Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation
Section snippets
Materials and Methods
Cell culture. 3T3-L1 cells were grown and maintained as fibroblasts in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Gaithersburg, MD) with high glucose containing 10% fetal bovine serum (FBS, HyClone Logan, UT) in a humidified atmosphere composed of 95% air and 5% CO2. Cells were differentiated into adipocytes by exposure to DMEM high glucose with 10% FBS, 0.4 μg/ml dexamethasone, 0.5 mmol/L isobutylmethylxanthine, and 5 μg/ml insulin. Following 3 days, the media were changed and
Insulin-, anisomycin-, and TNF-α-stimulated serine 307 phosphorylation is rapamycin-sensitive
Inhibition of mTOR with rapamycin is known to inhibit serine phosphorylation of IRS-1 in response to insulin [16], thus we tested whether insulin-induced serine 307 phosphorylation was rapamycin-sensitive. Insulin-stimulated serine 307 phosphorylation at 5 min, but more dramatically at 1 h (Fig. 1A). Phosphorylation of ser307 was accompanied by decreased electrophoretic mobility of IRS-1, consistent with increased serine phosphorylation. Pretreatment of the cells with rapamycin prevented the
Discussion
The results of the current experiments indicate that mTOR may be an important factor in the regulation of serine 307 phosphorylation. Serine 307 phosphorylation induced by insulin, anisomycin, or TNF-α was found to be quantitatively and temporally associated with mTOR activity (determined by p70 S6K phosphorylation). Because insulin and anisomycin can activate many signaling pathways, the observation that amino acids alone were able to stimulate serine 307 phosphorylation further demonstrates
Acknowledgements
C.J.C. was supported by a Northwestern University/Abbott Laboratories Post-Doctoral fellowship.
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