Timothy R. Peterson

The Rag GTPases Bind Raptor and Mediate Amino Acid Signaling to mTORC1

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins—a family of four related small guanosine triphosphatases (GTPases)—interact with mTORC1 in an amino acid–sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate–bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.

Regulation of the mTOR Complex 1 Pathway by Nutrients, Growth Factors, and Stress

The large serine/threonine protein kinase mTOR regulates cellular and organismal homeostasis by coordinating anabolic and catabolic processes with nutrient, energy, and oxygen availability and growth factor signaling. Cells and organisms experience a wide variety of insults that perturb the homeostatic systems governed by mTOR and therefore require appropriate stress responses to allow cells to continue to function. Stress can manifest from an excess or lack of upstream signals or as a result of genetic perturbations in upstream effectors of the pathway. mTOR nucleates two large protein complexes that are important nodes in the pathways that help buffer cells from stresses, and are implicated in the progression of stress-associated phenotypes and diseases, such as aging, tumorigenesis, and diabetes. This review focuses on the key components of the mTOR complex 1 pathway and on how various stresses impinge upon them.

The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling

A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

mTOR Complex 1 Regulates Lipin 1 Localization to Control the SREBP Pathway

The nutrient- and growth factor-responsive kinase mTOR complex 1 (mTORC1) regulates many processes that control growth, including protein synthesis, autophagy, and lipogenesis. Through unknown mechanisms, mTORC1 promotes the function of SREBP, a master regulator of lipo- and sterolgenic gene transcription. Here, we demonstrate that mTORC1 regulates SREBP by controlling the nuclear entry of lipin 1, a phosphatidic acid phosphatase. Dephosphorylated, nuclear, catalytically active lipin 1 promotes nuclear remodeling and mediates the effects of mTORC1 on SREBP target gene, SREBP promoter activity, and nuclear SREBP protein abundance. Inhibition of mTORC1 in the liver significantly impairs SREBP function and makes mice resistant, in a lipin 1-dependent fashion, to the hepatic steatosis and hypercholesterolemia induced by a high-fat and -cholesterol diet. These findings establish lipin 1 as a key component of the mTORC1-SREBP pathway.

mTORC1 controls fasting-induced ketogenesis and its modulation by ageing

The multi-component mechanistic target of rapamycin complex 1 (mTORC1) kinase is the central node of a mammalian pathway that coordinates cell growth with the availability of nutrients, energy and growth factors. Progress has been made in the identification of mTORC1 pathway components and in understanding their functions in cells, but there is relatively little known about the role of the pathway in vivo. Specifically, we have little knowledge regarding the role mTOCR1 has in liver physiology. In fasted animals, the liver performs numerous functions that maintain whole-body homeostasis, including the production of ketone bodies for peripheral tissues to use as energy sources. Here we show that mTORC1 controls ketogenesis in mice in response to fasting. We find that liver-specific loss of TSC1 (tuberous sclerosis 1), an mTORC1 inhibitor, leads to a fasting-resistant increase in liver size, and to a pronounced defect in ketone body production and ketogenic gene expression on fasting. The loss of raptor (regulatory associated protein of mTOR, complex 1) an essential mTORC1 component, has the opposite effects. In addition, we find that the inhibition of mTORC1 is required for the fasting-induced activation of PPARα (peroxisome proliferator activated receptor α), the master transcriptional activator of ketogenic genes, and that suppression of NCoR1 (nuclear receptor co-repressor 1), a co-repressor of PPARα, reactivates ketogenesis in cells and livers with hyperactive mTORC1 signalling. Like livers with activated mTORC1, livers from aged mice have a defect in ketogenesis, which correlates with an increase in mTORC1 signalling. Moreover, we show that the suppressive effects of mTORC1 activation and ageing on PPARα activity and ketone production are not additive, and that mTORC1 inhibition is sufficient to prevent the ageing-induced defect in ketogenesis. Thus, our findings reveal that mTORC1 is a key regulator of PPARα function and hepatic ketogenesis and suggest a role for mTORC1 activity in promoting the ageing of the liver.

ER stress inhibits mTORC2 and Akt signaling through GSK-3β-mediated phosphorylation of rictor

Cellular stress attenuates growth factor signaling through a phosphorylation event that blocks substrate access to the kinase complex mTORC2. No Access During Stressful Times Under conditions of cellular stress, cells tend to halt anabolic processes, such as cell growth and proliferation, to conserve resources. mTORC2 (mammalian target of rapamycin complex 2), which mediates its effects through activation of the kinase Akt, is a key signaling complex that promotes anabolic processes. Chen et al. investigated the mechanisms by which mTORC2 activity is inhibited by endoplasmic reticulum (ER) stress. They found that glycogen synthase kinase–3β (GSK-3β), which is activated by ER stress, phosphorylated rictor, a component of mTORC2 that helps to determine substrate specificity for the complex. This phosphorylation event decreased binding of Akt to mTORC2, resulting in reduced activation of Akt and cell proliferation. Furthermore, transformed cells expressing a mutant form of rictor lacking the GSK-3β phosphorylation site formed larger tumors in mice than did those expressing wild-type rictor or a rictor mutant that mimicked a constitutively phosphorylated form. These results define a pathway by which mTORC2 and Akt signaling can be attenuated by cellular stress and provide a potential therapeutic target for limiting cell proliferation (such as in cancer). In response to environmental cues, cells coordinate a balance between anabolic and catabolic pathways. In eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival through activation of the phosphoinositide 3-kinase (PI3K)–Akt pathway. Akt-mediated phosphorylation of glycogen synthase kinase–3β (GSK-3β) inhibits its enzymatic activity, thereby stimulating glycogen synthesis. We show that GSK-3β itself inhibits Akt by controlling the mammalian target of rapamycin complex 2 (mTORC2), a key activating kinase for Akt. We found that during cellular stress, GSK-3β phosphorylated the mTORC2 component rictor at serine-1235, a modification that interfered with the binding of Akt to mTORC2. The inhibitory effect of GSK-3β on mTORC2-Akt signaling and cell proliferation was eliminated by blocking phosphorylation of rictor at serine-1235. Thus, in response to cellular stress, GSK-3β restrains mTORC2-Akt signaling by specifically phosphorylating rictor, thereby balancing the activities of GSK-3β and Akt, two opposing players in glucose metabolism.

Rictor phosphorylation on the Thr-1135 site does not require mammalian target of rapamycin complex 2.

In animal cells, growth factors coordinate cell proliferation and survival by regulating the phosphoinositide 3-kinase/Akt signaling pathway. Deregulation of this signaling pathway is common in a variety of human cancers. The PI3K-dependent signaling kinase complex defined as mammalian target of rapamycin complex 2 (mTORC2) functions as a regulatory Ser-473 kinase of Akt. We find that activation of mTORC2 by growth factor signaling is linked to the specific phosphorylation of its component rictor on Thr-1135. The phosphorylation of this site is induced by the growth factor stimulation and expression of the oncogenic forms of ras or PI3K. Rictor phosphorylation is sensitive to the inhibition of PI3K, mTOR, or expression of integrin-linked kinase. The substitution of wild-type rictor with its specific phospho-mutants in rictor null mouse embryonic fibroblasts did not alter the growth factor–dependent phosphorylation of Akt, indicating that the rictor Thr-1135 phosphorylation is not critical in the regulation of the mTORC2 kinase activity. We found that this rictor phosphorylation takes place in the mTORC2-deficient cells, suggesting that this modification might play a role in the regulation of not only mTORC2 but also the mTORC2-independent function of rictor. Mol Cancer Res; 8(6); 896–906. ©2010 AACR.

Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing-bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here, we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

Identification and characterization of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates

Nitrogen-containing-bisphosphonates (N-BPs) are a class of drugs widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies have established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance.

Toward personalized calcium and vitamin D supplementation

Calcium and vitamin D (CaD) are essential components for building and maintaining strong bones throughout life. Although calcium is present in many food products and vitamin D can be synthesized in the skin by sunlight irradiation, for many people, especially elderly, frail, and institutionalized individuals, these sources are not sufficient to provide the necessary daily requirements. Chronic deficiency of CaD results in defective bone mineralization, secondary hyperparathyroidism, bone loss, and increased risk of falls and fractures (1). A beneficial effect of CaD supplementation on fracture risk has emerged from meta-analysis of large fracture trials (2, 3), and dietary CaD supplements and fortified food products are readily available and inexpensive. Hence, one would think that this aspect of bone health management is settled. Yet, it is hard to imagine any other topic in the bone metabolism field that has generated more animated and prolonged controversy. There is still disagreement on what constitutes vitamin D deficiency and on the recommended daily requirements of CaD, and whether calcium (not vitamin D) supplements increase cardiovascular disease risk is still hotly debated (4, 5). The heterogeneity of the populations studied in the different trials in terms of genetic and environmental risk factors might contribute to explain these inconsistencies. Therefore, a better understanding of who may most benefit from CaD supplementation would allow a more targeted approach, possibly resulting in improved outcomes. The study by Wang et al. (6), published in this issue of the Journal, represents a new step in this direction. The authors analyzed genome-wide association study data from the CaD trial in the Women’s Health Initiative database to determine genetic risk scores (GRSs) for fractures (Fx-GRS) and for bone mineral density (BMD-GRS) in relation to CaD intake. Their main finding was that there was no interaction between Fx-GRS and CaD on fracture risk; however, there was a significant multiplicative interaction between BMD-GRS and CaD assignment. Further analysis showed that the significant effects of CaD intake on fracture risk occurred only in women with the lowest genetic predisposition to low BMD. On first look, these results are somewhat counterintuitive, because in drug-effectiveness trials the higher the incidence (or prevalence) of the risk one tries to prevent, the higher the likelihood that the intervention may show an effect. For example, alendronate reduces the risk of hip fracture in women with osteoporosis, but not in those with higher bone density (7); likewise, statins have a greater effect in patients with higher cardiovascular disease risk (8). On more careful analysis, these results become clearer when considering the possibility that high-risk patients might not benefit from CaD supplementation if they have alterations in genetic factors that are involved in CaD responsiveness. The authors argue that possible variants in genes contributing to calcium, vitamin D, or other aspects of bone metabolism included in the genetic risk analysismight havemade subjects whowere at higher risk partially “resistant” to the effect of CaD. A corollary to this conclusion is that CaD is not a generalizable “one-size-fits-all” therapy for fracture risk prevention, but rather a risk modifier in genetically defined populations of postmenopausal women, hence supporting the rationale for personalized therapeutic interventions. In a way, the results of Wang et al. are reminiscent of the reality cable companies now face in the unbundling of their monolithic services. In increasingly more human population genetics studies, patient groups need to be “unbundled” to extract trends. To this end, it would be important to understand the pathobiological bases of CaD effects on low-risk subjects. One way of looking at the variants that confer low risk is that they confer hypersensitivity to CaD. In single nucleotide polymorphism (SNP) analysis, SNPs associated with 2 genes, dynamin 3 (DNM3) and osteoprotegerin (OPG/TNFRSF11B), were identified. Because osteoprotegerin is a major regulator of osteoclastogenesis, it should not be too surprising that OPG has emerged as a genetic factor for bone density and fracture risk (9, 10). However, if and how CaD regulates OPG remains to be determined. The role of DNM3 is intriguing considering that it has been linked to the regulation of the vitamin D receptor (11) and to skeletal development (12). Another interesting result is that the greatest risk reduction was seen in subjects taking not more than 1200 mg Ca. Although low power might explain the lack of effect of CaD in women taking higher amounts of supplemental calcium, this result supports the view that there are no extra benefits in taking calcium in excess of what is recommended for daily intake. There are some limitations to this study, as pointed out by the authors. First, most of the participants were not calciumand/or