Constitutive activity of glycogen synthase kinase-3beta: positive regulation of steady-state levels of insulin receptor substrates-1 and -2 in adrenal chromaffin cells.
In cultured bovine adrenal chromaffin cells, 12-h treatment with 1-20 mM LiCl, an inhibitor of glycogen synthase kinase-3 (GSK-3), increased Ser(9) phosphorylation of GSK-3beta by approximately 44%, while decreasing insulin receptor substrate-1 (IRS-1) and IRS-2 protein levels by approximately 38 and approximately 62% in a concentration-dependent manner. Treatment with SB216763 (0.1-30 microM for 12 h), a selective inhibitor of GSK-3, lowered IRS-1 and IRS-2 levels by approximately 38 and approximately 48%, while increasing beta-catenin protein level by approximately 47%, due to the prevention of GSK-3-induced degradation of beta-catenin by SB216763. Insulin (100 nM for 24 h) increased Ser(9) phosphorylation of GSK-3beta by approximately 104%, while decreasing IRS-1 and IRS-2 levels by approximately 41 and approximately 72%; the insulin-induced Ser(9) phosphorylation of GSK-3beta, as well as down-regulations of IRS-1 and IRS-2 levels were restored to the control levels of nontreated cells at 24 h after the washout of the insulin (100 nM for 12 h)-treated cells. Either clasto-lactacystin beta-lactone or lactacystin (an inhibitor of proteasome) prevented LiCl- or SB216763-induced decreases of IRS-1 and IRS-2 levels by approximately 100 and approximately 69%, respectively. In contrast, calpastatin (an inhibitor of calpain) and leupeptin (an inhibitor of lysosome) failed to prevent the decreases of IRS-1 and IRS-2 levels caused by LiCl or SB216763. LiCl or SB216763 lowered IRS-2 mRNA level, with no effect on IRS-1 mRNA level. These results suggest that constitutive activity of GSK-3beta in quiescent cells positively maintains steady-state levels of IRS-1 and IRS-2 via regulating proteasomal degradation and/or synthesis of IRS-1 and IRS-2 proteins.
Nemoto
Takayuki
T
Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki, Japan.
Yokoo
Hiroki
H
Satoh
Shinya
S
Yanagita
Toshihiko
T
Sugano
Takashi
T
Yoshikawa
Norie
N
Maruta
Toyoaki
T
Kobayashi
Hideyuki
H
Wada
Akihiko
A
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000311:Adrenal Glands / Q000166:cytology; D000818:Animals; D015152:Blotting, Northern / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000502:physiology*; D007211:Indoles / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D018021:Lithium Chloride / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D010750:Phosphoproteins / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D046988:Proteasome Endopeptidase Complex / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013997:Time Factors
D000276:Adjuvants, Immunologic; D015853:Cysteine Proteinase Inhibitors; D007211:Indoles; D007783:Lactones; D008301:Maleimides; D010750:Phosphoproteins; C417521:SB 216763; C067713:lactacystin; D012694:Serine; D038362:Glycogen Synthase Kinase 3; D046988:Proteasome Endopeptidase Complex; D018021:Lithium Chloride; D000111:Acetylcysteine