Proteasomal degradation of IRS-2, but not IRS-1 by calcineurin inhibition: Attenuation of insulin-like growth factor-I-induced GSK-3β 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 (≥3 h) treatment of cultured bovine adrenal chromaffin cells with cyclosporin A or FK506 decreased IRS-2 protein level by ∼50% (IC50 = 200 or 10 nM), without changing IRS-2 mRNA level, and insulin receptor, insulin-like growth factor-I (IGF-I) receptor, IRS-1, PI3K/PDK-1/Akt/GSK-3β 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 (IC50 = 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 (t1/2) from >24 to ∼4.2 h, 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 125I-IGF-I binding capacity was not changed in cyclosporin A- or FK506-treated cells; however, IGF-I-induced phosphorylations of GSK-3β and ERK1/ERK2 were attenuated by ∼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-3β and ERK pathways.