Serine 396 of PDK1 is required for maximal PKB activation.
Protein kinase B (PKB; also known as Akt) is important for mediating survival and proliferation signals. Following activation, PKB shuttles to various compartments of the cell, including the nucleus, where it phosphorylates an array of targets. PKB is phosphorylated at T308 by its activator PDK1. PDK1 is normally excluded from the nucleus via a nuclear exclusion sequence (NES), and our previous work suggested that nuclear exclusion can be attenuated by IGF-1-induced phosphorylation of S396 proximal to the NES. No studies have been done to test the significance of S396 phosphorylation or the impact of nuclear accumulation of PDK1 on PKB activation. To address these questions, we created isogenic embryonic stem cell (ESC) lines expressing various alleles of PDK1 within a PDK1-/- background. Disruption of the NES domain of PDK1 correlated with elevated PKB phosphorylation at both T308 and S473. In contrast, mutation of S396 to alanine reduced PDK1 nuclear localization and reduced PKB phosphorylation and activation. The loss of phosphorylation of PKB by S396A mutation was rescued by forcing nuclear PDK1 or by conversion of S396 to an aspartic acid. The phosphorylation of the PKB substrate FOXO3alpha was reduced in S396A PDK1 ESC. Other known and suspected PKB substrates, including GSK3 and Raf1, were unaffected. This study therefore reveals that S396 plays a role in the activation of PKB leading to the regulated phosphorylation of some PKB substrates including FOXO3alpha.
Moon
Zoe
Z
Department of Biology, York University, Toronto, Ontario, Canada.
Wang
Yanping
Y
Aryan
Nathan
N
Mousseau
Darrell D
DD
Scheid
Michael P
MP
D064413:3-Phosphoinositide-Dependent Protein Kinases; D020816:Amino Acid Motifs; D000818:Animals; D002460:Cell Line; D002999:Clone Cells; D053595:Embryonic Stem Cells / Q000201:enzymology; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D051379:Mice; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D045522:Recombinases / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; C474033:FoxO3 protein, mouse; D045522:Recombinases; D012694:Serine; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019893:Ribosomal Protein S6 Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020930:MAP Kinase Kinase Kinases; D038362:Glycogen Synthase Kinase 3