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. 2009 Feb;29(3):640-9.
doi: 10.1128/MCB.00980-08. Epub 2008 Dec 1.

The TSC-mTOR pathway mediates translational activation of TOP mRNAs by insulin largely in a raptor- or rictor-independent manner

Ilona Patursky-Polischuk  1 , Miri Stolovich-Rain, Mirit Hausner-Hanochi, Judith Kasir, Nadine Cybulski, Joseph Avruch, Markus A Rüegg, Michael N Hall, Oded Meyuhas
Affiliations

The TSC-mTOR pathway mediates translational activation of TOP mRNAs by insulin largely in a raptor- or rictor-independent manner

Ilona Patursky-Polischuk et al. Mol Cell Biol. 2009 Feb.

Erratum in

  • Mol Cell Biol. 2009 Mar;29(6):1670

Abstract

The stimulatory effect of insulin on protein synthesis is due to its ability to activate various translation factors. We now show that insulin can increase protein synthesis capacity also by translational activation of TOP mRNAs encoding various components of the translation machinery. This translational activation involves the tuberous sclerosis complex (TSC), as the knockout of TSC1 or TSC2 rescues TOP mRNAs from translational repression in mitotically arrested cells. Similar results were obtained upon overexpression of Rheb, an immediate TSC1-TSC2 target. The role of mTOR, a downstream effector of Rheb, in translational control of TOP mRNAs has been extensively studied, albeit with conflicting results. Even though rapamycin fully blocks mTOR complex 1 (mTORC1) kinase activity, the response of TOP mRNAs to this drug varies from complete resistance to high sensitivity. Here we show that mTOR knockdown blunts the translation efficiency of TOP mRNAs in insulin-treated cells, thus unequivocally establishing a role for mTOR in this mode of regulation. However, knockout of the raptor or rictor gene has only a slight effect on the translation efficiency of these mRNAs, implying that mTOR exerts its effect on TOP mRNAs through a novel pathway with a minor, if any, contribution of the canonical mTOR complexes mTORC1 and mTORC2. This conclusion is further supported by the observation that raptor knockout renders the translation of TOP mRNAs rapamycin hypersensitive.

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Figures

FIG. 1.
FIG. 1.
Insulin induces translational activation of TOP mRNAs. (A) NIH-IR cells were serum-deprived for 72 h and then kept starved (−S), serum refed (+S), or insulin-treated (Ins). Twenty-four hours later, the cell number was assessed by the methylene blue staining protocol (39). The number of cells following each treatment was normalized to that of serum-starved cells, which was arbitrarily set at 1 and is presented as the mean ± the standard error of the mean (SEM; n ≥ 10). *, P < 0.05. (B) NIH-IR cells were serum starved for 72 h and then insulin treated in the absence or presence (+Rapa) of rapamycin for the indicated time or LY294002 for the last 0.5 h (+LY [0.5 h]). Subsequently, cells were harvested and cytoplasmic extracts were prepared. These extracts were centrifuged through sucrose gradients and separated into polysomal (P) and subpolysomal (S) fractions. RNA from equivalent aliquots of these fractions was analyzed by Northern blot hybridization with cDNAs corresponding to TOP mRNAs (encoding rpL32 and rpS6) and a non-TOP mRNA (Actin). The radioactive signals were quantified, and the relative translational efficiency of each mRNA is numerically presented beneath the autoradiograms as a percentage of the mRNA engaged in polysomes. These figures are expressed as an average ± SEM of the number of determinations in parentheses or the average with the individual values in parentheses if only two determinations are presented. (C) NIH-IR cells were serum starved for 72 h (0) and then either insulin treated without (−) or with (+) rapamycin for the indicated amount of time. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (D) HEK 293 cells were serum starved for 48 h and insulin treated for the last 3 h without or with rapamycin (R) or with LY294002 (LY) for the last 0.5 h. Cells were harvested and subjected to polysomal analysis. (E) HEK 293 cells were serum starved for 48 h (0) and then treated with 100 nM insulin without (−) or with (+) rapamycin for the indicated amount of time. The cytoplasmic proteins were subjected to Western blot analysis. (F) Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate, and when more than 90% of the cells converted into adipocytes, they were serum starved for 48 h and then kept starved with or without insulin for up to 48 h. At the indicated time, cells were trypsinized and counted. Numbers of cells (average of at least three repetitions for each time point) were normalized to the number at time zero. (G) Adipocytes were serum starved for 48 h and then kept starved (−insulin) or treated with insulin for the indicated amount of time. The size of cells was determined as described in “Materials and Methods” and is presented as FSC-H. (H) Adipocytes were serum starved for 48 h and then harvested or insulin treated for 1 or 4 h. Subsequently, cells were harvested and subjected to polysomal analysis.
FIG. 2.
FIG. 2.
TSC2 or TSC1 deficiency rescues TOP mRNAs from translational repression in mitotically arrested cells. (A) TSC2+/+ and TSC2−/− MEFs were untreated (control), serum starved for 72 h (−serum), treated with aphidicolin for 48 h (+Aphi), LY294002 for 0.5 h (+LY), or rapamycin (+Rapa) for 2 h, or were serum starved and then refed for 3 h. Cells were harvested and subjected to polysomal analysis. (B) TSC2+/+ and TSC2−/− cells were seeded in 96-well plates at a density of 4 × 103 cells per well. Cells were either untreated (control) or serum starved (−serum) for the indicated amount of time. Proliferation was monitored by the methylene blue staining protocol (39). Absorbance measured 24 h after platting, set arbitrarily at 1, and measured at later time points (average ± SEM [n = 12] for each time point) was normalized to that value. (C) TSC2+/+ and TSC2−/− cells were seeded in 96-well plates at a density of 4 × 103 cells per well. Cells were either untreated (control) or aphidicolin-treated (Aphi) for the indicated amount of time. Proliferation was monitored and presented as described in panel B. (D) TSC2+/+ and TSC2−/− cells were untreated (control), serum starved for 72 h (−serum), serum refed for 3 h (refed), rapamycin treated for 2 h (rapa), or LY294002 treated for 0.5 h (LY), and then harvested. The cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies. (E and F) Experimental details are similar to those described in panels A and D, respectively, except for the usage of TSC1+/+ and TSC1+/+ MEFs.
FIG. 3.
FIG. 3.
Overexpression of Rheb can relieve translational repression of TOP mRNAs in mitotically arrested cells. (A) HEK 293 cells were transiently transfected with expression vectors encoding FLAG-Rheb (WT) or Myc-Rheb-5A, and 24 h later they were serum starved for 36 h. Subsequently, cells were harvested and subjected to polysomal analysis. (B) HEK 293 cells were transiently transfected with expression vectors encoding FLAG-Rheb (WT) or Myc-Rheb (5A), and 24 h later they were serum starved for 36 h. Subsequently, cells were harvested and cytoplasmic proteins were subjected to Western blot analysis using the indicated antibodies.
FIG. 4.
FIG. 4.
Knockdown of mTOR downregulates the translation efficiency of TOP mRNAs in insulin-treated cells. (A) HEK 293 cells were infected with viruses expressing Red shRNA (shRed) or mTOR shRNA (shmTOR). Cells were 48-h serum starved and then insulin stimulated for 3 h. The abundance of mTOR and its activity were monitored by Western blot analysis of cytoplasmic proteins with the indicated antibodies. The relative abundance of mTOR was normalized to that of actin, whereas the relative abundance of phospho rpS6 (P-rpS6) was normalized to that of rpS6. The results are numerically presented relative to those obtained for infected cells that expressed Red shRNA, which were arbitrarily set at 1. (B) Cytoplasmic extracts from cells described in panel A were subjected to polysomal analysis.
FIG. 5.
FIG. 5.
The translation efficiency of TOP mRNAs does not rely on insulin-treated cells. (A) HEK 293 cells were infected with viruses expressing Red shRNA (shRed) or raptor shRNA (shRaptor). Cells were treated as described in the legend to Fig. 4. The abundance of raptor- and mTOR-dependent proteins was monitored by Western blot analysis with the indicated antibodies. The relative abundance of raptor, phospho S6K1(Thr389) and phospho Akt(Ser473) was normalized to that of actin, whereas the relative abundance of phospho rpS6(Ser235/236) was normalized to that of rpS6. The results are numerically presented relative to those obtained with Red shRNA-expressing cells that were arbitrarily set at 1. (B) Cytoplasmic extracts from cells described in panel A were subjected to polysomal analysis. (C) iRapKO cells were either untreated (−) or treated (+) with tamoxifen for 4 days and were then further incubated for 3 h in the absence (−) or presence (+) of rapamycin. The relative abundance of the indicated proteins was assessed as described in panel A with the exception that results are numerically presented relative to those obtained with cells untreated with either tamoxifen or rapamycin. (D) iRapKO cells were either treated or untreated with tamoxifen (for 4 days) and subsequently were harvested and subjected to polysomal analysis.
FIG. 6.
FIG. 6.
TOP mRNAs are translationally activated by insulin in a rictor-independent fashion. (A) iRicKO cells were either untreated (−) or treated (+) with 1 μM tamoxifen for 4 days and then serum starved for the last 48 h followed by 3-h insulin treatment. The relative abundance of the indicated proteins was assessed as described in the legend to Fig. 5A. (B) iRicKO cells were either untreated (−) or treated (+) with 1 μM tamoxifen for 4 days and then serum starved for the last 48 h followed by 3 h without (−serum) or with (−serum + insulin) insulin treatment. Subsequently, cells were harvested and subjected to polysomal analysis.
FIG. 7.
FIG. 7.
Rapamycin represses the translation of TOP mRNAs in an FKBP-12-dependent and raptor-sensitive fashion. (A) 3T3-L1 preadipocytes were either untreated (Control), treated for 3 h with rapamycin, FK506, or both drugs, or treated for 0.5 h with LY294002. Subsequently, they were harvested and subjected to polysomal analysis. (B) 3T3-L1 preadipocytes were treated for 3 h with rapamycin, FK506, or both drugs. The cytoplasmic proteins were subjected to Western blot analysis. (C) HEK 293 cells were transiently transfected with pcDNA3-6myc-hFKBP12 (myc-FKBP12) or pcDNA3-6myc (myc tag). Forty-eight hours later, cells were incubated without (−) or with (+) rapamycin and were subsequently harvested and subjected to polysomal analysis.
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