Upstream and downstream of mTOR

Nissim Hay 1 , 3 and Nahum Sonenberg 2 , 4 1Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA; 2Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6

Next Section Abstract The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the last decade clearly demonstrates that TOR controls protein synthesis through a stunning number of downstream targets. Some of the targets are phosphorylated directly by TOR, but many are phosphorylated indirectly. In this review, we summarize some recent developments in this fast-evolving field. We describe both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis. We also summarize the roles of mTOR in the control of cell growth and proliferation, as well as its relevance to cancer and synaptic plasticity. Keywords Akt

TSC1/TSC2

Rheb

4E-BP

S6K

eIF4E

Previous Section Next Section mTOR targets involved in transcription Consistent with the critical role of mTOR in cell growth via the modulation of protein synthesis in yeast and mammals, it and its yeast homologs strongly stimulate transcription from all genes involved in ribosome biogenesis, transcription of rRNA genes by RNA polymerase I (Pol I), transcription of ribosomal protein genes by RNA polymerase II (Pol II), and transcription of tRNA and 5S genes by RNA polymerase III (Pol III; Mahajan 1994; Zaragoza et al. 1998; Powers and Walter 1999; Hannan et al. 2003). Recently, several studies identified two mammalian Pol I-specific transcription factors, TIF1A and USB, whose activity is modulated by rapamycin. Mayer et al. (2004) demonstrated that TIF-IA (the homolog of yeast Rrn3, an essential RNA PolI transcription factor [Claypool et al. 2004]) is sufficient to rescue rapamycin-mediated inhibition of rDNA transcription. Also, in yeast the TOR pathway regulates Rrn3p-dependent recruitment of yeast Pol I to its promoter (Claypool et al. 2004). Thus, at least some of the downstream effectors of mTOR that regulate rDNA transcription appear to be conserved in evolution. However, Hannan and colleagues could not demonstrate that TIF-IA is regulated by mTOR (Hannan et al. 2003). Instead, they demonstrated that the rDNA transcription factor UBF (upstream binding factor) is responsible for the stimulation of rDNA transcription by mTOR, which is dependent on S6K activity. Treatment with rapamycin inhibits the phosphorylation of UBF in its C-terminal region, and this phosphorylation is required for the activity of UBF. Interestingly, UBF does not appear to be a direct substrate for S6K1, implying the existence of a novel kinase upstream of UBF.

Previous Section Next Section mTOR translational control, cell growth, and proliferation As introduced above, under most circumstances, the rate-limiting step in mammalian translation initiation is the binding of the ribosome to mRNA. Strikingly, almost all of the factors involved in recruiting the ribosome, including eIF4E, eIF4B, and eIF4G, are phosphoproteins whose phosphorylation states are directly proportional to the translation and growth rates of the cell. In addition, the repressor proteins, 4E-BPs, are similarly phosphorylated under the same circumstances. Thus, increased phosphorylation of these factors in response to numerous extracellular stimuli correlates with increased translation of a subset of mRNAs (see below) and accelerated growth and proliferation (for review, see Raught et al. 2000b; Gingras et al. 2001a). It is striking that the mTOR pathway mediates the phosphorylation of all of these factors, except for eIF4E. How does phosphorylation of these factors affect translation, and consequently, cell growth? An attractive hypothesis is based on the finding that eIF4E is limiting in the cell (Duncan et al. 1987), which might underlie the finding that ribosome binding is the rate-limiting step during translation initiation (Mathews et al. 2000). Because eIF4E is part of the eIF4F complex, it stands to reason that an increase in any of the components of eIF4F would enhance translation initiation rates. Inasmuch as the eIF4F complex functions to recognize the mRNA 5′ cap and unwind the mRNA 5′ secondary structure, it has been postulated that the translation of mRNAs containing extensive secondary structure would be preferably stimulated by increased eIF4E activity (Sonenberg 1993). eIF4E overexpression in cells enabled efficient translation of a reporter mRNA in which more secondary structure had been inserted in the mRNA 5′ UTR (Koromilas et al. 1992). Subsequently, several groups identified mRNAs whose translation was preferentially stimulated in eIF4E-overexpressing NIH-3T3 cells as well as other cell lines. These mRNAs include, among others, ODC (ornithine decarboxylase), FGF (fibroblast growth factor), and VEGF (vascular endothelial growth factor). Two common features of these mRNAs are (1) a relatively long and structured 5′ UTR, and (2) most importantly, their protein products function in controlling cell growth and proliferation. Hence, the translational activation of these mRNAs is expected to promote cell growth and proliferation. ODC has been studied in some detail, as it is a model par excellence for studying translational control by eIF4E. It contains a G/C-rich 5′ UTR of ∼300 nt and is not well translated in vivo or in vitro. In response to insulin stimulation, which activates eIF4E, its translation increases by ∼30-fold (Manzella et al. 1991). Consistent with these findings, the translation of ODC in eIF4E-overexpressing NIH-3T3 cells is also increased by ∼30-fold (Shantz and Pegg 1994). Experimentally induced elevation in the levels of other components of eIF4F and eIF4B would be expected to elicit similar effects. Several studies have directly measured and documented the effects of eIF4E on cell growth and proliferation. One study showed that eIF4E overexpression increases cell size, and that eIF4E and S6K cooperate downstream of TOR to control cell size (Fingar et al. 2002). A subsequent study (Fingar et al. 2004) reported that eIF4E and S6K also promote cell cycle progression. This is not surprising, because cell growth and cell division are generally tightly coupled in yeast as well as in mammals, under most circumstances. These results are also consistent with the earlier finding that eIF4E overexpression in NIH-3T3 cells promotes malignant transformation (see below), which requires an increase in both growth and proliferation. Thus, the S6K and eIF4E/4E-BP pathways promote proliferation by coupling cell growth with cell cycle progression.

Previous Section Next Section Concluding remarks and perspectives The mTOR pathway is emerging as a critical player in the etiology of cancer and metabolic diseases, including diabetes and obesity. The recent breathtaking advances in the understanding of the upstream and downstream targets of mTOR provide rational explanations for the origins and progression of these diseases. For example, insulin is a major upstream effector of mTOR that increases protein synthesis as part of the modulation of anabolic processes in response to glucose. Thus, deficiencies in mTOR signaling might play a role in the development of glucose- and insulin-resistant type II diabetes (Pende et al. 2000). As discussed above, a link between the mTOR pathway and cancer is also clearly evident, as most of the upstream and downstream components of mTOR are directly implicated in cancer initiation and progression. The enhanced understanding of the mTOR signaling pathway should lead to the design of drugs to treat diabetes and cancer. The success of rapamycin in clinical trials for cancer, restenosis in heart valves (Marks 2003), and arthritis (Forre et al. 2000) highlights the multitude of diseases whose origins stem from aberrant proliferation and that are linked to mTOR. Other drugs that act on other components of the pathway are also sought. Studies are in progress to develop drugs that inhibit upstream mTOR effectors such as Akt/PKB or downstream targets such as eIF4E. Several important details related to the regulation of mTOR activity remain unresolved. In particular, the mechanism by which Rheb activates mTOR is still elusive, and future studies will be likely directed toward resolving this link. In addition, as discussed earlier, there needs to be clarification on the interplay between the regulation of mTOR activity by growth factors and by nutrients. Another important and unresolved question concerns the identity of the downstream target(s) of S6K, which activates the translation of TOP mRNAs to stimulate cell growth. As described above, eIF4B could be a candidate (Raught et al. 2004), although other as yet undiscovered proteins could also play a role (e.g., see Fingar et al. 2004). An important avenue for future studies is the understanding of the cross-talk between the PI3K-Akt/PKB-mTOR signaling pathway and the signaling pathway leading to the activation of ERK. It is clear that both pathways cooperate to effect many cellular functions. These interactions have critical consequences for the control of cell growth, memory, and learning. These two signaling pathways activate key components of the translational machinery involved in recruiting ribosomes to mRNA. The ERK pathway is responsible for phosphorylating eIF4E (Waskiewicz et al. 1997; Pyronnet et al. 1999; Radimerski et al. 2002), a modification that is thought to increase its activity; whereas, as described above, the mTOR pathway phosphorylates 4E-BPs, which, in turn, stimulate eIF4E activity and enhance ribosome recruitment. Recent experiments show that the ERK and mTOR pathways cooperate to stimulate translation and induce glioblastomas in a mouse model (Rajasekhar et al. 2003). As both pathways become activated in neurons in response to experience, they likely cooperate to promote new protein synthesis required for learning and memory.

Previous Section Next Section Acknowledgments We thank Davide Ruggero and So Young Kim for helpful comments on this review. Work in the authors' laboratories was supported by grants from the NIH (N.H. and N.S.) and the National institute of Canada, Canadian Institute of Health Research (CIHR), and Howard Hughes Medical Institute (HHMI; N.S.). N.S. is a CIHR Distinguished Scientist and an HHMI International Scholar.