3-phosphoinositide-dependent protein kinase 1, an Akt1 kinase, is involved in dephosphorylation of Thr-308 of Akt1 in Chinese hamster ovary cells.
To investigate the role of 3-phosphoinositide-dependent protein kinase 1 (PDK1) in the Akt1 phosphorylation state, wild-type (wt) PDK1 and its kinase dead (kd) mutant were expressed using an adenovirus gene transduction system in Chinese hamster ovary cells stably expressing insulin receptor. Immunoblotting using anti-phosphorylated Akt1 antibody revealed Thr-308 already to be maximally phosphorylated at 1 min but completely dephosphorylated at 5 min, with insulin stimulation, whereas insulin-induced Akt1 activation was maintained even after dephosphorylation of Thr-308. Overexpression of wt-PDK1 further increased insulin-stimulated phosphorylation of Thr-308, also followed by rapid dephosphorylation. The insulin-stimulated Akt1 activity was also enhanced by wt-PDK1 expression but was maintained even at 15 min. Thus, phosphorylation of Thr-308 is not essential for maintaining the Akt1 activity once it has been achieved. Interestingly, the insulin-stimulated phosphorylation state of Thr-308 was maintained even at 15 min in cells expressing kd-PDK1, suggesting that kd-PDK1 has a dominant negative effect on dephosphorylation of Thr-308 of Akt1. Calyculin A, an inhibitor of PP1 and PP2A, also prolonged the insulin-stimulated phosphorylation state of Thr-308. In addition, in vitro experiments revealed PP2A, but not PP1, to dephosphorylate completely Thr-308 of Akt1. These findings suggest that a novel pathway involving dephosphorylation of Akt1 at Thr-308 by a phosphatase, possibly PP2A, originally, identified as is regulated downstream from PDK1, an Akt1 kinase.
Yamada
T
T
Third Department of Internal Medicine, Yamaguchi University School of Medicine, Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan.
Katagiri
H
H
Asano
T
T
Inukai
K
K
Tsuru
M
M
Kodama
T
T
Kikuchi
M
M
Oka
Y
Y
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D029681:Arabidopsis Proteins; D016466:CHO Cells; D006224:Cricetinae; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008387:Marine Toxins; D009154:Mutation; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D010769:Phosphothreonine / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D014161:Transduction, Genetic
D029681:Arabidopsis Proteins; D004791:Enzyme Inhibitors; D007328:Insulin; D008387:Marine Toxins; D010080:Oxazoles; D010940:Plant Proteins; D015221:Potassium Channels; D010769:Phosphothreonine; C074500:AKT1 protein, Arabidopsis; D010768:Phosphoserine; C059041:calyculin A; D064413:3-Phosphoinositide-Dependent Protein Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases