Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation

Abstract

The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycin-resistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.

Fig. 1.

Rapamycin does not functionally mimic hypophosphorylation of 4E-BP1. (A) HEK293 cells were serum-starved for 24 h, pretreated with rapamycin (20 nM) for 30 min, and stimulated with 10% FBS, insulin (100 nM), or PMA (100 ng/mL). 4E-BP1 cap binding activity was measured with m7GTP Sepharose association. (B) S6K1 activity was measured with GST-S6 as a substrate. (C) In vivo cap-dependent translational assays were conducted with a dual Renilla/firefly luciferase assay with the Polio virus IRES driving firefly expression. One microgram of DNA was transfected into HEK293 cells in 6-well plates and 24 h later were either starved or treated with rapamycin for 24 more hours. (D) Dominant negative 4E-BP1 (Thr-37/46 AA), WT 4E-BP1, or control plasmids were cotransfected with the translational vector at a 1:2 ratio, and rapamycin or ethanol was treated in other samples. Luciferase activity was measured and is shown as relative cap-dependent translation. Phospho-S6 and 4E-BP1 expression is also shown.

Fig. 2.

Rapamycin exhibits differential effects toward S6Ks versus 4E-BP1. (A) HEK293 cells were treated with rapamycin for 1, 24, and 48 h or ethanol for 48 h and were analyzed for the binding of 4E-BP1 to the cap complex. (B) Rapamycin or ethanol was readded to the 24-h-treated sample 1 h before lysis, and it was analyzed for 4E-BP1 phosphorylation. (C) Lysates from rapamycin-treated samples were blotted for the phosphorylation status of 4E-BP1. The sites analyzed were Ser-65, Thr-70, Thr-37/46, and total 4E-BP1. Gel shifts can be observed in samples treated with rapamycin for 24 or 48 h. The α-β-γ isoforms represent the phosphorylation status of 4E-BP1 with α being hypophosphorylated and γ being hyperphosphorylated. (D) The kinetics of rapamycin-induced 4E-BP1 was measured in HEK293 and treated according to the time listed. 4E-BP1 binding to the m7GTP Sepharose and gel shifts on lysates are shown.

Fig. 3.

Rapamycin-induced 4E-BP1 phosphorylation requires rap-resistant mTORC1 but is insensitive to inhibitors of the PI3K and MEK-ERK pathways. (A) The conformation of the mTORC1 complex was determined with mTOR coimmunoprecipitation experiments. The association of Raptor to mTOR is shown. (B) siRNAs against mTOR and Raptor were transfected into HEK293 cells and incubated for 24 h. Thereafter, the cells were treated with rapamycin for 24 more hours or 1 h before lysis and were analyzed. C, Scrambled control siRNA; R, siRNA against Raptor; T, siRNA against mTOR. (C) WT, TOS motif mutant (F114A), and 37/46 AA 4E-BP1s were transfected (3 μg) into a 10-cm plate of HEK293 cells, treated with rapamycin for 24 h, and analyzed for 4E-BP1 phosphorylation. (D) TSC2^−/− MEFs were treated with control (DMSO) or rapamycin (20 nM) for either 1 or 24 h. In the 24 h rapamycin-treated samples, either control, rapamycin (20 nM), Wortmannin (50 and 500 nM), or PI-103 (1 μM) was added for 1 h before lysis. The samples were analyzed for 4E-BP1 phosphorylation.

Fig. 4.

Rapamycin-induced 4E-BP1 phosphorylation stimulates cap-dependent translation and regulates cell-specific inhibition of translation. (A) The interaction between eIF4E and eIF4G was measured in coimmunoprecipitation experiments in HEK293 cells treated with rapamycin for 1 or 24 h. (B) In vivo cap-dependent translational assays were performed except rapamycin was added 3 h after transfection for 24, 48, or 72 h. (C) Phospho-S6 and actin were blotted for normalization. (D) The experiments were completed in the same way as in C, except WT 4E-BP1, AA 4E-BP1, or a control vector was cotransfected. Phospho-S6, actin, and HA (4E-BP1) blots are shown. Black, control vector; white, WT 4E-BP1; gray, AA 4E-BP1. (E) Different cell lines that differentially exhibit rapamycin-induced 4E-BP1 hyperphosphorylation were transfected with the HIF-1α 5′ UTR-driven translational vector and were treated with rapamycin or ethanol 3 h after transfection for 72 h. Black, control; gray, rapamycin (20 nM). The 4E-BP1 and S6(P) Western blots are shown in conjunction with the translational data.