The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogen and nutrient signals to control cell proliferation and cell size. Hence, mTORC1 is implicated in a large number of human diseases--including diabetes, obesity, heart disease, and cancer--that are characterized by aberrant cell growth and proliferation. Although eukaryotic translation initiation factor 4E-binding proteins (4E-BPs) are critical mediators of mTORC1 function, their precise contribution to mTORC1 signaling and the mechanisms by which they mediate mTORC1 function have remained unclear. We inhibited the mTORC1 pathway in cells lacking 4E-BPs and analyzed the effects on cell size, cell proliferation, and cell cycle progression. Although the 4E-BPs had no effect on cell size, they inhibited cell proliferation by selectively inhibiting the translation of messenger RNAs that encode proliferation-promoting proteins and proteins involved in cell cycle progression. Thus, control of cell size and cell cycle progression appear to be independent in mammalian cells, whereas in lower eukaryotes, 4E-BPs influence both cell growth and proliferation.The mammalian target of rapamycin complex 1 (mTORC1) controls growth (increase in cell mass) and proliferation (increase in cell number) by modulating mRNA translation through phosphorylation of the eukaryotic translation initiation factor 4E (eIF4E)-binding proteins (4E-BP1, 2, and 3) and the ribosomal protein S6 kinases (S6K1 and 2) (1,2). 4E-BPs regulate the translation of a subset of mRNAs by competing with eIF4G for binding to eIF4E, thus preventing the assembly of the eIF4F complex, whereas the S6Ks control the phosphorylation status of a number of translational components (1-3). Rapamycin has been an important tool in understanding mTORC1 signaling; however, it inefficiently and transiently inhibits 4E-BP phosphorylation (4)( fig. S1A). Moreover, we found that rapamycin inhibited proliferation and G1/S cell cycle progression of WT and 4E-BP double knock-out (DKO) mouse embryonic fibroblasts (MEFs) to the same extent, which suggests that its effects are not mediated by 4E-BPs ( fig. S1, B to D). To directly address the role of 4E-BPs in mTORC1 signaling, we depleted raptor, a component of mTORC1 required for substrate binding (5), in these MEFs. 4E-BP DKO MEFs lack all three 4E-BPs as they do not express 4E-BP3 ( fig. S2A) 1A). Wild-type MEFs in which raptor was depleted proliferated more slowly than control cells, whereas raptor-depleted 4E-BP DKO MEFs proliferated at a rate indistinguishable from that of control cells (Fig. 1B). Similarly, in human embryonic kidney (HEK) 293T cells, raptor silencing had a pronounced effect on mTORC1 signaling and proliferation (Figs. 1C and D). The effect of raptor silencing on proliferation, but not mTOR signaling, was attenuated by codepletion of 4E-BPs (Fig. 1D). Thus, mTORC1-dependent proliferation requires 4E-BPs.To further assess the role of 4E-BPs in mTORC1-mediated cell proliferation, we depleted mTOR or rictor (an mTORC2 specific component), i...

Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LARP1 is a target of mTORC1 that associates with TOP mRNAs via their 5ЈTOP motif to repress their translation. Conclusion: LARP1 represses TOP mRNA translation downstream of mTORC1. Significance: We elucidate an important novel signaling pathway downstream of mTORC1 that controls the production of ribosomes and translation factors in eukaryotic cells.

No abstract

BackgroundMammalian target of rapamycin complex 1 (mTORC1) is a protein kinase that relays nutrient availability signals to control numerous cellular functions including autophagy, a process of cellular self-eating activated by nutrient depletion. Addressing the therapeutic potential of modulating mTORC1 signaling and autophagy in human disease requires active chemicals with pharmacologically desirable properties.Methodology/Principal FindingsUsing an automated cell-based assay, we screened a collection of >3,500 chemicals and identified three approved drugs (perhexiline, niclosamide, amiodarone) and one pharmacological reagent (rottlerin) capable of rapidly increasing autophagosome content. Biochemical assays showed that the four compounds stimulate autophagy and inhibit mTORC1 signaling in cells maintained in nutrient-rich conditions. The compounds did not inhibit mTORC2, which also contains mTOR as a catalytic subunit, suggesting that they do not inhibit mTOR catalytic activity but rather inhibit signaling to mTORC1. mTORC1 inhibition and autophagosome accumulation induced by perhexiline, niclosamide or rottlerin were rapidly reversed upon drug withdrawal whereas amiodarone inhibited mTORC1 essentially irreversibly. TSC2, a negative regulator of mTORC1, was required for inhibition of mTORC1 signaling by rottlerin but not for mTORC1 inhibition by perhexiline, niclosamide and amiodarone. Transient exposure of immortalized mouse embryo fibroblasts to these drugs was not toxic in nutrient-rich conditions but led to rapid cell death by apoptosis in starvation conditions, by a mechanism determined in large part by the tuberous sclerosis complex protein TSC2, an upstream regulator of mTORC1. By contrast, transient exposure to the mTORC1 inhibitor rapamycin caused essentially irreversible mTORC1 inhibition, sustained inhibition of cell growth and no selective cell killing in starvation.Conclusion/SignificanceThe observation that drugs already approved for human use can reversibly inhibit mTORC1 and stimulate autophagy should greatly facilitate the preclinical and clinical testing of mTORC1 inhibition for indications such as tuberous sclerosis, diabetes, cardiovascular disease and cancer.