Abstract
Mammalian target of rapamycin (mTOR) is a core component of raptor-mTOR (mTORC1) and rictor-mTOR (mTORC2) complexes that control diverse cellular processes. Both mTORC1 and mTORC2 regulate several elements downstream of type I insulin-like growth factor receptor (IGF-IR) and insulin receptor (InsR). However, it is unknown whether and how mTOR regulates IGF-IR and InsR themselves. Here we show that mTOR possesses unexpected tyrosine kinase activity and activates IGF-IR/InsR. Rapamycin induces the tyrosine phosphorylation and activation of IGF-IR/InsR, which is largely dependent on rictor and mTOR. Moreover, mTORC2 promotes ligand-induced activation of IGF-IR/InsR. IGF- and insulin-induced IGF-IR/InsR phosphorylation is significantly compromised in rictor-null cells. Insulin receptor substrate (IRS) directly interacts with SIN1 thereby recruiting mTORC2 to IGF-IR/InsR and promoting rapamycin- or ligand-induced phosphorylation of IGF-IR/InsR. mTOR exhibits tyrosine kinase activity towards the general tyrosine kinase substrate poly(Glu-Tyr) and IGF-IR/InsR. Both recombinant mTOR and immunoprecipitated mTORC2 phosphorylate IGF-IR and InsR on Tyr1131/1136 and Tyr1146/1151, respectively. These effects are independent of the intrinsic kinase activity of IGF-IR/InsR, as determined by assays on kinase-dead IGF-IR/InsR mutants. While both rictor and mTOR immunoprecitates from rictor+/+ MCF-10A cells exhibit tyrosine kinase activity towards IGF-IR and InsR, mTOR immunoprecipitates from rictor−/− MCF-10A cells do not induce IGF-IR and InsR phosphorylation. Phosphorylation-deficient mutation of residue Tyr1131 in IGF-IR or Tyr1146 in InsR abrogates the activation of IGF-IR/InsR by mTOR. Finally, overexpression of rictor promotes IGF-induced cell proliferation. Our work identifies mTOR as a dual-specificity kinase and clarifies how mTORC2 promotes IGF-IR/InsR activation.
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Acknowledgements
We thank Prof Wei Zhang (Cancer Genomics Core Laboratory, MD Anderson Cancer Center, USA) for generously providing pcDNA3.1-IGF-IR plasmid. This work was supported by the intramural research funding from West China Hospital, Sichuan University, China.
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Supplementary information
Supplementary information, Figure S1
mTORC1 inhibition leads to mTORC2-dependent phosphorylation of IGF-IR/InsR. (PDF 1031 kb)
Supplementary information, Figure S2
Rictor and mTOR were required for IGF-IR/InsR phosphorylation resulting from rapamycin treatment and ligand stimulation. (PDF 593 kb)
Supplementary information, Figure S3
Western blot analysis of the efficacy and specificity of rictor, IRS1 and IRS2 knockdown. (PDF 219 kb)
Supplementary information, Figure S4
The effects of rapamycin, AICAR and insulin on IGF-IR/InsR-rictor-mTOR complexes. (PDF 287 kb)
Supplementary information, Figure S5
IRS1/2 promotes rapamycin-and ligand-induced phosphorylation of IGF-IR/InsR. (PDF 421 kb)
Supplementary information, Figure S6
Torin2 inhibits IGF/insulin signaling. (PDF 163 kb)
Supplementary information, Figure S7
The rictor immunoprecipitates induces kinase-dead IGF-IR (KD-IGF-IR) and InsR (KD-InsR) phosphorylation in vitro. (PDF 148 kb)
Supplementary information, Figure S8
mTOR promotes IGF-IR phosphorylation. (PDF 191 kb)
Supplementary information, Figure S9
Torin2 does not directly inhibit IGF-IR/InsR activity but inhibit mTOR-induced IGF-IR/InsR activation. (PDF 249 kb)
Supplementary information, Figure S10
In vitro kinase assays for the effects of mTOR on the activity of IGF-IR/InsR towards their substrate. (PDF 346 kb)
Supplementary information, Figure S11
Roles of PI3K in IGF-I- and rapamycin-induced IGF-IR/InsR phosphorylation. (PDF 146 kb)
Supplementary information, Figure S12
The effects of Ser/Thr phosphorylation and phosphatase inhibitors on IGF-IR/InsR tyrosine phosphorylation. (PDF 188 kb)
Supplementary information, Figure S13
mTOR induces tyrosine phosphorylation. (PDF 182 kb)
Supplementary information, Figure S14
The effects of mTOR and InsR on wild-type IGF-IR/InsR (WT-IGF-IR/InsR) peptide and mutated IGF-IR/InsR peptides. (PDF 275 kb)
Supplementary information, Figure S15
Mass spectrometric analysis of mTOR-induced phosphorylation in kinase-dead IGF-IR (K1033R). (PDF 490 kb)
Supplementary information, Figure S16
The effect of rapamycin and AG1024 on HepG2 cell proliferation. (PDF 120 kb)
Supplementary information, Table S1
Mass spectrometric identification of IGF-IR, InsR, IRS1, IRS2, mTOR and other proteins in rictor immunoprecipitates. (PDF 114 kb)
Supplementary information, Table S2
The molecular weight of proteins in western blot analysis. (PDF 133 kb)
Supplementary information, Data S1
Supplementary Methods (PDF 132 kb)
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Yin, Y., Hua, H., Li, M. et al. mTORC2 promotes type I insulin-like growth factor receptor and insulin receptor activation through the tyrosine kinase activity of mTOR. Cell Res 26, 46–65 (2016). https://doi.org/10.1038/cr.2015.133
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DOI: https://doi.org/10.1038/cr.2015.133
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