phosphorylated at a similar rate in serum-treated cells and serum-starved cells treated with the phosphoinositide 3-kinase inhibitors, LY294002 and wortmannin. Under the same conditions, the PDK-1-catalyzed phosphorylation of another substrate, Akt/protein kinase B, is abolished by these inhibitors. Our data are consistent with a model in which PDK-1 phosphorylates newly synthesized PKC by a mechanism that is independent of 3'-phosphoinositides.
authors.affiliation: Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0640, USA.
authors.forename: E D
authors.initials: ED
authors.lastname: Sonnenburg
authors.forename: T
authors.initials: T
authors.lastname: Gao
authors.forename: A C
authors.initials: AC
authors.lastname: Newton
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D016475:3T3 Cells; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism* / Q000494:pharmacology; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism* / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013347:Subcellular Fractions; D013997:Time Factors; D000077191:Wortmannin
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D000730:Androstadienes; D002867:Chromones; D016895:Culture Media, Serum-Free; D004791:Enzyme Inhibitors; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D020033:Protein Isoforms; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins; D000077191:Wortmannin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one