Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis.

Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes, including growth, proliferation, and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.

Sense and sensibility: nutritional response and signal integration in yeast.

Yeast cells respond to the quantity and quality of carbon and nitrogen sources in the environment both by adjusting their transcriptional and metabolic profiles to make optimum use of the available nutrients and by selecting a developmental program--budding, pseudohyphal differentiation, quiescence or sporulation--that maximizes their potential for survival under the existing nutrient conditions. Recent studies fueled by genomic tools have refined our knowledge of the components and connections within individual pathways and the interconnections between pathways. More significantly, these studies begin to paint an as yet inchoate portrait of the yeast cells' means of processing its environmental information, in which specific transcription factors and chromatin modifying activities coordinate input from several signaling pathways to yield an appropriate and coherent response of genes involved in mass accumulation and metabolism.

Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway.

BACKGROUND: The signaling pathways imposing hormonal control over adipocyte differentiation are poorly understood. While insulin and Akt signaling have been found previously to be essential for adipogenesis, the relative importance of their many downstream branches have not been defined. One direct substrate that is inhibited by Akt-mediated phosphorylation is the tuberous sclerosis complex 2 (TSC2) protein, which associates with TSC1 and acts as a critical negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Loss of function of the TSC1-TSC2 complex results in constitutive mTORC1 signaling and, through mTORC1-dependent feedback mechanisms and loss of mTORC2 activity, leads to a concomitant block of Akt signaling to its other downstream targets. METHODOLOGY/PRINCIPAL FINDINGS: We find that, despite severe insulin resistance and the absence of Akt signaling, TSC2-deficient mouse embryo fibroblasts and 3T3-L1 pre-adipocytes display enhanced adipocyte differentiation that is dependent on the elevated mTORC1 activity in these cells. Activation of mTORC1 causes a robust increase in the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master transcriptional regulator of adipocyte differentiation. In examining the requirements for different Akt-mediated phosphorylation sites on TSC2, we find that only TSC2 mutants lacking all five previously identified Akt sites fully block insulin-stimulated mTORC1 signaling in reconstituted Tsc2 null cells, and this mutant also inhibits adipogenesis. Finally, renal angiomyolipomas from patients with tuberous sclerosis complex contain both adipose and smooth muscle-like components with activated mTORC1 signaling and elevated PPARgamma expression. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that activation of mTORC1 signaling is a critical step in adipocyte differentiation and identifies TSC2 as a primary target of Akt driving this process. Therefore, the TSC1-TSC2 complex regulates the differentiation of mesenchymal cell lineages, at least in part, through its control of mTORC1 activity and PPARgamma expression.

Polyadenylation of rRNA- and tRNA-based yeast transcripts cleaved by internal ribozyme activity.

Polyadenylation, an important step in 3' end-processing of mRNA in eukaryotes, results in a poly(A) tail that ensures RNA transport into the cytoplasm and subsequent translation. Addition of a poly(A) tail is restricted to transcripts that are synthesized by RNA polymerase II. Here, we demonstrate that the 3' ends of yeast transcripts based on rRNA and tRNA, respectively, can be polyadenylated in vivo. The transcripts were modified by insertion of a self-cleaving hammerhead ribozyme sequence in the corresponding gene. Both the rDNA-based transcript and the tRNA transcript were cleaved efficiently by the hammerhead ribozyme, resulting in two stable cleavage products. The 5' cleavage product was found to be polyadenylated in both cases. This demonstrates that, in yeast, transcripts that are usually synthesized by RNA polymerase I or III can be polyadenylated if the 3' end of the transcript has been generated independently by a ribozyme.

Multiple factors prevent transcriptional interference at the yeast ARO4-HIS7 locus.

Increased transcriptional activity may cause transcriptional interference in organisms with compact genomes such as the yeast Saccharomyces cerevisiae. Replacement of the yeast ARO4 promoter by the stronger ACT1 promoter increases ARO4 transcription and simultaneously reduces the basal transcription of the downstream HIS7 gene. The open reading frames of ARO4 and HIS7 are tandemly transcribed and are separated by 416 bp. In wild-type cells, a nuclease-resistant site suggests that the two genes are separated by a single positioned nucleosome. Transcriptional interference correlates with Micrococcus nuclease accessibility of this otherwise nuclease-resistant site. Deletion analyses of the region between the two open reading frames revealed that transcriptional interference increases upon removal of either parts of the ARO4 3' end or HIS7 promoter sequences. The abolishment of the Abf1p-binding site within the HIS7 promoter significantly enhances transcriptional interference, resulting in a histidine auxotrophic strain. Our data suggest that the yeast cell prevents transcriptional interference by the combined action of efficient ARO4 transcription termination, the positioning of a fixed nucleosome, and transcription factor binding to the HIS7 promoter.

PP2A phosphatase activity is required for stress and Tor kinase regulation of yeast stress response factor Msn2p.

In response to stress and nutrient starvation, the Saccharomyces cerevisiae transcription factor Msn2p accumulates in the nucleus and activates expression of a broad array of genes. Here, we analyze the role of the Tor (target of rapamycin) signaling pathway in mediating these responses. Inactivation of the Tor pathway component Tap42p using tap42(Ts) alleles causes a sustained nuclear localization similar to that after the addition of the Tor kinase inhibitor rapamycin. Effects of Tap42p inactivation and rapamycin addition could be suppressed by deletion of TIP41, which encodes a Tap42p-interacting protein. These results support the notion that rapamycin affects Msn2p by inactivating Tap42p function. Tap42p interacts with the catalytic subunit of PP2A (protein phosphatase 2A) and PP2A-like phosphatases. Deletion of either the catalytic or regulatory subunit that forms the PP2A phosphatase complex prevents nuclear accumulation of Msn2p in the tap42(Ts) strain and in wild-type strains treated with rapamycin. These results suggest that Tap42p is an inhibitor of PP2A phosphatase, which in turn inhibits nuclear export of Msn2p. Interestingly, PP2A function is also required for nuclear accumulation of Msn2p in response to stresses, such as heat and osmotic shock, as well as nitrogen (but not glucose) starvation. Thus, PP2A and the Tor kinase pathway transduce stress and nitrogen starvation signals to Msn2p. Finally, Msn2p localization is unaffected by conditional loss of 14-3-3 protein function, ruling out the possibility that 14-3-3 proteins act as a scaffold to sequester Msn2p in the cytoplasm.

Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast.

Tor proteins, targets of the antiinflammatory drug rapamycin, mediate a conserved signaling pathway required for cell growth and proliferation in eukaryotes. By global transcriptional analysis of Saccharomyces cerevisiae, we have examined the role of the essential protein Tap42 in transcriptional regulation by Tor. We find that Tap42 inactivation, like rapamycin addition, prolongs activation of stress response genes. In contrast, Tap42 inactivation, as does inactivation of the protein phosphatases Sit4 and Pph21/22, blocks rapamycin induction of nitrogen discrimination pathway genes. Tap42 inactivation neither affects ribosomal protein gene expression nor blocks rapamycin-induced repression of these genes. These results indicate that Tap42 can both inhibit and activate protein phosphatases and provide insight into the complex events underlying TOR regulation of transcription.

Replacement of the yeast TRP4 3' untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail.

The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 3' untranslated region (3' UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 3' UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.