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 rapamycinresistant 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.cap-dependent translation ͉ mTORC1 ͉ rapamycin resistance T he mammalian translational initiation machinery governs the recruitment of ribosomes to mRNA to commence the production of protein synthesis. This machinery consists of various eukaryotic initiation factors (eIFs) that tightly regulate protein synthesis based on environmental cues. Importantly, initiation is an important step for cellular control because it is the ratelimiting step of translation (1).Two predominant pathways translate mammalian mRNA through cap-dependent and independent mechanisms. The capping of the 5Ј end of mRNA by m 7 GTP allows the recruitment of the eIF4F complex, eIF3, and the 40S ribosomal subunit to the 5Ј mRNA cap. Capindependent translation is mediated by an internal RNA structure called internal ribosome entry site (IRES), which recruits the ribosome independent of both the cap and the entire eIF4F complex (2).The initiation of cap-dependent translation is tightly regulated by extracellular conditions including glucose, nutrient, and growth factor levels. These factors control cap-dependent translation by regulating the evolutionarily conserved mTORC1 (mTOR, Raptor, mLST8) pathway (3). Activation of mTORC1 positively stimulates mRNA translation via its downstream substrates S6Ks and 4E-BP1/ eIF4E (4-7). Phosphorylation of 4E-BP1 by mTORC1 results in its dissociation from eIF4E, promoting assembly of the eIF4F complex. It is thought that S6K1 can phosphorylate translational regulators such as eIF4B and PDCD4 to enhance the translational efficiency of mRNAs with highly structured 5Ј UTRs (8-10).Therefore, growth factors positively regulate cap-dependent translation via mTORC...

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis

“…However, rapamycin was unable to inhibit Rheb-induced phosphorylation of the mTORC1 downstream target 4EBP1. This was consistent with recent findings indicating that 4EBP1 phosphorylation may become rapamycin resistant in some cell types and under certain experimental conditions (Choo et al, 2008;Thoreen et al, 2009). Therefore, to confirm that Rheb can regulate AMPK activity independently of mTORC1, we used Torin1, an ATP-competitive mTOR inhibitor, which effectively inhibits mTORC1 activity (Thoreen et al, 2009).…”

Combination of dual mTORC1/2 inhibition and immune-checkpoint blockade potentiates anti-tumour immunity

“…Phosphorylation of the alternative mTORC1 target 4Ebp1 (Thr36/45) was previously shown to be less sensitive to rapamycin-mediated inhibition compared to pS6 (Ser240/244). 16 Comparably vistusertib was capable of inhibiting p4Ebp1 (Thr36/45) to a greater extent than rapamycin (Fig. S1C).…”

The Effect of L-Ornithine on the Phosphorylation of mTORC1 Downstream Targets in Rat Liver

“…Rapamycin influenced the phosphorylation state of mTORC1 downstream factors; however, the extent of inhibition was different. Previous studies have shown that S6 activity is completely inhibited by rapamycin, but 4EBP1 is only partially inhibited depending on the cell type or duration of rapamycin treatment (23). Thus the restricted inhibition of 4EBP1 shown in the present study was due to the potential of rapamycin.…”