Amino acid regulation of TOR complex 1 Avruch, Joseph; Long, Xiaomeng; Ortiz-Vega, Sara ...
American journal of physiology: endocrinology and metabolism,
04/2009, Letnik:
296, Številka:
4
Journal Article
Recenzirano
Odprti dostop
Department of Molecular Biology and Diabetes Unit, Medical Services, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts
Submitted 22 February ...2008
; accepted in final form 28 August 2008
ABSTRACT
TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GβL, controls cell growth in all eukaryotes in response to nutrient availability and in metazoans to insulin and growth factors, energy status, and stress conditions. This review focuses on the biochemical mechanisms that regulate mTORC1 kinase activity, with special emphasis on mTORC1 regulation by amino acids. The dominant positive regulator of mTORC1 is the GTP-charged form of the ras-like GTPase Rheb. Insulin, growth factors, and a variety of cellular stressors regulate mTORC1 by controlling Rheb GTP charging through modulating the activity of the tuberous sclerosis complex, the Rheb GTPase activating protein. In contrast, amino acids, especially leucine, regulate mTORC1 by controlling the ability of Rheb-GTP to activate mTORC1. Rheb binds directly to mTOR, an interaction that appears to be essential for mTORC1 activation. In addition, Rheb-GTP stimulates phospholipase D1 to generate phosphatidic acid, a positive effector of mTORC1 activation, and binds to the mTOR inhibitor FKBP38, to displace it from mTOR. The contribution of Rheb's regulation of PL-D1 and FKBP38 to mTORC1 activation, relative to Rheb's direct binding to mTOR, remains to be fully defined. The rag GTPases, functioning as obligatory heterodimers, are also required for amino acid regulation of mTORC1. As with amino acid deficiency, however, the inhibitory effect of rag depletion on mTORC1 can be overcome by Rheb overexpression, whereas Rheb depletion obviates rag's ability to activate mTORC1. The rag heterodimer interacts directly with mTORC1 and may direct mTORC1 to the Rheb-containing vesicular compartment in response to amino acid sufficiency, enabling Rheb-GTP activation of mTORC1. The type III phosphatidylinositol kinase also participates in amino acid-dependent mTORC1 activation, although the site of action of its product, 3'OH-phosphatidylinositol, in this process is unclear.
mammalian target of rapamycin; Rheb; rag; FKBP38; phospholipase D; guanosine 5'-triphosphatase
Address for reprint requests and other correspondence: J. Avruch, Diabetes Research Lab, Dept. of Molecular Biology, Massachusetts General Hospital, Simches Research Center, 185 Cambridge St., Boston, MA 02114 (E-mail: avruch{at}molbio.mgh.harvard.edu )
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and ...promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to RasSer-17 abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from 32P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive 32PGTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
Background: Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers.
Results: mTOR complex 1-Rag binding in vitro is independent of Rag guanyl nucleotide charging, and withdrawal of amino acids from cells does not alter Rag GTP charging.
Conclusion: Amino acids promote Rag binding to mTOR complex 1 without changing Rag GTP charging.
Significance: Amino acids promote Rag-mTORC1 binding by undefined mechanisms.
Variants in the IMP2 (insulin-like growth factor 2 IGF2 mRNA-binding protein 2) gene are implicated in susceptibility to type 2 diabetes. We describe the ability of mammalian target of rapamycin ...(mTOR) to regulate the cap-independent translation of IGF2 mRNA through phosphorylation of IMP2, an oncofetal RNA-binding protein. IMP2 is doubly phosphorylated in a rapamycin-inhibitable, amino acid-dependent manner in cells and by mTOR in vitro. Double phosphorylation promotes IMP2 binding to the IGF2 leader 3 mRNA 5' untranslated region, and the translational initiation of this mRNA through eIF-4E- and 5' cap-independent internal ribosomal entry. Unexpectedly, the interaction of IMP2 with mTOR complex 1 occurs through mTOR itself rather than through raptor. Whereas depletion of mTOR strongly inhibits IMP2 phosphorylation in cells, comparable depletion of raptor has no effect; moreover, the ability of mTOR to phosphorylate IMP2 in vitro is unaffected by the elimination of raptor. Dual phosphorylation of IMP2 at the mTOR sites is evident in the mouse embryo, likely coupling nutrient sufficiency to IGF2 expression and fetal growth. Doubly phosphorylated IMP2 is also widely expressed in adult tissues, including islets of Langerhans.
mTOR complex1, the major regulator of mRNA translation in all eukaryotic cells, is strongly activated in most cancers. We performed a genome-wide RNAi screen in a human cancer cell line, seeking ...genes that regulate S6 phosphorylation, readout of mTORC1 activity. Applying a stringent selection, we retrieved nearly 600 genes wherein at least two RNAis gave significant reduction in S6-P. This cohort contains known regulators of mTOR complex 1 and is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. We next examined the effect of RNAi pools directed at 534 of these gene products on S6-P in TSC1 null mouse embryo fibroblasts. 76 RNAis reduced S6 phosphorylation significantly in 2 or 3 replicates. Surprisingly, among this cohort of genes the only elements previously associated with the maintenance of mTORC1 activity are two subunits of the vacuolar ATPase and the CUL4 subunit DDB1. RNAi against a second set of 84 targets reduced S6-P in only one of three replicates. However, an indication that this group also bears attention is the presence of rpS6KB1 itself, Rac1 and MAP4K3, a protein kinase that supports amino acid signaling to rpS6KB1. The finding that S6 phosphorylation requires a previously unidentified, functionally diverse cohort of genes that participate in fundamental cellular processes such as mRNA translation, RNA processing, DNA repair and metabolism suggests the operation of feedback pathways in the regulation of mTORC1 operating through novel mechanisms.
Nek6 and Nercc1 (also known as Nek9) belong to the NIMA family of protein kinases. Nercc1 is activated in mitosis, whereupon it binds, phosphorylates and activates Nek6. Interference with Nek6 or ...Nercc1 in mammalian cells causes prometaphase-metaphase arrest, and depletion of Nercc1 from Xenopus egg extracts prevents normal spindle assembly. Herein we show that Nek6 is constitutively associated with Eg5 (also known as Kinesin-5 and Kif11), a kinesin that is necessary for spindle bipolarity. Nek6 phosphorylated Eg5 at several sites in vitro and one of these sites, Ser1033, is phosphorylated in vivo during mitosis. Whereas CDK1 phosphorylates nearly all Eg5 at Thr926 during mitosis, Nek6 phosphorylates ~3% of Eg5, primarily at the spindle poles. Eg5 depletion caused mitotic arrest, resulting in cells with a monopolar spindle. This arrest could be rescued by wild-type Eg5 but not by Eg5Thr926Ala. Despite substantial overexpression, Eg5Ser1033Ala rescued 50% of cells compared with wild-type Eg5, whereas an Eg5Ser1033Asp mutant was nearly as effective as wild type. Thus, during mitosis Nek6 phosphorylates a subset of Eg5 polypeptides at a conserved site, the phosphorylation of which is crucial for the mitotic function of Eg5.
Insulin activation of mTOR complex 1 is accompanied by enhanced binding of substrates. We examined the mechanism and contribution of this enhancement to insulin activation of mTORC1 signaling in 293E ...and HeLa cells. In 293E, insulin increased the amount of mTORC1 retrieved by the transiently expressed nonphosphorylatable 4E-BP5A to an extent that varied inversely with the amount of PRAS40 bound to mTORC1. RNAi depletion of PRAS40 enhanced 4E-BP5A binding to ∼70% the extent of maximal insulin, and PRAS40 RNAi and insulin together did not increase 4E-BP5A binding beyond insulin alone, suggesting that removal of PRAS40 from mTORC1 is the predominant mechanism of an insulin-induced increase in substrate access. As regards the role of increased substrate access in mTORC1 signaling, RNAi depletion of PRAS40, although increasing 4E-BP5A binding, did not stimulate phosphorylation of endogenous mTORC1 substrates S6K1(Thr389) or 4E-BP (Thr37/Thr46), the latter already ∼70% of maximal in amino acid replete, serum-deprived 293E cells. In HeLa cells, insulin and PRAS40 RNAi also both enhanced the binding of 4E-BP5A to raptor but only insulin stimulated S6K1 and 4E-BP phosphorylation. Furthermore, Rheb overexpression in 293E activated mTORC1 signaling completely without causing PRAS40 release. In the presence of Rheb and insulin, PRAS40 release is abolished by Akt inhibition without diminishing mTORC1 signaling. In conclusion, dissociation of PRAS40 from mTORC1 and enhanced mTORC1 substrate binding results from Akt and mTORC1 activation and makes little or no contribution to mTORC1 signaling, which rather is determined by Rheb activation of mTOR catalytic activity, through mechanisms that remain to be fully elucidated.
Background: Insulin-stimulated dissociation of PRAS40 from mammalian target of rapamycin (mTOR) complex 1 is proposed to promote signaling by enabling increased substrate binding.
Results: PRAS40 release from mTOR complex 1 does promote 4E-BP binding but not mTOR signaling.
Conclusion: Increased substrate binding is the result and not a cause of mTOR complex 1 signaling.
Significance: The mechanism of mTOR activation and the functions of PRAS40 phosphorylation remain to be defined.
Abstract
To identify the cellular components that participate in the regulation of mTOR complex 1 (mTORC1), the amino acid-dependent, rapamycin-inhibitable complex, we carried out a genome-wide RNAi ...depletion screen. We employed a rabbit monoclonal antibody specific for RPS6 Ser235P/Ser236P and high content microscopy to quantify rpS6 phosphorylation in the pancreatic ductal adenocarcinoma cancer cell line (PDAC) MiaPaCa-2. Applying a stringent selection, we retrieved over 600 genes wherein at least two RNAi gave significant reduction in S6 phosphorylation. This cohort is significantly enriched in genes whose depletion affects the proliferation/viability of the large set of cancer cell lines in the Achilles database in a manner paralleling that caused by mTOR depletion. To identify mTORC1 regulators that did not require the TSC, we examined which of the positives identified in MiaPaCa-2 cells were also required for S6 phosphorylation in TSC1 null mouse embryo fibroblasts. Among the 541 such gene products examined in TSC1 null MEFs, RNAi pools directed against 79 were found to reduce S6 phosphorylation significantly in 2 or 3 replicates. Among these, GPCRs, other transmembrane proteins and several signaling proteins comprised 30%, elements concerned with DNA structure and transcription, RNA processing and translation together comprised 28%, and the remainder distributed among proteins concerned with membrane transport and traffic, protein traffic and modification, mitochondria, metabolism and the cytoskeleton. Surprisingly, the only elements among this cohort previously associated with the maintenance of mTORC1 activity are two of the numerous subunits of the vacuolar ATPase subunits and the CUL4 subunit DDB1. RNAis against another 84 TSC1null MEF targets were observed to reduce S6 phosphorylation in only one of three replicates, however an indication that this group also bears attention is the presence of RPS6KB1 itself, as well as Rac1 and MAP4K3, a protein kinase that supports amino acid signaling of mTORC1 to RPS6KB1. Our screen confirmed previously known and proposed regulators of S6 phosphorylation and mTORC1 in addition to previously unidentified mTORC1 related proteins. Our results suggest the potential operation of feedback pathways from mRNA translation, RNA processing and DNA repair in the regulation of mTORC1. Since many cancers exhibit mTOR hyperactivation, identifying previously unappreciated proteins needed for maintenance of mTORC1 activity may provide new drug targets and lead to the development of beneficial therapies for tumors sensitive to mTOR inhibition. In addition, we found 43 genes that matched the mTOR essentiality profile in 216 different cancer cell lines represented in the Achilles dataset. These genes may help delineate a more general cellular state characterized by co-dependency of mTOR dependency and the respective P-S6 positive genes therefore providing the rationale for selecting specific proteins for further analysis as candidate drug targets.
Citation Format: Angela Papageorgiou, Pablo Tamayo, Jill Mesirov, Joseph Avruch, Joseph Rapley. A genome-wide siRNA screen in mammalian cells for regulators of S6 phosphorylation. abstract. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A30.
The signalling function of mTOR complex 1 is activated by Rheb-GTP, which controls the catalytic competence of the mTOR (mammalian target of rapamycin) kinase domain by an incompletely understood ...mechanism. Rheb can bind directly to the mTOR kinase domain, and association with inactive nucleotide-deficient Rheb mutants traps mTOR in a catalytically inactive state. Nevertheless, Rheb-GTP targets other than mTOR, such as FKBP38 (FK506-binding protein 38) and/or PLD1 (phospholipase D(1)), may also contribute to mTOR activation. Once activated, the mTOR catalytic domain phosphorylates substrates only when they are bound to raptor (regulatory associated protein of mTOR), a separate polypeptide within the complex. The mechanism of insulin/nutrient stimulation of mTOR complex 1 signalling, in addition to Rheb-GTP activation of the mTOR catalytic function, also involves a stable modification of the configuration of mTORC1 (mTOR complex 1) that increases access of substrates to their binding site on the raptor polypeptide. The mechanism underlying this second step in the activation of mTORC1 is unknown.
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