The evolutionarily conserved Ser-Thr kinase mTOR plays a critical role in regulating many pathophysiological processes. Functional characterization of the mTOR signaling pathways, however, has been hampered by the paucity of known substrates. We used large-scale quantitative phospho-proteomics experiments to define the signaling networks downstream of mTORC1 and mTORC2. Characterization of one mTORC1 substrate, the growth factor receptor-bound protein 10 (Grb10), showed that mTORC1-mediated phosphorylation stabilized Grb10, leading to feedback inhibition of the phosphatidylinositol-3-kinase (PI3K) and extracellular signal-regulated, mitogen-activated protein kinase (ERK-MAPK) pathways. Grb10 expression is frequently downregulated in various cancers, and loss of Grb10 and loss of the well-established tumor suppressor phosphatase PTEN appear to be mutually exclusive events, suggesting that Grb10 might be a tumor suppressor regulated by mTORC1.

SUMMARY Messenger RNAs (mRNAs) can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of untranslated regions, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

The mammalian target of rapamycin (mTOR), a critical modulator of cell growth, acts to integrate signals from hormones, nutrients, and growth-promoting stimuli to downstream effector mechanisms involved in the regulation of protein synthesis. Dexamethasone, a synthetic glucocorticoid that represses protein synthesis, acts to inhibit mTOR signaling as assessed by reduced phosphorylation of the downstream targets S6K1 and 4E-BP1. Dexamethasone has also been shown in one study to up-regulate the expression of REDD1 (also referred to RTP801, a novel stress-induced gene linked to repression of mTOR signaling) in lymphoid, but not nonlymphoid, cells. In contrast to the findings of that study, here we demonstrate that REDD1, but not REDD2, mRNA expression is dramatically induced following acute dexamethasone treatment both in rat skeletal muscle in vivo and in L6 myoblasts in culture. In L6 myoblasts, the effect of the drug on mTOR signaling is efficiently blunted in the presence of REDD1 RNA interference oligonucleotides. Moreover, the dexamethasone-induced assembly of the mTOR regulatory complex Tuberin⅐Hamartin is disrupted in L6 myoblasts following small interfering RNA-mediated repression of REDD1 expression. Finally, overexpression of Rheb, a downstream target of Tuberin function and a positive upstream effector of mTOR, reverses the effect of dexamethasone on phosphorylation of mTOR substrates. Overall, the data support the conclusion that REDD1 functions upstream of Tuberin and Rheb to down-regulate mTOR signaling in response to dexamethasone.In contrast to the anabolic actions of growth-promoting hormones such as insulin and insulin-like growth factor 1, glucocorticoids act to repress protein synthesis in skeletal muscle of animals in vivo (1), in perfused hind limb preparations (2, 3), and in isolated muscle preparations (4, 5). In part, the repression of protein synthesis in response to glucocorticoids is a result of decreased signaling through a protein kinase referred to as the mammalian target of rapamycin (mTOR).2 mTOR phosphorylates at least two proteins involved in the regulation of mRNA translation, the eukaryotic initiation factor (eIF)-4E-binding protein 1 (4E-BP1) and the ribosomal protein S6 kinase 1 (S6K1) (6). 4E-BP1 acts to repress mRNA translation by sequestering the mRNA cap-binding protein eIF4E into an inactive complex (7,8). Phosphorylation of 4E-BP1 by mTOR initiates a series of phosphorylation events that ultimately result in the release of eIF4E from the inactive 4E-BP1⅐eIF4E complex, allowing it to bind to eIF4G to form the active eIF4F complex. Phosphorylation of S6K1 by mTOR generates a docking site for a second protein kinase, phosphoinositide-dependent protein kinase 1 (PDK1), allowing PDK1 to phosphorylate and activate S6K1 (9, 10). Subsequently, S6K1 phosphorylates ribosomal protein rpS6 (11), eIF4B (12, 13), and eukaryotic elongation factor (eEF2) kinase (14). Thus, repression of mTOR signaling results in a reduction in both the initiation and elongation phases of mRNA translati...

The contribution of mammalian target of rapamycin (mTOR) signaling to the resistance exercise-induced stimulation of skeletal muscle protein synthesis was assessed by administering rapamycin to Sprague-Dawley rats 2 h prior to a bout of resistance exercise. Animals were sacrificed 16 h postexercise, and gastrocnemius protein synthesis, mTOR signaling, and biomarkers of translation initiation were assessed. Exercise stimulated the rate of protein synthesis; however, this effect was prevented by pretreatment with rapamycin. The stimulation of protein synthesis was mediated by an increase in translation initiation, since exercise caused an increase in polysome aggregation that was abrogated by rapamycin administration. Taken together, the data suggest that the effect of rapamycin was not mediated by reduced phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein 1 (BP1), because exercise did not cause a significant change in 4E-BP1(Thr-70) phosphorylation, 4E-BP1-eIF4E association, or eIF4F complex assembly concomitant with increased protein synthetic rates. Alternatively, there was a rapamycinsensitive decrease in relative eIF2B⑀(Ser-535) phosphorylation that was explained by a significant increase in the expression of eIF2B⑀ protein. The proportion of eIF2B⑀ mRNA in polysomes was increased following exercise, an effect that was prevented by rapamycin treatment, suggesting that the increase in eIF2B⑀ protein expression was mediated by an mTOR-dependent increase in translation of the mRNA encoding the protein. The increase in eIF2B⑀ mRNA translation and protein abundance occurred independent of similar changes in other eIF2B subunits. These data suggest a novel link between mTOR signaling and eIF2B⑀ mRNA translation that could contribute to the stimulation of protein synthesis following acute resistance exercise.