Regulation of Akt mRNA and protein levels by glycogen synthase kinase-3beta in adrenal chromaffin cells: effects of LiCl and SB216763.
In cultured bovine adrenal chromaffin cells, where Akt1 is the predominant isoform over Akt2 and Akt3, chronic (> or =12 h) treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3, decreased Akt1 level by approximately 52% (EC50=3.7 mM; t1/2=l2 h); it was associated with LiCl-induced increased levels of Ser9-phosphorylated glycogen synthase kinase-3beta (approximately 37%) and beta-catenin (approximately 59%), two hallmarks of glycogen synthase kinase-3beta inhibition. The same LiCl treatment did not change phosphoinositide 3-kinase, phosphoinositide-dependent kinase 1, and extracellular signal-regulated kinase-1/2 levels. Treatment with SB216763 [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione], a selective inhibitor of glycogen synthase kinase-3, lowered Akt1 level by approximately 67% (EC50=2 microM; t1/2=l2 h), when SB216763 caused concentration- and time-dependent increase of beta-catenin level by approximately 76%. LiCl- or SB216763-induced Akt1 decrease, as well as increases of Ser9-phosphorylated glycogen synthase kinase-3beta and beta-catenin were restored to the control levels of nontreated cells after the washout of LiCl (20 mM for 24 h)- or SB216763 (30 microM for 24 h)-treated cells. LiCl-induced Akt1 reduction was not prevented by beta-lactone, lactacystin (two inhibitors of proteasome), calpastatin (an inhibitor of calpain), or leupeptin (an inhibitor of lysosome). LiCl decreased Akt1 mRNA level by 20% at 6 h, with no effect on Akt1 mRNA stability. These results suggest that glycogen synthase kinase-3beta inhibition caused down-regulation of Akt1 mRNA and Akt1 protein levels; conversely, constitutive activity of glycogen synthase kinase-3beta maintains steady-state level of Akt1 in quiescent adrenal chromaffin cells.
Nemoto
Takayuki
T
Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan.
Kanai
Tasuku
T
Yanagita
Toshihiko
T
Satoh
Shinya
S
Maruta
Toyoaki
T
Yoshikawa
Norie
N
Kobayashi
Hideyuki
H
Wada
Akihiko
A
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D007211:Indoles / Q000494:pharmacology*; D018021:Lithium Chloride / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000096:biosynthesis*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*; D051176:beta Catenin / Q000378:metabolism
D064413:3-Phosphoinositide-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D007211:Indoles; D018021:Lithium Chloride; D008301:Maleimides; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D051058:Oncogene Protein v-akt; D019869:Phosphatidylinositol 3-Kinases; D011480:Protease Inhibitors; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D051176:beta Catenin; C417521:SB 216763