Glucose addiction of TSC null cells is caused by failed mTORC1-dependent balancing of metabolic demand with supply.

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.

Down-regulation of CMTM8 induces epithelial-to-mesenchymal transition-like changes via c-MET/extracellular signal-regulated kinase (ERK) signaling.

The acquisition of an invasive phenotype is a critical turning point for malignant tumor cells. CMTM8, a potential tumor suppressor, is frequently down-regulated in solid tumors, and its overexpression induces tumor cell apoptosis. Here, we identify a new role for CMTM8 in regulating tumor cell migration. Reducing CMTM8 expression in HepG2 hepatocellular carcinoma cells results in the acquisition of epithelial-to-mesenchymal transition (EMT) features, including a morphological change from organized epithelial sheets to scattered fibroblast-like shapes, reduction of the epithelial marker E-cadherin, and an increased invasive and migratory ability. These phenotypic changes are mediated in large part by the ERK-MAPK pathway, as the MEK inhibitor U0126 and shRNA-mediated knockdown of ERK2 significantly reversed these phenotypes. Hepatocyte growth factor binding to the c-MET receptor is known to induce EMT in HepG2 cells. We found that CMTM8 knockdown in HepG2 cells induced c-MET signaling and ERK activation. Inhibition of c-MET signaling with the small molecule inhibitor SU11274 or c-MET RNAi blocked the EMT-like changes following CMTM8 knockdown. CMTM8 overexpression in HepG2 cells inhibited hepatocyte growth factor-induced EMT-like morphological changes and cell motility. Down-regulation of CMTM8 also promoted an EMT-like change in MCF-10A cells, indicating a broader role for CMTM8 in regulating cellular transformation.

A novel role for Gtb1p in glucose trimming of N-linked glycans.

Glucosidase II (GluII) is a glycan-trimming enzyme active on nascent glycoproteins in the endoplasmic reticulum (ER). It trims the middle and innermost glucose residues (Glc2 and Glc1) from N-linked glycans. The monoglucosylated glycan produced by the first GluII trimming reaction is recognized by calnexin/calreticulin and serves as the signal for entry into this folding pathway. GluII is a heterodimer of alpha and beta subunits corresponding to yeast Gls2p and Gtb1p, respectively. While Gls2p contains the glucosyl hydrolase active site, the Gtb1p subunit has previously been shown to be essential for the Glc1 trimming event. Here we demonstrate that Gtb1p also determines the rate of Glc2 trimming. In order to further dissect these activities we mutagenized a number of conserved residues across the protein. Our data demonstrate that both the MRH and G2B domains of Gtb1p contribute to the Glc2 trimming event but that the MRH domain is essential for Glc1 trimming.

Discovery of Small-Molecule Selective mTORC1 Inhibitors via Direct Inhibition of Glucose Transporters.

The mechanistic target of rapamycin (mTOR) is a central regulator of cellular metabolic processes. Dysregulation of this kinase complex can result in a variety of human diseases. Rapamycin and its analogs target mTORC1 directly; however, chronic treatment in certain cell types and in vivo results in the inhibition of both mTORC1 and mTORC2. We have developed a high-throughput cell-based screen for the detection of phosphorylated forms of the mTORC1 (4E-BP1, S6K1) and mTORC2 (Akt) substrates and have identified and characterized a chemical scaffold that demonstrates a profile consistent with the selective inhibition of mTORC1. Stable isotope labeling of amino acids in cell culture-based proteomic target identification revealed that class I glucose transporters were the primary target for these compounds yielding potent inhibition of glucose uptake and, as a result, selective inhibition of mTORC1. The link between the glucose uptake and selective mTORC1 inhibition are discussed in the context of a yet-to-be discovered glucose sensor.

A small molecule inhibitor of Rheb selectively targets mTORC1 signaling.

The small G-protein Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) in response to growth factor signals. mTORC1 is a master regulator of cellular growth and metabolism; aberrant mTORC1 signaling is associated with fibrotic, metabolic, and neurodegenerative diseases, cancers, and rare disorders. Point mutations in the Rheb switch II domain impair its ability to activate mTORC1. Here, we report the discovery of a small molecule (NR1) that binds Rheb in the switch II domain and selectively blocks mTORC1 signaling. NR1 potently inhibits mTORC1 driven phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) but does not inhibit phosphorylation of AKT or ERK. In contrast to rapamycin, NR1 does not cause inhibition of mTORC2 upon prolonged treatment. Furthermore, NR1 potently and selectively inhibits mTORC1 in mouse kidney and muscle in vivo. The data presented herein suggest that pharmacological inhibition of Rheb is an effective approach for selective inhibition of mTORC1 with therapeutic potential.

Cell signaling in protein synthesis ribosome biogenesis and translation initiation and elongation.

Protein synthesis is a highly energy-consuming process that must be tightly regulated. Signal transduction cascades respond to extracellular and intracellular cues to phosphorylate proteins involved in ribosomal biogenesis and translation initiation and elongation. These phosphorylation events regulate the timing and rate of translation of both specific and total mRNAs. Alterations in this regulation can result in dysfunction and disease. While many signaling pathways intersect to control protein synthesis, the mTOR and MAPK pathways appear to be key players. This chapter briefly reviews the mTOR and MAPK pathways and then focuses on individual phosphorylation events that directly control ribosome biogenesis and translation.