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...

Summary Hyperactivation of Ras-ERK1/2 signaling is critical to the development of many human malignancies but little is known regarding the specific contribution of ERK1 or ERK2 to oncogenic processes. We demonstrate that ERK2 but not ERK1 signaling is necessary for Ras-induced epithelial-to-mesenchymal transformation (EMT). Further, ERK2 but not ERK1 overexpression is sufficient to induce EMT. Many ERK1/2 interacting proteins contain amino acid motifs, e.g. DEF or D-motifs, which regulate docking with ERK1/2. Remarkably, ERK2 signaling to DEF motif-containing targets is required to induce EMT and correlates with increased migration, invasion and survival. Importantly, the late response gene product Fra1 is necessary for Ras- and ERK2-induced EMT through up-regulation of ZEB1/2 proteins. Thus, an apparent critical role for ERK2 DEF motif signaling during tumorigenesis is the regulation of Fra1, and the subsequent induction of ZEB1/2, suggesting a potential therapeutic target for Ras-regulated tumorigenesis.

We report that the activity of glycogen synthase kinase-3 (GSK-3) is necessary for the maintenance of the epithelial architecture. Pharmacological inhibition of its activity or reducing its expression using small interfering RNAs in normal breast and skin epithelial cells results in a reduction of E-cadherin expression and a more mesenchymal morphology, both of which are features associated with an epithelial–mesenchymal transition (EMT). Importantly, GSK-3 inhibition also stimulates the transcription of Snail, a repressor of E-cadherin and an inducer of the EMT. We identify NFκB as a transcription factor inhibited by GSK-3 in epithelial cells that is relevant for Snail expression. These findings indicate that epithelial cells must sustain activation of a specific kinase to impede a mesenchymal transition.

Summary The mTORC1 signaling pathway integrates environmental conditions into distinct signals for cell growth by balancing anabolic and catabolic processes. Accordingly, energetic stress inhibits mTORC1 signaling predominantly through AMPK-dependent activation of TSC1/2. Thus, TSC1/2-/- cells are hypersensitive to glucose deprivation and this has been linked to increased p53 translation and activation of apoptosis. Herein, we show that mTORC1 inhibition during glucose deprivation prevented not only the execution of death, but also induction of energetic stress. mTORC1 inhibition during glucose deprivation decreased AMPK activation and allowed ATP to remain high, which was both necessary and sufficient for protection. This effect was not due to increased catabolic activities such as autophagy, but rather exclusively due to decreased anabolic processes, reducing energy consumption. Specifically, TSC1/2-/- cells become highly dependent on glutamate dehydrogenase-dependent glutamine metabolism via the TCA cycle for survival. Therefore, mTORC1 inhibition during energetic stress is primarily to balance metabolic demand with supply.