title: In vivo role of the PIF-binding docking site of PDK1 defined by knock-in mutation.
abstract: PKB/Akt, S6K, SGK and RSK are mediators of responses triggered by insulin and growth factors and are activated following phosphorylation by 3-phosphoinositide-dependent protein kinase-1 (PDK1). To investigate the importance of a substrate-docking site in the kinase domain of PDK1 termed the 'PIF-pocket', we generated embryonic stem (ES) cells in which both copies of the PDK1 gene were altered by knock-in mutation to express a form of PDK1 retaining catalytic activity, in which the PIF-pocket site was disrupted. The knock-in ES cells were viable, mutant PDK1 was expressed at normal levels and insulin-like growth factor 1 induced normal activation of PKB and phosphorylation of the PKB substrates GSK3 and FKHR. In contrast, S6K, RSK and SGK were not activated, nor were physiological substrates of S6K and RSK phosphorylated. These experiments establish the importance of the PIF-pocket in governing the activation of S6K, RSK, SGK, but not PKB, in vivo. They also illustrate the power of knock-in technology to probe the physiological roles of docking interactions in regulating the specificity of signal transduction pathways.
authors.affiliation: MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK. b.j.collins@dundee.ac.uk
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Collins
authors.forename: Maria
authors.initials: M
authors.lastname: Deak
authors.forename: J Simon C
authors.initials: JS
authors.lastname: Arthur
authors.forename: Laura J
authors.initials: LJ
authors.lastname: Armit
authors.forename: Dario R
authors.initials: DR
authors.lastname: Alessi
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D048868:Adaptor Proteins, Signal Transducing; D000483:Alleles; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008954:Models, Biological; D009154:Mutation; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D013234:Stem Cells / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; C491819:FRAT1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D007334:Insulin-Like Growth Factor I; D064413:3-Phosphoinositide-Dependent Protein Kinases; C494918:AKT1 protein, human; C578562:PDPK1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt