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PubMed2004

Molecular mechanisms through which amino acids mediate signaling through the mammalian target of rapamycin.

PURPOSE OF REVIEW: The purpose of this review is to provide a summary of the current state of knowledge concerning one of the intracellular signal transduction pathways through which amino acids, and in particular leucine, regulate the initiation phase of mRNA translation. The primary focus is on a protein kinase, termed the mammalian target of rapamycin (mTOR), that is a point of convergence between amino acid and growth factor signaling to mRNA translation and thereby to cell growth. RECENT FINDINGS: Until recently the pathway through which amino acids signal to mTOR was completely undefined. Several recent reports, however, describe the identification of proteins that modulate amino acid signaling through mTOR, that is the tuberous sclerosis complex proteins 1 and 2 and the Ras homolog enriched in brain (Rheb) protein. Tuberous sclerosis complex protein 2 is a GTPase activator protein for Rheb that is inhibited by amino acids, allowing Rheb to activate mTOR through a mechanism still to be delineated. In addition, two proteins that interact with mTOR to target it to two important substrates, eukaryotic initiation factor 4E binding protein 1 and ribosomal protein S6 kinase, have been identified. Both proteins, that is the regulatory associated protein of mTOR and G protein beta-subunit-like protein, are required for optimal signaling through mTOR by amino acids. SUMMARY: Studies reported in the past 18 months have greatly expanded our knowledge of one of the signaling pathways through which amino acids act to regulate mTOR and also the molecular interactions that mediate the interaction between mTOR and two downstream substrates, eukaryotic initiation factor 4E binding protein 1 and ribosomal protein S6 kinase.

Amino Acids↗

PubMed2003

Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins.

Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumour syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP, suggesting that a GTPase might be the physiological target of Tsc2. Here we show that the small GTPase Rheb (Ras homologue enriched in brain) is a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupted its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. Our studies identify Rheb as a molecular target of the TSC tumour suppressors.

Animals↗

PubMed2004

Temporal control of differentiation by the insulin receptor/tor pathway in Drosophila.

Multicellular organisms must integrate growth and differentiation precisely to pattern complex tissues. Despite great progress in understanding how different cell fates are induced, it is poorly understood how differentiation decisions are temporally regulated. In a screen for patterning mutants, we isolated alleles of tsc1, a component of the insulin receptor (InR) growth control pathway. We find that loss of tsc1 disrupts patterning due to a loss of temporal control of differentiation. tsc1 controls the timing of differentiation downstream or in parallel to the RAS/MAPK pathway. Examination of InR, PI3K, PTEN, Tor, Rheb, and S6 kinase mutants demonstrates that increased InR signaling leads to precocious differentiation while decreased signaling leads to delays in differentiation. Importantly, cell fates are unchanged, but tissue organization is lost upon loss of developmental timing controls. These data suggest that intricate developmental decisions are coordinated with nutritional status and tissue growth by the InR signaling pathway.

Animals↗

PubMed2003

Tuberous sclerosis complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb.

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Measurements of TSC2 GAP activity toward Rheb.

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either the tsc1 or tsc2 tumor suppressor genes. TSC1 and TSC2 protein form a physical and functional complex in vivo. Recent studies have demonstrated that TSC2 displays GTPase activating protein (GAP) activity specifically toward the small G protein Rheb (Ras homolog enriched in brain) and inhibits its ability to stimulate the mammalian target of rapamycin (mTOR) signaling pathway. We have presented three methods to determine the activity of TSC2 as a GAP toward the Rheb GTPase. The first involves the isolation of TSC2 from cells and measurement of its activity toward Rheb substrate in vitro. The second involves the measurement of Rheb-associated guanine nucleotides as measure of TSC2 GAP activity on Rheb in vivo. The last method is to determine the phosphorylation of S6K1 (ribosomal S6 kinase), which is a downstream target of mTOR, as an indirect assay for TSC2 GAP activity in vivo.

Cells, Cultured↗

PubMed2003

Rheb promotes cell growth as a component of the insulin/TOR signalling network.

Insulin signalling is a potent stimulator of cell growth and has been proposed to function, at least in part, through the conserved protein kinase TOR (target of rapamycin) [corrected]. Recent studies suggest that the tuberous sclerosis complex Tsc1-Tsc2 may couple insulin signalling to Tor activity [corrected]. However, the regulatory mechanism involved remains unclear, and additional components are most probably involved. In a screen for novel regulators of growth, we identified Rheb (Ras homologue enriched in brain), a member of the Ras superfamily of GTP-binding proteins. Increased levels of Rheb in Drosophila melanogaster promote cell growth and alter cell cycle kinetics in multiple tissues. In mitotic tissues, overexpression of Rheb accelerates passage through G1-S phase without affecting rates of cell division, whereas in endoreplicating tissues, Rheb increases DNA ploidy. Mutation of Rheb suspends larval growth and prevents progression from first to second instar. Genetic and biochemical tests indicate that Rheb functions in the insulin signalling pathway downstream of Tsc1-Tsc2 and upstream of TOR. Levels of rheb mRNA are rapidly induced in response to protein starvation, and overexpressed Rheb can drive cell growth in starved animals, suggesting a role for Rheb in the nutritional control of cell growth.

Animals↗

PubMed2004

The Rheb family of GTP-binding proteins.

Rheb proteins represent a novel and unique family of the Ras superfamily GTP-binding proteins that is conserved from yeast to human. Biochemical studies establish that they bind and hydrolyze GTP. Molecular modeling studies reveal a few structural differences between Rheb and Ras, which may suggest that residues involved in biochemical activities differ between the two G-proteins. The function of Rheb has been studied in a number of organisms that point to the involvement of Rheb in cell growth and cell cycle progression. In addition, studies in fungi suggest that Rheb is involved in arginine uptake. Further studies in Drosophila and mammalian cells have shown that the effects of Rheb on growth and cell cycle progression are mediated by the effect on the insulin/TOR/S6K signaling pathway. These studies have also shown that a complex consisting of the tuberous sclerosis gene products, Tsc1/Tsc2, serves as a GTPase activating protein (GAP) for Rheb, implying Rheb's role in this genetic disorder. Finally, Rheb proteins have been shown to be farnesylated and small molecule inhibitors of protein farnesyltransferase can block the ability of Rheb to activate the TOR/S6K signaling.

Alkyl and Aryl Transferases↗

PubMed2004

Inappropriate activation of the TSC/Rheb/mTOR/S6K cassette induces IRS1/2 depletion, insulin resistance, and cell survival deficiencies.

Tuberous sclerosis is a largely benign tumor syndrome derived from the acquisition of somatic lesions in genes encoding the tumor suppressor products, TSC1 or TSC2. Loss of function of the TSC1-TSC2 complex, which acts as a Rheb GAP, yields constitutive, unrestrained signaling from the cell growth machinery comprised of Rheb, mTOR, and S6K. We demonstrate herein that constitutive activation of the Rheb/mTOR/S6K cassette, whether by genetic deletion of TSC1 or TSC2 or by ectopic expression of Rheb, is sufficient to induce insulin resistance. This is the result of downregulation of the insulin receptor substrates, IRS1 and IRS2, which become limiting for signal transmission from the insulin receptor to PI3K. Downstream of PI3K, the survival kinase, Akt, is completely refractory to activation by IRS-dependent growth factor pathways such as insulin or IGF-I in TSC1- or TSC2-deficient cells but not to activation by IRS-independent pathways such as those utilized by PDGF. The antiapoptotic program induced by IGF-I but not PDGF is severely compromised in TSC2 null cells. Our results suggest that inappropriate activation of the Rheb/mTOR/S6K pathway imposes a negative feedback program to attenuate IRS-dependent processes such as cell survival.

Blotting, Northern↗

PubMed2004

Tuberous sclerosis complex: from Drosophila to human disease.

Tuberous sclerosis complex (TSC) is a human syndrome characterized by a widespread development of benign tumors. This disease is caused by mutations in the TSC1 or TSC2 tumor suppressor genes; the molecular mechanisms underlying the activity of these have long been elusive. Recent studies of Drosophila and mammalian cells demonstrate that the TSC1-TSC2 complex functions as GTPase activating protein against Rheb - a Ras-like small GTPase, which in turn regulates TOR signaling in nutrient-stimulated cell growth. These findings provide a new paradigm for how proteins involved in nutrient sensing could function as tumor suppressors and suggest novel therapeutic targets against TSC. Here, we review these exciting developments with an emphasis on Drosophila studies and discuss how Drosophila can be a powerful model system for an understanding of the molecular mechanisms of the activity of human disease genes.

Animals↗

PubMed2003

Rheb fills a GAP between TSC and TOR.

There has been much interest in determining the molecular and cellular functions of hamartin and tuberin, which are encoded by the genes TSC1 and TSC2 that are mutated in the tuberous sclerosis complex disease. Recently, several laboratories have independently reported a major breakthrough in this field. Together, these genetic, biochemical and cell-biological studies have demonstrated that the tuberin-hamartin complex inhibits target of rapamycin (TOR) signaling by acting as a GTPase-activating protein for the Ras-related small G protein Rheb.

GTPase-Activating Proteins↗

PubMed2003

Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2.

Tumor suppressor genes evolved as negative effectors of mitogen and nutrient signaling pathways, such that mutations in these genes can lead to pathological states of growth. Tuberous sclerosis (TSC) is a potentially devastating disease associated with mutations in two tumor suppressor genes, TSC1 and 2, that function as a complex to suppress signaling in the mTOR/S6K/4E-BP pathway. However, the inhibitory target of TSC1/2 and the mechanism by which it acts are unknown. Here we provide evidence that TSC1/2 is a GAP for the small GTPase Rheb and that insulin-mediated Rheb activation is PI3K dependent. Moreover, Rheb overexpression induces S6K1 phosphorylation and inhibits PKB phosphorylation, as do loss-of-function mutations in TSC1/2, but contrary to earlier reports Rheb has no effect on MAPK phosphorylation. Finally, coexpression of a human TSC2 cDNA harboring a disease-associated point mutation in the GAP domain, failed to stimulate Rheb GTPase activity or block Rheb activation of S6K1.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling.

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.

Antibiotics, Antineoplastic↗

PubMed2006

Turnover of the active fraction of IRS1 involves raptor-mTOR- and S6K1-dependent serine phosphorylation in cell culture models of tuberous sclerosis.

The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity.

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.

Binding Sites↗

PubMedDate not supplied

Rhebbing up mTOR: new insights on TSC1 and TSC2, and the pathogenesis of tuberous sclerosis.

Tuberous sclerosis is an autosomal dominant human genetic disorder in which distinctive tumors called hamartomas develop. Germline mutations in either TSC1 or TSC2 cause this syndrome, and hamartomas typically display second hit events with loss of the remaining normal allele. Studies initiated in Drosophila have identified a role for the Tsc1 and Tsc2 genes in the regulation of cell and organ size, and genetic interaction studies have placed them in the PI3K-Akt-mTOR-S6K pathway. Biochemical studies have shown that activated Akt phosphorylates TSC2 in the TSC1/TSC2 protein complex, inactivating it; while TSC1/TSC2 has GAP activity for the Rheb GTPase (a member of the ras family), and activated Rheb-GTP activates mTOR. Thus, in cells lacking TSC1 or TSC2 there are increased levels of Rheb-GTP which leads to activation of mTOR, leading to cell size increase and growth. These developments provide enhanced understanding of this signaling pathway and fundamental insights into the pathogenesis of tuberous sclerosis, and open the possibility of treatment for hamartomas by several pharmacologic approaches.

Drosophila Proteins↗

PubMed1997

Ras-like GTPases.

Explore the source record for details and available documents.

3T3 Cells↗

PubMed1996

Isolation of cDNA and genomic clones of a human Ras-related GTP-binding protein gene and its chromosomal localization to the long arm of chromosome 7, 7q36.

A Ras-related GTP-binding protein cDNA has been isolated from a human skin fibroblast cDNA library using a genomic subclone derived from a YAC clone as a probe. The polypeptide, consisting of 184 amino acids deduced from nucleotide sequences, contains five repeats of the Ras-related GTP-binding region and is highly homologous to the rat RHEB (Ras homologue enriched in brain) gene, which encodes a Ras-related growth factor- and synaptic activity-regulated protein, with 98.9% amino acid identity. Therefore, it is suggested to be a human homologue of the rat RHEB protein, and we have designated it human RHEB. Using fluorescence in situ hybridization, we concluded that this human RHEB gene was localized to band q36 on chromosome 7. Considering the chromosomal localization as well as the potential function of this protein, it will be very important to investigate whether it may play a role in the etiopathogenesis of holoprosencephaly type 3 or hereditary sacral agenesis, in which the disease susceptible locus is linked to the microsatellite marker, D7S22, in this chromosomal region, 7q36.

Amino Acid Sequence↗

PubMed1997

The Ras-related protein Rheb is farnesylated and antagonizes Ras signaling and transformation.

Presently, nothing is known about the function of the Ras-related protein Rheb. Since Rheb shares significant sequence identity with the core effector domains of Ras and KRev-1/Rap1A, it may share functional similarities with these two structurally related, yet functionally distinct, small GTPases. Furthermore, since like Ras, Rheb terminates with a COOH terminus that is likely to signal for farnesylation, it may be a target for the farnesyltransferase inhibitors that block Ras processing and function. To compare Rheb function with those of Ras and KRev-1, we introduced mutations into Rheb that generate constitutively active or dominant negative forms of Ras and Ras-related proteins and were designated Rheb(64L) and Rheb(20N), respectively. Expression of wild type or mutant Rheb did not alter the morphology or growth properties of NIH 3T3 cells. Thus, aberrant Rheb function is distinct from that of Ras and fails to cause cellular transformation. Instead, similar to KRev-1, co-expression of Rheb antagonized oncogenic Ras transformation and signaling. In vitro and in vivo analyses showed that like Ras, Rheb proteins are farnesylated and are sensitive to farnesyltransferase inhibition. Thus, it is possible that Rheb function may be inhibited by farnesyltransferase inhibitors treatment and, consequently, may contribute to the ability of these inhibitors to impair Ras transformation.

3T3 Cells↗