Activation of Mammalian Target of Rapamycin (mTOR) by Insulin Is Associated with Stimulation of 4EBP1 Binding to Dimeric mTOR Complex 1

Lifu Wang,Christopher J Rhodes,John C Lawrence

doi:10.1074/jbc.m603566200

Abstract

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.

Highlights

The finding that the effects of insulin and insulin-like growth factor 1 on 4EBP1 were attenuated by rapamycin provided the first evidence that mammalian target of rapamycin (mTOR) controlled 4EBP1 [16, 17]
The 3-fold increase in mTORC1 activity produced by insulin (Fig. 1B) is comparable to the increase in phosphorylation of endogenous 4EBP1 when 32Plabeled 3T3-L1 adipocytes are incubated with the hormone [17]. mTORC1 isolated with C-Rap Ab was not able to phosphorylate F113A (Fig. 1C), a 4EBP1 protein having a point mutation that disrupts the TOR signaling (TOS) motif [22]
The stimulatory effects of insulin on mTOR activity and binding occurred rapidly and at physiological concentrations of the hormone. Both effects of insulin were dependent upon the TOS motif in the substrate, 4EBP1, and it was essential to preserve intact mTORC1 to detect the effect of insulin on kinase activity and 4EBP1 binding

Summary

EXPERIMENTAL PROCEDURES

Adipocyte Culture and Extract Preparation—3T3-L1 fibroblasts were grown in Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum. Antibodies (designated N-Rap Ab) to the region in raptor (amino acids 36 –53) originally targeted by Kim et al [7] were generated as described previously [22]. The resulting raptor antibodies (C-Rap Ab) were purified using a column containing an affinity resin prepared by coupling the peptide to Sulfolink beads (Pierce). Phosphospecific antibodies to the Ser-473 site in Akt, the Thr389 site in S6K1, and the activating sites in the ERK1 and ERK2 isoforms of mitogen-activated protein kinase were from Cell Signaling Technology Inc. Purification of Recombinant Proteins—His-tagged forms of wild type 4EBP1 and a 4EBP1 protein having Ala at position 113 (F113A) were expressed in bacteria and purified as described previously [22]. Insulin-like growth factor 1, Nonidet P-40, Triton X-100, and wortmannin were from Sigma

RESULTS

Time Course and Concentration

Stimulation of Kinase Activity and Substrate Binding by Insulin

DISCUSSION