Identification of Dominant Negative Mutants of Rheb GTPase and Their Use to Implicate the Involvement of Human Rheb in the Activation of p70S6K (original) (raw)
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Recent progress in the study of the Rheb family GTPases
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In this review we highlight recent progress in the study of Rheb family GTPases. Structural studies using X-ray crystallography and NMR have given us insight into unique features of this GTPase. Combined with mutagenesis studies, these works have expanded our understanding of residues that affect Rheb GTP/GDP bound ratios, effector protein interactions, and stimulation of mTORC1 signaling. Analysis of cancer genome databases has revealed that several human carcinomas contain activating mutations of the protein. Rheb's role in activating mTORC1 signaling at the lysosome in response to stimuli has been further elucidated. Rheb has also been suggested to play roles in other cellular pathways including mitophagy and peroxisomal ROS response. A number of studies in mice have demonstrated the importance of Rheb in development, as well as in a variety of functions including cardiac protection and myelination. We conclude with a discussion of future prospects in the study of Rheb family...
Analysis of mTOR signaling by the small G-proteins, Rheb and RhebL1
FEBS Letters, 2005
The small G protein Rheb (Ras homologue enriched in brain) is known to promote mammalian target of rapamycin (mTOR) signaling. In this study, we show that Rheb like-1 protein (RhebL1) rescues mTOR signaling during nutrient withdrawal and that tuberous sclerosis complex-1 (TSC) and TSC2 impairs RhebL1-mediated signaling through mTOR. We identify critical residues within the switch I region (N41) and ÔconstitutiveÕ effector (Ec) region (Y/F54 and L56) of Rheb and RhebL1, which are required for their efficient activation of mTOR signaling. Mutation of Rheb and RhebL1 at N41 impaired their interaction with mTOR, which identifies mTOR as a common downstream target of both Rheb and RhebL1.
Rheb Binds and Regulates the mTOR Kinase
Current Biology, 2005
raptor [3, 4] (KOG1 in S. cerevisiae [5]) and LST8 [5] (also called GβL [6]), and its output is inhibited by rapa-Kazuyoshi Yonezawa, 2,3 and Joseph Avruch 1, * mycin. The TOR complex 2 (TORC2) contains TOR in 1 Diabetes Unit and Medical Services and association with the proteins AVO3/rictor and LST8 [5, Department of Molecular Biology 7, 8]. TORC2 controls the actin cytoskeleton; its output Massachusetts General Hospital and is insensitive to rapamycin and will not be considered Department of Medicine further here. Harvard Medical School A major target of TORC1 regulation in all cells is Boston, Massachusetts 02114 mRNA translation [9]. In mammalian cells, mTOR stimu-2 Biosignal Research Center lates translational initiation through the phosphoryla-Kobe University tion of 4E-BP1, an inhibitor of the binding of the mRNA-Kobe 657-8501 cap binding protein eIF-4E to the eIF-4G scaffold. Japan Phosphorylation of 4E-BP1 promotes its dissociation 3 CREST from eIF-4E, enabling recruitment of the latter into the Japan Science and Technology Agency eIF-4F complex. mTOR also directly phosphorylates Kawaguchi 332-0012 and, in collaboration with PDK1, activates the p70 S6 Japan kinase [10]; the latter regulates cell size [11] through incompletely defined mechanisms. The ability of the mTOR kinase to phosphorylate these targets is depen-Summary dent on the association of TOR with raptor, inasmuch as raptor binds the TOR substrates 4E-BP1 and Background: The target of rapamycin (TOR), in com-p70S6K and presents them to mTOR [3]. In fact, the plex with the proteins raptor and LST8 (TOR complex ability of mTOR to phosphorylate 4E-BP1 is nearly elim-1), phosphorylates the p70S6K and 4E-BP1 to promote inated by removal of raptor in vitro [3, 12], or in vivo by mRNA translation. Genetic evidence establishes that mutation of the 4E-BP1 "TOS" motif [13], the 4E-BP1 TOR complex activity in vivo requires the small GTPase segment that mediates binding to raptor [12, 14-16]. Rheb, and overexpression of Rheb can rescue TOR The ability of TOR to regulate p70S6K is also strongly from inactivation in vivo by amino-acid withdrawal. The dependent on the association of p70S6K with raptor. Tuberous Sclerosis heterodimer (TSC1/TSC2) functions Mutation of the p70S6K TOS motif reduces mTOR-catas a Rheb GTPase activator and inhibits TOR signaling alyzed phosphorylation of p70S6K in vitro by approxiin vivo. mately 75% and renders p70S6K insensitive to inhibi-Results: Here, we show that Rheb binds to the TOR tion in vivo by rapamycin or to regulation by ambient complex specifically, independently of its ability to bind amino acids [12]. LST8, the third component of the TSC2, through separate interactions with the mTOR cat-TORC1 complex [5, 6], is a 36 kDa polypeptide whose alytic domain and with LST8. Rheb binding to the TOR predicted structure is composed entirely of seven WD complex in vivo and in vitro does not require Rheb guarepeats, presumably arrayed in a so-called "β propelnyl nucleotide charging but is modulated by GTP and lor" [17]. LST8 plays an important but incompletely deimpaired by certain mutations (Ile39Lys) in the switch 1 fined role in TOR regulation. The polypeptide binds loop. Nucleotide-deficient Rheb mutants, although catightly to the TOR catalytic domain and enhances the pable of binding mTOR in vivo and in vitro, are inhibiassociation of raptor with mTOR [6]; however, other tory in vivo, and the mTOR polypeptides that associate roles remain likely. with nucleotide-deficient Rheb in vivo lack kinase activ-In mammalian cells, mTOR output is sensitive to ity in vitro. Reciprocally, mTOR polypeptides bound to amino-acid (especially leucine and arginine) sufficiency Rheb(Gln64Leu), a mutant that is nearly 90% GTP but is also controlled by overall energy supply through charged, exhibit substantially higher protein kinase the AMP-activated protein kinase (AMPK) and by inputs specific activity than mTOR bound to wild-type Rheb. from cell surface receptors through the PI-3 Kinase Conclusions: The TOR complex 1 is a direct target of (PI3K)-PKB pathway [2]. Recent evidence indicates that Rheb-GTP, whose binding enables activation of the the tuberous sclerosis complex (a TSC1/TSC2 hetero-TOR kinase. dimer) acts as a negative regulator upstream of mTOR and is a major target through which PKB, AMPK, and
Rheb is an essential regulator of S6K in controlling cell growth in Drosophila
Nature Cell Biology, 2003
Understanding the mechanisms through which multicellular organisms regulate cell, organ and body growth is of relevance to developmental biology and to research on growth-related diseases such as cancer. Here we describe a new effector in growth control, the small GTPase Rheb (Ras homologue enriched in brain). Mutations in the Drosophila melanogaster Rheb gene were isolated as growth-inhibitors, whereas overexpression of Rheb promoted cell growth. Our genetic and biochemical analyses suggest that Rheb functions downstream of the tumour suppressors Tsc1 (tuberous sclerosis 1)-Tsc2 in the TOR (target of rapamycin) signalling pathway to control growth, and that a major effector of Rheb function is ribosomal S6 kinase (S6K).
Ras and Rheb Signaling in Survival and Cell Death
Cancers, 2013
One of the most obvious hallmarks of cancer is uncontrolled proliferation of cells partly due to independence of growth factor supply. A major component of mitogenic signaling is Ras, a small GTPase. It was the first identified human protooncogene and is known since more than three decades to promote cellular proliferation and growth. Ras was shown to support growth factor-independent survival during development and to protect from chemical or mechanical lesion-induced neuronal degeneration in postmitotic neurons. In contrast, for specific patho-physiological cases and cellular systems it has been shown that Ras may also promote cell death. Proteins from the Ras association family (Rassf, especially Rassf1 and Rassf5) are tumor suppressors that are activated by Ras-GTP, triggering apoptosis via e.g., activation of mammalian sterile 20-like (MST1) kinase. In contrast to Ras, their expression is suppressed in many types of tumours, which makes Rassf proteins an exciting model for understanding the divergent effects of Ras activity. It seems likely that the outcome of Ras signaling depends on the balance between the activation of its various downstream effectors, thus determining cellular fate towards either proliferation or apoptosis. Ras homologue enriched in brain (Rheb) is a protein from the Ras superfamily that is also known to promote proliferation, growth, and regeneration through the mammalian target of rapamycin (mTor) pathway. However, recent evidences indicate that the Rheb-mTor pathway may switch its function from a pro-growth into a cell death pathway, depending on the cellular situation. In contrast to Ras signaling, for Rheb,
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