The Experts below are selected from a list of 5307 Experts worldwide ranked by ideXlab platform
Kunliang Guan - One of the best experts on this subject based on the ideXlab platform.
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Measurements of TSC2 GAP activity toward RHEB.
Methods in Enzymology, 2020Co-Authors: Yong Li, Ken Inoki, Haris G. Vikis, Kunliang GuanAbstract: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.
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amino acids license kinase mtorc1 activity and treg cell function via small g proteins rag and RHEB
Immunity, 2019Co-Authors: Nicole M Chapman, Lingyun Long, Yogesh Dhungana, Sherri Rankin, Stephane Pelletier, Peter Vogel, Hong Wang, Junmin Peng, Kunliang GuanAbstract:Summary Regulatory T (Treg) cells are critical mediators of immune tolerance whose activity depends upon T cell receptor (TCR) and mTORC1 kinase signaling, but the mechanisms that dictate functional activation of these pathways are incompletely understood. Here, we showed that amino acids license Treg cell function by priming and sustaining TCR-induced mTORC1 activity. mTORC1 activation was induced by amino acids, especially arginine and leucine, accompanied by the dynamic lysosomal localization of the mTOR and Tsc complexes. Rag and RHEB GTPases were central regulators of amino acid-dependent mTORC1 activation in effector Treg (eTreg) cells. Mice bearing RagA-RagB- or RHEB1-RHEB2-deficient Treg cells developed a fatal autoimmune disease and had reduced eTreg cell accumulation and function. RagA-RagB regulated mitochondrial and lysosomal fitness, while RHEB1-RHEB2 enforced eTreg cell suppressive gene signature. Together, these findings reveal a crucial requirement of amino acid signaling for licensing and sustaining mTORC1 activation and functional programming of Treg cells.
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tsc1 tsc2 and RHEB have different effects on torc1 and torc2 activity
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Qian Yang, Ken Inoki, Kunliang GuanAbstract:Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (RHEB) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of RHEB in TORC1 activation remains to be determined. However, the function of RHEB in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRHEB has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRHEB on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that RHEB does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of RHEB, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and RHEB have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.
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TSC1/TSC2 and RHEB have different effects on TORC1 and TORC2 activity
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Qian Yang, Ken Inoki, Kunliang GuanAbstract:Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (RHEB) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of RHEB in TORC1 activation remains to be determined. However, the function of RHEB in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRHEB has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRHEB on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that RHEB does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of RHEB, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and RHEB have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.
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structural basis for the unique biological function of small gtpase RHEB
Journal of Biological Chemistry, 2005Co-Authors: Yadong Yu, Edward Arnold, Xiang Xu, Kunliang Guan, Yong Li, Sheng Li, Jianping DingAbstract:Abstract The small GTPase RHEB displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human RHEB in complexes with GDP, GTP, and GppNHp (5′-(β,γ-imide)triphosphate), which reveal novel structural features of RHEB and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of RHEB undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the α-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of RHEB. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in RHEB is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that RHEB forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of RHEB as the catalytic residue.
Elizabeth P Henske - One of the best experts on this subject based on the ideXlab platform.
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notch transactivates RHEB to maintain the multipotency of tsc null cells
Nature Communications, 2017Co-Authors: B J Patel, Elizabeth P Henske, Santosh Bonala, Sasikanth Manne, Yan Zhou, Surya Kumari Vadrevu, Jalpa Patel, Marco Peronaci, Shanawaz M Ghouse, Fabrice RoegiersAbstract:Differentiation abnormalities are a hallmark of tuberous sclerosis complex (TSC) manifestations; however, the genesis of these abnormalities remains unclear. Here we report on mechanisms controlling the multi-lineage, early neuronal progenitor and neural stem-like cell characteristics of lymphangioleiomyomatosis (LAM) and angiomyolipoma cells. These mechanisms include the activation of a previously unreported RHEB-Notch-RHEB regulatory loop, in which the cyclic binding of Notch1 to the Notch-responsive elements (NREs) on the RHEB promoter is a key event. This binding induces the transactivation of RHEB. The identified NRE2 and NRE3 on the RHEB promoter are important to Notch-dependent promoter activity. Notch cooperates with RHEB to block cell differentiation via similar mechanisms in mouse models of TSC. Cell-specific loss of Tsc1 within nestin-expressing cells in adult mice leads to the formation of kidney cysts, renal intraepithelial neoplasia, and invasive papillary renal carcinoma. Tuberous sclerosis complex (TSC) is a rare genetic condition causing tumours with differentiation abnormalities; however the molecular mechanisms causing these defects are unclear. Here the authors show that Notch cooperates with RHEB to block cell differentiation forming a regulatory loop that could underlie TSC tumorigenesis.
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non canonical functions of the tuberous sclerosis complex RHEB signalling axis
Embo Molecular Medicine, 2011Co-Authors: Nicole A Neuman, Elizabeth P HenskeAbstract:The protein products of the tuberous sclerosis complex (TSC) genes, TSC1 and TSC2, form a complex, which inhibits the small G-protein, Ras homolog enriched in brain (RHEB). The vast majority of research regarding these proteins has focused on mammalian Target of Rapamycin (mTOR), a target of RHEB. Here, we propose that there are clinically relevant functions and targets of TSC1, TSC2 and RHEB, which are independent of mTOR. We present evidence that such non-canonical functions of the TSC-RHEB signalling network exist, propose a standard of evidence for these non-canonical functions, and discuss their potential clinical and therapeutic implications for patients with TSC and lymphangioleiomyomatosis (LAM).
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Non‐canonical functions of the tuberous sclerosis complex‐RHEB signalling axis
Embo Molecular Medicine, 2011Co-Authors: Nicole A Neuman, Elizabeth P HenskeAbstract:The protein products of the tuberous sclerosis complex (TSC) genes, TSC1 and TSC2, form a complex, which inhibits the small G-protein, Ras homolog enriched in brain (RHEB). The vast majority of research regarding these proteins has focused on mammalian Target of Rapamycin (mTOR), a target of RHEB. Here, we propose that there are clinically relevant functions and targets of TSC1, TSC2 and RHEB, which are independent of mTOR. We present evidence that such non-canonical functions of the TSC-RHEB signalling network exist, propose a standard of evidence for these non-canonical functions, and discuss their potential clinical and therapeutic implications for patients with TSC and lymphangioleiomyomatosis (LAM).
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differential requirement of caax mediated posttranslational processing for RHEB localization and signaling
Oncogene, 2010Co-Authors: Ariella B Hanker, Natalia Mitin, Rhonda S Wilder, Elizabeth P Henske, Fuyuhiko TamanoiAbstract:The RHEB1 and RHEB2 small GTPases and their effector mTOR are aberrantly activated in human cancer and are attractive targets for anti-cancer drug discovery. RHEB is targeted to endomembranes via its C-terminal CAAX (C = cysteine, A = aliphatic, X = terminal amino acid) motif, a substrate for posttranslational modification by a farnesyl isoprenoid. Following farnesylation, RHEB undergoes two additional CAAX-signaled processing steps, Rce1-catalyzed cleavage of the AAX residues and Icmt-mediated carboxylmethylation of the farnesylated cysteine. However, whether these post-prenylation processing steps are required for RHEB signaling through mTOR is not known. We found that RHEB1 and RHEB2 localize primarily to the endoplasmic reticulum and Golgi apparatus. We determined that Icmt and Rce1 processing is required for RHEB localization, but is dispensable for RHEB-induced activation of the mTOR substrate p70 S6 kinase (S6K). Finally, we evaluated whether farnesylthiosalicylic acid (FTS) blocks RHEB localization and function. Surprisingly, FTS prevented S6K activation induced by a constitutively active mTOR mutant, indicating that FTS inhibits mTOR at a level downstream of RHEB. We conclude that inhibitors of Icmt and Rce1 will not block RHEB function, but FTS could be a promising treatment for RHEB- and mTOR-dependent cancers.
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RHEB inhibits c raf activity and b raf c raf heterodimerization
Journal of Biological Chemistry, 2006Co-Authors: Magdalena Karbowniczek, Gavin P Robertson, Elizabeth P HenskeAbstract:Abstract The Ras-Raf-MEK signaling cascade is critical for normal development and is activated in many forms of cancer. We have recently shown that B-Raf kinase interacts with and is inhibited by RHEB, the target of the GTPase-activating domain of the tuberous sclerosis complex 2 gene product tuberin. Here, we demonstrate for the first time that activation of RHEB is associated with decreased B-Raf and C-Raf phosphorylation at residues Ser-446 and Ser-338, respectively, concomitant with a decrease in the activities of both kinases and decreased heterodimerization of B-Raf and C-Raf. Importantly, the impact of RHEB on B-Raf/C-Raf heterodimerization and kinase activity are rapamycin-insensitive, indicating that they are independent of RHEB activation of the mammalian target of rapamycin-Raptor complex. In addition, we found that RHEB inhibits the association of B-Raf with H-Ras. Taken together, these results support a central role of RHEB in the regulation of the Ras/B-Raf/C-Raf/MEK signaling network.
Fuyuhiko Tamanoi - One of the best experts on this subject based on the ideXlab platform.
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A Genetic Approach to Define the Importance of RHEB in Tuberous Sclerosis
2020Co-Authors: Fuyuhiko TamanoiAbstract:Abstract : This report summarizes our accomplishments in characterizing the TSC/RHEB/mTOR signaling pathway that is altered in tuberous sclerosis. We have generated mice with decreased expression of RHEB1. We have succeeded in raising an antibody against mouse RHEB2. Effects of the TSC/RHEB/mTOR signaling on cell cycle progression have been investigated and we have obtained results suggesting the involvement of p27 and AMPK. Novel activating mutations of mTOR have been identified and they were used to investigate the consequences of the activation of the TSC/RHEB/mTOR signaling pathway on cell physiology. Our study makes significant contribution to understand how the TSC/RHEB/mTOR signaling pathway is regulated. The results we obtained make important contribution to the understanding of tuberous sclerosis.
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Using Drosophila and yeast genetics to investigate a role for the RHEB GTPase in cell growth.
Methods in Enzymology, 2020Co-Authors: Parthive H. Patel, Fuyuhiko TamanoiAbstract:Abstract The small, Ras‐like GTPase RHEB plays an important role in the regulation of cell growth by the insulin/PI3K and nutrient/TOR pathways in eukaryotic systems. Studies in genetically tractable organisms such as Drosophila melanogaster and fission yeast ( S. pombe ) were critical for establishing the significance of RHEB in cell growth. In Drosophila , we find that overexpression of Drosophila RHEB (dRHEB) in S2 cells causes their accumulation in S phase and an increase in cell size. In contrast, treatment of S2 cells with double‐stranded RNA (RNAi) toward dRHEB results in G1 arrest and a reduction in cell size. These altered cell size phenotypes observed in culture are also recapitulated in vivo . Overexpression of dRHEB results in increased cell and tissue size without an increase in cell number; reduction of dRHEB function results in reduced cell and tissue size. In S. pombe , inhibition of RHEB (SpRHEB) expression also results in small, rounded cells that arrest in G0/G1. We will discuss here how we use Drosophila and S. pombe to explain a mechanism by which RHEB promotes cell growth.
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RHEB G-Proteins and the Activation of mTORC1.
The Enzymes, 2020Co-Authors: Nitika Parmar, Fuyuhiko TamanoiAbstract:I. Abstract RHEB belongs to a unique family within the Ras superfamily of G-proteins. Although initially identified in rat brain, this G-protein is highly conserved from yeast to human. While only one RHEB is present in lower eukaryotes, two RHEB proteins exist in mammalian cells. A number of studies establish that one of the functions of RHEB is to activate mTOR leading to growth. In particular, the ability of RHEB to activate mTORC1 in vitro points to direct interaction of RHEB with the mTORC1 complex. Additional functions of RHEB that are independent of mTOR have also been suggested.
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Characterization of RHEB functions using yeast and mammalian systems.
Methods in Enzymology, 2020Co-Authors: Jun Urano, Geoffrey J. Clark, Chad A. Ellis, Fuyuhiko TamanoiAbstract:Publisher Summary This chapter presents the methods used in the study of ScRHEB in the yeast, S. cerevisiae , as well as those used to study mammalian RHEB. The chapter also describes methods used to address the C-terminal farnesylation of RHEB proteins and the requirement of this modification in RHEB function. RHEB (Ras homolog enriched in brain) is a new member of the Ras superfamily of G proteins that is highly conserved in a wide range of organisms. Homologs have been identified in human, rat, Saccharomyces cerevisiae , Schizosaccharomyces pombe , fruitfly, zebrafish, sea squirt, Botrvtis cinerea , and Candida albicans . RHEB shares some of the biological properties of Rap proteins, such as binding nonproductively to Raf-I and antagonizing Ras transformation: however, RHEB also has unique attributes. RHEB shows immediate-early gene characteristics. The RHEB transcript is increased in response to maximal electroconvulsive seizures as well as to N-methyl-D-aspartate (NMDA)-mediated synaptic activity and growth factors. In addition, the protein is farnesylated and the protein is localized to the plasma membrane.
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an oncogenic mutant of RHEB RHEB y35n exhibits an altered interaction with braf resulting in cancer transformation
BMC Cancer, 2018Co-Authors: Jeffrey J Heard, Fuyuhiko Tamanoi, Ivy Phung, Mark I PotesAbstract:RHEB is a unique member of the RAS superfamily of small GTPases expressed in all tissues and conserved from yeast to humans. Early studies on RHEB indicated a possible RHEB-RAF interaction, but this has not been fully explored. Recent work on cancer genome databases has revealed a reoccurring mutation in RHEB at the Tyr35 position, and a recent study points to the oncogenic potential of this mutant that involves activation of RAF/MEK/ERK signaling. These developments prompted us to reassess the significance of RHEB effect on RAF, and to compare mutant and wild type RHEB. To study RHEB-RAF interaction, and the effect of the Y35N mutation on this interaction, we used transfection, immunoprecipitation, and Western blotting techniques. We generated cell lines stably expressing RHEB WT, RHEB Y35N, and KRAS G12V, and monitored cellular transforming properties through cell proliferation, anchorage independent growth, cell cycle analysis, and foci formation assays. We observe a strong interaction between RHEB and BRAF, but not with CRAF. This interaction is dependent on an intact RHEB effector domain and RHEB-GTP loading status. RHEB overexpression decreases RAF activation of the RAF/MEK/ERK pathway and RHEB knockdown results in an increase in RAF/MEK/ERK activation. RHEB Y35N mutation has decreased interaction with BRAF, and RHEB Y35N cells exhibit greater BRAF/CRAF heterodimerization resulting in increased RAF/MEK/ERK signaling. This leads to cancer transformation of RHEB Y35N stably expressing cell lines, similar to KRAS G12 V expressing cell lines. RHEB interaction with BRAF is crucial for inhibiting RAF/MEK/ERK signaling. The RHEB Y35N mutant sustains RAF/MEK/ERK signaling due to a decreased interaction with BRAF, leading to increased BRAF/CRAF heterodimerization. RHEB Y35N expressing cells undergo cancer transformation due to decreased interaction between RHEB and BRAF resulting in overactive RAF/MEK/ERK signaling. Taken together with the previously established function of RHEB to activate mTORC1 signaling, it appears that RHEB performs a dual function; one is to suppress the RAF/MEK/ERK signaling and the other is to activate mTORC1 signaling.
Ken Inoki - One of the best experts on this subject based on the ideXlab platform.
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Measurements of TSC2 GAP activity toward RHEB.
Methods in Enzymology, 2020Co-Authors: Yong Li, Ken Inoki, Haris G. Vikis, Kunliang GuanAbstract: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.
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tsc1 tsc2 and RHEB have different effects on torc1 and torc2 activity
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Qian Yang, Ken Inoki, Kunliang GuanAbstract:Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (RHEB) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of RHEB in TORC1 activation remains to be determined. However, the function of RHEB in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRHEB has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRHEB on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that RHEB does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of RHEB, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and RHEB have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.
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TSC1/TSC2 and RHEB have different effects on TORC1 and TORC2 activity
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Qian Yang, Ken Inoki, Kunliang GuanAbstract:Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (RHEB) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of RHEB in TORC1 activation remains to be determined. However, the function of RHEB in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRHEB has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRHEB on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that RHEB does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of RHEB, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and RHEB have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.
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biochemical and functional characterizations of small gtpase RHEB and tsc2 gap activity
Molecular and Cellular Biology, 2004Co-Authors: Yong Li, Ken Inoki, Kunliang GuanAbstract: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.
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RHEB gtpase is a direct target of tsc2 gap activity and regulates mtor signaling
Genes & Development, 2003Co-Authors: Ken Inoki, Tian Xu, Yong Li, Kunliang GuanAbstract: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.
Yong Li - One of the best experts on this subject based on the ideXlab platform.
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Measurements of TSC2 GAP activity toward RHEB.
Methods in Enzymology, 2020Co-Authors: Yong Li, Ken Inoki, Haris G. Vikis, Kunliang GuanAbstract: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.
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structural basis for the unique biological function of small gtpase RHEB
Journal of Biological Chemistry, 2005Co-Authors: Yadong Yu, Edward Arnold, Xiang Xu, Kunliang Guan, Yong Li, Sheng Li, Jianping DingAbstract:Abstract The small GTPase RHEB displays unique biological and biochemical properties different from other small GTPases and functions as an important mediator between the tumor suppressor proteins TSC1 and TSC2 and the mammalian target of rapamycin to stimulate cell growth. We report here the three-dimensional structures of human RHEB in complexes with GDP, GTP, and GppNHp (5′-(β,γ-imide)triphosphate), which reveal novel structural features of RHEB and provide a molecular basis for its distinct properties. During GTP/GDP cycling, switch I of RHEB undergoes conformational change while switch II maintains a stable, unusually extended conformation, which is substantially different from the α-helical conformation seen in other small GTPases. The unique switch II conformation results in a displacement of Gln64 (equivalent to the catalytic Gln61 of Ras), making it incapable of participating in GTP hydrolysis and thus accounting for the low intrinsic GTPase activity of RHEB. This rearrangement also creates space to accommodate the side chain of Arg15, avoiding its steric hindrance with the catalytic residue and explaining its noninvolvement in GTP hydrolysis. Unlike Ras, the phosphate moiety of GTP in RHEB is shielded by the conserved Tyr35 of switch I, leading to the closure of the GTP-binding site, which appears to prohibit the insertion of a potential arginine finger from its GTPase-activating protein. Taking the genetic, biochemical, biological, and structural data together, we propose that RHEB forms a new group of the Ras/Rap subfamily and uses a novel GTP hydrolysis mechanism that utilizes Asn1643 of the tuberous sclerosis complex 2 GTPase-activating protein domain instead of Gln64 of RHEB as the catalytic residue.
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biochemical and functional characterizations of small gtpase RHEB and tsc2 gap activity
Molecular and Cellular Biology, 2004Co-Authors: Yong Li, Ken Inoki, Kunliang GuanAbstract: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.
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RHEB gtpase is a direct target of tsc2 gap activity and regulates mtor signaling
Genes & Development, 2003Co-Authors: Ken Inoki, Tian Xu, Yong Li, Kunliang GuanAbstract: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.
