Mammalian target of rapamycin regulates IRS-1 serine 307 phosphorylation

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Abstract

Insulin signaling can be negatively regulated by phosphorylation of serine 307 of the insulin receptor substrate (IRS)-1. Rapamycin, an inhibitor of the kinase mTOR, can prevent serine 307 phosphorylation and the development of insulin resistance. We further investigated the role of mTOR in regulating serine 307 phosphorylation, demonstrating that serine 307 phosphorylation in response to insulin, anisomycin, or tumor necrosis factor was quantitatively and temporally associated with activation of mTOR and could be inhibited by rapamycin. Amino acid stimulation activated mTOR and resulted in IRS-1 serine 307 phosphorylation without activating PKB or JNK. Okadaic acid, an inhibitor of the phosphatase PP2A, activated mTOR and stimulated the phosphorylation of serine 307 in a rapamycin-sensitive manner, indicating serine 307 phosphorylation requires mTOR activity but not PP2A, suggesting that mTOR itself may be responsible for phosphorylating serine 307. Finally, we demonstrated that serine 307 phosphorylated IRS-1 is detected primarily in the cytosolic fraction.

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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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