Proteasomal degradation of IRS-2, but not IRS-1 by calcineurin inhibition: attenuation of insulin-like growth factor-I-induced GSK-3beta and ERK pathways in adrenal chromaffin cells.
The ability of calcineurin to regulate IRS-1 and IRS-2 levels has not been examined in any given cells, although calcineurin inhibition by therapeutic immunosuppressants produced cytoprotective and cytotoxic effects (e.g., new-onset of diabetes mellitus, seizure). Chronic (>or=3h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by approximately 50% (IC(50)=200 or 10nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3beta and ERK1/ERK2 protein levels. When the cells were washed to remove the test drug, the decreased IRS-2 level restored to the control level. Cyclosporin A or FK506 treatment inhibited calcineurin activity (IC(50)=500 or 40 nM, in vitro assay). Rapamycin, an FK506-binding protein ligand unable to inhibit calcineurin, failed to decrease IRS-2, but reversed FK506-induced decreases of calcineurin activity and IRS-2 level. Pulse-label followed by polyacrylamide gel electrophoresis revealed that cyclosporin A or FK506 accelerated IRS-2 degradation rate (t(1/2)) from >24 to approximately 4.2h, without altering IRS-2 synthesis. IRS-2 reduction by cyclosporin A or FK506 was prevented by lactacystin (proteasome inhibitor), but not by calpeptin (calpain inhibitor) or leupeptin (lysosome inhibitor). Cyclosporin A or FK506 increased serine-phosphorylation and ubiquitination of IRS-2. Cell surface (125)I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3beta and ERK1/ERK2 were attenuated by approximately 50%, which were prevented by rapamycin or lactacystin. Thus, calcineurin inhibition decreased IRS-2 level via proteasomal IRS-2 degradation, attenuating IGF-I-induced GSK-3beta and ERK pathways.
Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan.
Shinya
S
Satoh
Toshihiko
T
Yanagita
Toyoaki
T
Maruta
Takayuki
T
Nemoto
Norie
N
Yoshikawa
Hideyuki
H
Kobayashi
Tetsuya
T
Tono
Akihiko
A
Wada
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000311:Adrenal Glands / Q000166:cytology*; D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000187:drug effects*; D016572:Cyclosporine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D047468:Immunoprecipitation; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D016559:Tacrolimus / Q000494:pharmacology; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D004791:Enzyme Inhibitors; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D007334:Insulin-Like Growth Factor I; D016572:Cyclosporine; D000071679:Glycogen Synthase Kinase 3 beta; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D046988:Proteasome Endopeptidase Complex; D016559:Tacrolimus