Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency

The removal of extracellular amino acids or leucine alone inhibits the ability of the mammalian target of rapamycin (mTOR) to signal to the raptor-dependent substrates, p70 S6 kinase and 4E-BP. This inhibition can be overcome by overexpression of the Rheb GTPase. Rheb binds directly to the amino-terminal lobe of the mTOR catalytic domain, and activates mTOR kinase in a GTP-dependent manner. Herein we show that the binding of Rheb to endogenous and recombinant mTOR is reversibly inhibited by withdrawal of all extracellular amino acids or just leucine. The effect of amino acid withdrawal is not attributable to changes in Rheb-GTP charging; amino acid withdrawal does not alter the GTP charging of recombinant Rheb. Moreover, the binding of mTOR to Rheb mutants that are unable to bind guanyl nucleotide in vivo is also inhibited by amino withdrawal. The inhibitory effect of amino acid withdrawal is exerted through an action on mTOR, at a site largely distinct from that responsible for the binding of Rheb; deletion of the larger, carboxyl-terminal lobe of the mTOR catalytic domain eliminates the inhibitory effect of amino acid withdrawal on Rheb binding, without altering Rheb binding per se. The lesser ability of the mTOR catalytic domain to bind Rheb after amino acid withdrawal does not persist after extraction and purification of the mTOR polypeptide. Amino acid withdrawal may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1.     

mTOR, the mammalian target of rapamycin

The discovery of rapamycin from a soil sample on Easter Island in the mid 60's marked the beginning of an exciting field of research in cell biology and medicine. While it was first used as an antifungal and as an immunosuppressive drug, more recent studies confirmed rapamycin's antiproliferative properties over a variety of solid tumors. Research aimed at identifying its mechanism of action uncovered mTOR (mammalian target of rapamycin), a protein kinase that regulates mRNA translation and protein synthesis, an essential step in cell division and proliferation. Recent evidence suggests a more complex role for mTOR in the regulation of several growth factor-stimulated protein kinases, including the proto-oncogene Akt. This article reviews mTOR function and regulation, and briefly details the future challenges for anti-cancer therapies based on mTOR inhibition.     

Regulation of B-Raf kinase activity by tuberin and Rheb is mammalian target of rapamycin (mTOR)-independent

Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome with manifestations that can include seizures, mental retardation, autism, and tumors in the brain, retina, kidney, heart, and skin. The products of the TSC1 and TSC2 genes, hamartin and tuberin, respectively, heterodimerize and inhibit the mammalian target of rapamycin (mTOR). We found that tuberin expression increases p42/44 MAPK phosphorylation and B-Raf kinase activity. Short interfering RNA down-regulation of tuberin decreased the p42/44 MAPK phosphorylation and B-Raf activity. Expression of Rheb, the target of the GTPase-activating domain of tuberin, inhibited wild-type B-Raf kinase but not activated forms of B-Raf. The interaction of endogenous Rheb with B-Raf was enhanced by serum and by Ras overexpression. A farnesylation-defective mutant of Rheb co-immunoprecipitated with and inhibited B-Raf but did not activate ribosomal protein S6 kinase, indicating that farnesylation is not required for B-Raf inhibition by Rheb and that B-Raf inhibition and S6 kinase activation are separable activities of Rheb. Consistent with this, inhibition of B-Raf and p42/44 MAPK by Rheb was resistant to rapamycin in contrast to Rheb activation of S6 kinase, which is rapamycin-sensitive. Taken together these data demonstrate that inhibition of B-Raf kinase via Rheb is an mTOR-independent function of tuberin.     

Thr2446 is a novel mammalian target of rapamycin (mTOR) phosphorylation site regulated by nutrient status

The mammalian target of rapamycin (mTOR) is a key regulator of protein translation. Signaling via mTOR is increased by growth factors but decreased during nutrient deprivation. Previous studies have identified Ser2448 as a nutrient-regulated phosphorylation site located in the mTOR catalytic domain, insulin stimulates Ser2448 phosphorylation via protein kinase B (PKB), while Ser2448 phosphorylation is attenuated with amino acid starvation. Here we have identified Thr2446 as a novel nutrient-regulated phosphorylation site on mTOR. Thr2446 becomes phosphorylated when CHO-IR cells are nutrient-deprived, but phosphorylation is reduced by insulin stimulation. Nutrient deprivation activates AMP-activated protein kinase (AMPK). To test whether this could be involved in regulating phoshorylation of mTOR, we treated cultured murine myotubes with 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or dinitrophenol (DNP). Both treatments activated AMPK and also caused a concomitant increase in phosphorylation of Thr2446 and a parallel decrease in insulin's ability to phosphorylate p70 S6 kinase. In vitro kinase assays using peptides based on the sequence in amino acids 2440-2551 of mTOR found that PKB and AMPK are capable of phosphorylating sites in this region. However, phosphorylation by PKB is restricted when Thr2446 is mutated to an acidic residue mimicking phosphorylation. Conversely, AMP-kinase-induced phosphorylation is reduced when Ser2448 is phosphorylated. These data suggest differential phosphorylation Thr2446 and Ser2448 could act as a switch mechanism to integrate signals from nutrient status and growth factors to control the regulation of protein translation.     

Chlorophyll accumulation is enhanced by osmotic stress in graminaceous chlorophyllic cells

We have developed a new chlorophyllic cell line ('TADH-XO') from the highly water stress tolerant grass Bouteloua gracilis (blue grama). When grown under normal (non-stress) conditions, this new cell line accumulates higher levels of chlorophyll (up to 368.1 microg total chlorophyll g(-1) FW) than a previously obtained cell line ('TIANSJ98'). Both cell lines are capable of developing substantially higher amounts of chlorophyll when subjected to osmotic stress. In order to explain these changes in the chlorophyll kinetics of the chlorophyllic cells, we analyzed the following population variables in cells subjected to polyethylene glycol 8000-induced osmotic stress: growth, viability, chlorophyll (total, 'a' and 'b'), cell size, percentage of green cells and chloroplast (number and size). Although previous studies in some chlorophyllic cells of dicots have already reported that chlorophyll increases under saline stress, in this report we show that, at least in this graminaceous cell line, the increase in chlorophyll is an immediate and proportional response to the osmotic stress applied and not the result of a progressive adaptation process. Consistent with previous studies, the increase in chlorophyll accumulation could be the result of chloroplast development (increased thylakoid number per chloroplast). On the basis of our results, the increases in chlorophyll accumulation previously observed in salt-adapted dicot cells may be the result of the osmotic shock (water deficit), rather than the ionic effect of salt on the physiology of chlorophyllic cells of dicots. Under the cell population experimental approach we followed, our study provides important insights related to the physiological behavior of chlorophyllic cells subjected to osmotic stress.     

Activation of mammalian target of rapamycin (mTOR) by insulin is associated with stimulation of 4EBP1 binding to dimeric mTOR complex 1

Insulin stimulates protein synthesis by promoting phosphorylation of the eIF4E-binding protein, 4EBP1. This effect is rapamycin-sensitive and mediated by mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a signaling complex containing mTOR, raptor, and mLST8. Here we demonstrate that insulin produces a stable increase in the kinase activity of mTORC1 in 3T3-L1 adipocytes. The response was associated with a marked increase in 4EBP1 binding to raptor in mTORC1, and it was abolished by disrupting the TOR signaling motif in 4EBP1. The stimulatory effects of insulin on both 4EBP1 kinase activity and binding occurred rapidly and at physiological concentrations of insulin, and both effects required an intact mTORC1. Results of experiments involving size exclusion chromatography and coimmunoprecipitation of epitope-tagged subunits provide evidence that the major insulin-responsive form is dimeric mTORC1, a structure containing two heterotrimers of mTOR, raptor, and mLST8.     

Mammalian target of rapamycin (mTOR) is activated in cutaneous vascular malformations in vivo

BACKGROUND: Vascular malformation signaling pathways are the least understood out of all cutaneous endothelial lesions. The overexpression of Akt is known to cause vascular malformations in endothelial cells of mice. Since there are no Akt inhibitors approved for clinical use, we examined phosphorylated S6 expression, a downstream target of Akt. Phosphorylated S6 indicates potential sensitivity to rapamycin. METHODS AND RESULTS: Immunohistochemistry for phospho-s6k against phospho-S6 ribosomal protein was performed on specimens of vascular malformations taken from Sturge- Weber patients. Of the specimens, 70.8% were immunopositive for phospho-s6k. CONCLUSION: Endothelial expression of Akt is responsible for tumor responsiveness to rapamycin. We demonstrate that expression of phosphorylated S6 is elevated in specimens. Our findings provide a rationale for clinical trials of rapamycin on Sturge-Weber or Klippel-Trenaunay-Weber patients.     

Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR

The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (I(glu)), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, I(glu) was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased I(glu), an effect reversed by rapamycin (100 nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of I(glu) following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100 nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.     

Phosphorylation of mammalian target of rapamycin (mTOR) at Ser-2448 is mediated by p70S6 kinase

The mammalian target of rapamycin (mTOR) coordinates cell growth with the growth factor and nutrient/energy status of the cell. The phosphatidylinositol 3-kinase-AKT pathway is centrally involved in the transmission of mitogenic signals to mTOR. Previous studies have shown that mTOR is a direct substrate for the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. In this study, we demonstrate that rapamycin, a specific inhibitor of mTOR function, blocks serum-stimulated Ser-2448 phosphorylation and that this drug effect is not explained by the inhibition of AKT. Furthermore, the phosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggesting that mTOR itself or a protein kinase downstream from mTOR was responsible for the modification of Ser-2448. Here we show that p70S6 kinase phosphorylates mTOR at Ser-2448 in vitro and that ectopic expression of rapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation in rapamycin-treated cells. In addition, we show that cellular amino acid status, which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway, regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediated depletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Taken together, these results suggest that p70S6 kinase is a major effector of mTOR phosphorylation at Ser-2448 in response to both mitogen- and nutrient-derived stimuli.     

The mTOR (mammalian target of rapamycin) kinase maintains integrity of mTOR complex 2

In higher eukaryotes, growth factors promote anabolic processes and stimulate cell growth, proliferation, and survival by activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway. Deregulation of PI3K/Akt signaling is linked to human diseases, including cancer and metabolic disorders. The PI3K-dependent signaling kinase complex mTORC2 (mammalian target of rapamycin complex 2) has been defined as the regulatory Ser-473 kinase of Akt. The regulation of mTORC2 remains very poorly characterized. We have reconstituted mTORC2 by its assembly in vitro or by co-expression its four essential components (rictor, SIN1, mTOR, mLST8). We show that the functional mTOR kinase domain is required for the mTORC2 activity as the Ser-473 kinase of Akt. We also found that mTOR by phosphorylation of SIN1 prevents its lysosomal degradation. Thus, the kinase domain of mTOR is required for the functional activity of mTORC2, and it controls integrity of mTORC2 by maintaining the protein stability of SIN1.     

Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells.

We have studied the effect of plasminogen activator inhibitors PAI-1 and PAI-2 on the binding of urokinase-type plasminogen activator (u-PA) to its receptor in the human choriocarcinoma cell line JAR. With 125I-labeled ligands in whole-cell binding assays, both uncomplexed u-PA and u-PA-inhibitor complexes bound to the receptor with a Kd of approximately 100 pM at 4 degrees C. Transferring the cells to 37 degrees C led to degradation to amino acids of up to 50% of the cell-bound u-PA-inhibitor complexes, whereas the degradation of uncomplexed u-PA was 15%; the remaining ligand was recovered in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function for the removal of u-PA after its complex formation with a specific inhibitor. The data suggest a novel mechanism by which receptor-mediated endocytosis is initiated by the binding of a secondary ligand.     

The mTOR inhibitor rapamycin suppresses DNA double-strand break repair

mTOR (mammalian target of rapamycin) signaling plays a key role in the development of many tumor types. Therefore, mTOR is an attractive target for cancer therapeutics. Although mTOR inhibitors are thought to have radiosensitization activity, the molecular bases remain largely unknown. Here we show that treating MCF7 breast cancer cells with rapamycin (an mTOR inhibitor) results in significant suppression of homologous recombination (HR) and nonhomologous end joining (NHEJ), two major mechanisms required for repairing ionizing radiation-induced DNA DSBs. We observed that rapamycin impaired recruitment of BRCA1 and Rad51 to DNA repair foci, both essential for HR. Moreover, consistent with the suppressive role of rapamycin on both HR and NHEJ, persistent radiation-induced DSBs were detected in cells pretreated with rapamycin. Furthermore, the frequency of chromosome and chromatid breaks was increased in cells treated with rapamycin before and after irradiation. Thus our results show that radiosensitization by mTOR inhibitors occurs via disruption of the major two DNA DSB repair pathways.     

Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine synthetase

To investigate rate-limiting factors for glutathione and phytochelatin (PC) production and the importance of these compounds for heavy metal tolerance, Indian mustard (Brassica juncea) was genetically engineered to overexpress the Escherichia coli gshI gene encoding gamma-glutamylcysteine synthetase (gamma-ECS), targeted to the plastids. The gamma-ECS transgenic seedlings showed increased tolerance to Cd and had higher concentrations of PCs, gamma-GluCys, glutathione, and total non-protein thiols compared with wild-type (WT) seedlings. When tested in a hydroponic system, gamma-ECS mature plants accumulated more Cd than WT plants: shoot Cd concentrations were 40% to 90% higher. In spite of their higher tissue Cd concentration, the gamma-ECS plants grew better in the presence of Cd than WT. We conclude that overexpression of gamma-ECS increases biosynthesis of glutathione and PCs, which in turn enhances Cd tolerance and accumulation. Thus, overexpression of gamma-ECS appears to be a promising strategy for the production of plants with superior heavy metal phytoremediation capacity.     

The mTOR pathway is highly activated in diabetic nephropathy and rapamycin has a strong therapeutic potential

Diabetic nephropathy (DN) associated with type 2 diabetes is the most common cause of end-stage renal disease (ESRD) and a serious health issue in the world. Currently, molecular basis for DN has not been established and only limited clinical treatments are effective in abating the progression to ESRD associated with DN. Here we found that diabetic db/db mice which lack the leptin receptor signaling can be used as a model of ESRD associated with DN. We demonstrated that p70S6-kinase was highly activated in mesangial cells in diabetic obese db/db mice. Furthermore, systemic administration of rapamycin, a specific and potent inhibitor of mTOR, markedly ameliorated pathological changes and renal dysfunctions. Moreover, rapamycin treatment shows a significant reduction in fat deposits and attenuates hyperinsulinemia with few side effects. These results indicate that mTOR activation plays a pivotal role in the development of ESRD and that rapamycin could be an effective therapeutic agent for DN.     

Targeting mTOR globally in cancer: Thinking beyond rapamycin

The mammalian target of rapamycin (mTOR) is centrally involved in growth, survival and metabolism. In cancer, mTOR is frequently hyperactivated and is a clinically validated target for drug development. Until recently, we have relied largely on the use of rapamycin to study mTOR function and its anticancer potential. Recent insights now indicate that rapamycin is a partial inhibitor of mTOR through allosteric inhibition of mTOR complex-1 (mTORC1) but not mTOR complex-2 (mTORC2). Both the mechanism of action and the cellular response to mTORC1 inhibition by rapamycin and related drugs may limit the effectiveness of these compounds as antitumor agents. We and others have recently reported the discovery of second-generation ATP-competitive mTOR kinase inhibitors (TKIs) that bind to the active sites of mTORC1 and mTORC2, thereby targeting mTOR signaling function globally (see refs. 1-4). The discovery of specific, active-site mTOR inhibitors has opened a new chapter in the 40-plus year old odyssey that began with the discovery of rapamycin from a soil sample collected on Easter Island (see Vézina C, et al. J Antibiot 1975). Here, we discuss recent studies that highlight the emergence of rapamycin-resistant mTOR function in protein synthesis, cell growth, survival and metabolism. It is shown that these rapamycin-resistant mTOR functions are profoundly inhibited by TKIs. A more complete suppression of mTOR global signaling network by the new inhibitors is expected to yield a deeper and broader antitumor response in the clinic.