Sara Ortiz-Vega

TOR complex 1 (TORC1), an oligomer of the mTOR (mammalian target of rapamycin) protein kinase, its substrate binding subunit raptor, and the polypeptide Lst8/GbetaL, 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.

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 withdrawl may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1. 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 withdrawl may generate an inhibitor of the Rheb-mTOR interaction that interferes with the signaling function of TOR complex 1. IntroductionThe target of rapamycin (TOR) 1The abbreviations used are: TOR, target of rapamycin; mTOR, mammalian TOR; PKB, protein kinase B; TSC, tuberous sclerosis complex; S6K, S6 kinase; GST, glutathione S-transferase; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; GMPPNP, guanosine 5′-(β,γ-imido)triphosphate. is the founding member of the phosphatidylinositol 3′-OH kinase-related protein (Ser/Thr) kinase (PIKK) family (1Jacinto E. Hall M.N. Nat. Rev. Mol. Cell Biol. 2003; 4: 117-126Crossref PubMed Scopus (511) Google Scholar). The TOR polypeptides in Drosophila and mammalian cells are now known to be major regulators of cell growth in part through their ability to phosphorylate and control the activity of the translational regulators, the p70 S6 kinases (S6Ks), and the 4E-BPs. TOR signaling is effected by two TOR-containing complexes; TOR phosphorylation of these translational regulators is mediated by a rapamycin-sensitive complex of TOR with the polypeptides LST8 and raptor, known as TOR complex 1 (2Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 2: 158-166Crossref Scopus (440) Google Scholar). LST8 binds to and stimulates the kinase activity of the TOR catalytic domain (3Kim Do-Hung. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V.P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar), whereas raptor binds the TOR substrates 4E-BP and p70 S6K and is critical for their effective phosphorylation by the TOR catalytic domain in vivo (4Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). In turn, signaling by TOR is controlled by multiple upstream inputs provided by receptor tyrosine kinases, through their control of PtdIns 3′OH kinase and the protein kinase B (PKB), by energy sufficiency through regulation of the AMP-activated protein kinase, and by amino acid sufficiency, whose effectors in this pathway are as yet unknown (5Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3398) Google Scholar, 6Li Y. Corradetti M.N. Inoki K. Guan K-L. Trends Biochem. Sci. 2003; 29: 32-38Abstract Full Text Full Text PDF Scopus (328) Google Scholar, 7Findlay G.M. Harrington L.S. Lamb R.F. Curr. Opin. Genet. Dev. 2005; 15: 69-76Crossref PubMed Scopus (33) Google Scholar). Genetic evidence from Drosophila, fortified by genetic and biochemical data in mammalian systems, identified the critical regulators situated between PKB and TOR as the tuberous sclerosis complex (TSC), an obligatory heterodimer of the polypeptides Hamartin (TSC1) and Tuberin (TSC2), and Rheb, a Ras-like small GTPase. Rheb is a positive regulator of TOR signaling in vivo; the action of Rheb is opposed by the TSC complex, by virtue of the ability of the TSC complex to act as a Rheb GTPase activator, directly promoting the conversion of Rheb-GTP to Rheb-GDP. The inhibitory action of the TSC complex on Rheb is attenuated by PKB-catalyzed TSC2 phosphorylation, whereas the TSC-Rheb-GTPase activator activity is enhanced by AMP-activated protein kinase-catalyzed TSC2 phosphorylation (5Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3398) Google Scholar, 6Li Y. Corradetti M.N. Inoki K. Guan K-L. Trends Biochem. Sci. 2003; 29: 32-38Abstract Full Text Full Text PDF Scopus (328) Google Scholar, 7Findlay G.M. Harrington L.S. Lamb R.F. Curr. Opin. Genet. Dev. 2005; 15: 69-76Crossref PubMed Scopus (33) Google Scholar). Thus the TSC complex is a major site at which RTKs and energy sufficiency control TOR signaling.As regards the mechanisms by which Rheb acts as a positive regulator of TOR signaling, we recently demonstrated (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar) that Rheb binds directly to the smaller, amino-terminal lobe of the mTOR catalytic domain, and the kinase activity of the TOR polypeptides bound to Rheb is determined by the state of Rheb nucleotide charging. TOR polypeptides bound to mutant Rhebs that are unable to bind any guanyl nucleotide are essentially devoid of protein kinase activity; conversely, TOR polypeptides bound to RhebQ64L, a mutant that is 90% GTP-bound in vivo (9Li Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (187) Google Scholar), exhibit greater kinase activity than TOR polypeptides bound to wild type Rheb. Thus the mTOR polypeptide itself is a direct target of the Rheb GTPase. These findings do not preclude the operation of other Rheb effectors that may promote mTOR signaling indirectly, e.g. as by increasing intracellular amino acid levels. Notably, we also observed Rheb to be capable of interacting with LST8 and with raptor, independent of its ability to bind to TOR; this finding raised the possibility that, in addition to its ability to promote TOR catalytic activity, Rheb may also play a role in configuring TOR complex 1.The mechanisms and site of action of amino acids in the control of TOR signaling and its relation to the mechanisms of Rheb action are poorly understood. Withdrawal of extracellular amino acids inhibits TOR signaling in vivo (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar), as reflected by the progressive dephosphorylation of specific sites on p70 S6K (especially Thr412, a major site of direct phosphorylation by mTOR; Ref. 11Isotani S. Hara K. Tokunaga C. Inoue H. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 34493-34498Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and 4E-BP, over a period of 1–2 h. Although this response suggests an inhibition of TOR kinase activity, mTOR immunoprecipitates from amino acid-deprived cells exhibit kinase activity in vitro indistinguishable from that of mTOR isolated from amino acid-replete cells. Moreover, mutations of p70 S6K that eliminate the binding of the S6K polypeptide to raptor (when combined with a deletion of the S6K psuedosubstrate/autoinhibitory domain) render it resistant to dephosphorylation after amino acid depletion (as well as to rapamycin) (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar). Together, these results suggest that amino acid withdrawal may not alter TOR catalytic activity but rather may interfere with the ability of the TOR catalytic domain to phosphorylate raptor-bound substrates. Whatever the mechanism by which amino acid withdrawal inhibits TOR signaling, this inhibition can be overcome by excess active (i.e. GTP-charged) Rheb. Thus, overexpression of recombinant Rheb can restore the phosphorylation of p70 S6K and 4E-BP despite the lack of extracellular amino acids; this effect of Rheb is inhibited by rapamycin (12Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell. Biol. 2003; 5: 566-571Crossref PubMed Scopus (527) Google Scholar, 13Stocker H. Radimerski P. E. Nat. Cell. Biol. 2003; 5: PubMed Scopus Google Scholar, Cell. 2004; Scopus Google of the finding that Rheb can directly with the of TOR complex we the interaction of Rheb with any of these is by amino acid Herein we show that the binding of recombinant Rheb to the mTOR catalytic domain in vivo is inhibited by withdrawal of extracellular amino and all and (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar, K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar). The two mTOR by of after the the The from Cell from and from and the for amino acid or leucine withdrawal and are also by Hara (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google the of interaction and the of Rheb guanyl nucleotide charging are by (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google of extracellular amino acids results in the dephosphorylation of recombinant p70 S6K at to in p70 S6K of wild type Rheb with S6K the phosphorylation of in a despite the withdrawal of extracellular amino acids The ability of Rheb to restore phosphorylation that Rheb an 1 and domain and be capable of GTP charging in vivo (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). the effect of Rheb is to inhibition by rapamycin recently that recombinant Rheb can bind directly to mTOR and that the protein kinase activity of mTOR is by Rheb-GTP (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). Although the specific mechanism by which Rheb-GTP activates the mTOR kinase is not we amino acid sufficiency the interaction of Rheb with binds the endogenous mTOR complex 1 X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google and we show that amino acid withdrawal the of endogenous mTOR with of the with for to the of endogenous or recombinant mTOR with Rheb; removal of alone effect on the of with 1 with whereas the addition of an amino acid alone to is to restore the mTOR to the from cells in 1 with Rheb binds to the carboxyl-terminal of mTOR, to the mTOR catalytic of its ability to bind to mTOR, Rheb also binds directly to LST8 and to the carboxyl-terminal of raptor (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). the effect of amino acid withdrawal on the binding of to these polypeptides Notably, whereas the binding of to the mTOR carboxyl-terminal is inhibited by amino acid the binding of Rheb to LST8 and the raptor is that removal of of the amino acids inhibition of mTOR signaling to p70 S6K, the inhibition withdrawal of a amino acid with removal of and removal of is inhibitory (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar). we that the inhibitory on the Rheb-mTOR interaction by removal of all amino acids is largely by removal of leucine alone a response is observed with withdrawal not with its effect on mTOR signaling, the inhibitory effect of amino acid withdrawal on the Rheb-mTOR interaction is of amino acids to largely mTOR binding to Rheb is observed with to leucine regulation of Rheb-mTOR binding Thus the amino acid regulation of the a to the amino acid regulation of mTOR of Rheb to mTOR is reversibly inhibited by withdrawal of extracellular amino acids or cells with or wild type and cells to D-PBS, and all cells and the on The and of the by for mTOR two of the is in the with in cells. of the cells to or without or to amino acid the cells in isolated on the bound polypeptides and and of the cell to and and and the effect of amino acid withdrawal on Rheb binding to raptor carboxyl-terminal to and to the mTOR carboxyl-terminal of and and as for B; of the cells from to to and a of the effect of withdrawal of leucine or all amino acids on Rheb binding to cells with and or to and or to a of amino acids at a to in DMEM, leucine amino acids and and or and after cells in with all amino acids whereas of in amino acids with leucine for and as for of the binding of Rheb to its mTOR, and that with the interaction of other Ras-like with their known is that the Rheb-mTOR interaction does not Rheb GTP charging (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). Rheb polypeptides bind to mTOR than does Rheb, and Rheb-GTP it mTOR kinase activity in the of the Rheb-mTOR interaction in vivo and in vitro (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar). Notably, amino acid withdrawal interferes with the ability of mTOR to bind to wild type Rheb and to the 1 and mutants of Rheb to a in the binding of these Rheb mutants to the mTOR is also inhibited by amino acid withdrawal amino acid withdrawal also the binding of mTOR to a mutant that 90% GTP charging in vivo This that the effect of amino acid withdrawal is not mediated by changes in Rheb guanyl nucleotide charging. In amino acid withdrawal does not alter the guanyl nucleotide charging of wild type recombinant Rheb as observed by Y. Gao X. L.J. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: PubMed Scopus Google Scholar), but in to the findings of J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google inhibitory effect of amino acid withdrawal on the binding of to Rheb is not to a in Rheb GTP with wild type or mutant as a of the cells to and all The polypeptides and on and the and and of the to and and or Rheb GTP charging Amino acid withdrawal by to with to The show from a of the polypeptides for of the the effect of deletion of the mTOR on the ability of amino acids to Rheb the mTOR and bind wild type Rheb with to the binding of Rheb to the is not inhibited by amino acid whereas the binding of Rheb to the other two mTOR inhibition by amino acid withdrawal This that the regulation of the interaction by amino acid sufficiency is exerted and through an effect on mTOR, through the which to the larger, carboxyl-terminal lobe of the mTOR catalytic as to the ability of amino acid sufficiency to the ability of the to bind Rheb is in in a of which to amino acid withdrawal to The cells in the of after the of with or with the polypeptides by in binds in the mTOR polypeptides are from amino acid-replete or cells. Thus the ability of amino acid withdrawal to the ability of to bind to Rheb is not to a of the mTOR polypeptide that cell inhibitory effect of amino acid withdrawal on interaction is exerted on TOR through a site that is distinct from the amino-terminal lobe of TOR catalytic cells with and or or and and or and to and and as in the to the mTOR in cells for leucine and for The mTOR and from of in vitro with or also after and in vitro with the polypeptides on by and of the with the is in mechanism by which amino acid withdrawal acts the carboxyl-terminal lobe of the mTOR catalytic domain to interfere with Rheb binding to the amino-terminal lobe is not with mTOR kinase activity (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar), the of the inhibitory effect of amino acid withdrawal to cell a mechanism that of the mTOR polypeptide. this suggests that the in vivo inhibition of mTOR signaling and the interaction by amino acid withdrawal is to the of an inhibitor that binds to the mTOR catalytic domain and interferes by a mechanism with the S6K phosphorylation the TOR complex as well as with the ability of mTOR to bind Rheb. The inhibitor is on cell the mTOR kinase activity in as well as the ability of the mTOR catalytic domain to bind Rheb in The inhibitory effect of amino acid withdrawal on mTOR signaling may be directly to the inhibition of Rheb binding to mTOR; the ability of Rheb to overcome the inhibitory effect of amino acid withdrawal may be to the cell with an excess of Rheb-GTP that is to overcome the effect of the The ability of Rheb to bind to raptor and the other of the TOR complex may also be to the mechanism of Rheb action the Amino acid withdrawal to the of raptor with mTOR but in that LST8 (3Kim Do-Hung. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V.P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar, Do-Hung. Sarbassov D.D. Ali S.M. Latek R.R. H. Tempst P. Sabatini D.M. Cell. Full Text Full Text PDF PubMed Scopus Google that is to the ability of amino acid withdrawal to Rheb binding to mTOR is IntroductionThe target of rapamycin (TOR) 1The abbreviations used are: TOR, target of rapamycin; mTOR, mammalian TOR; PKB, protein kinase B; TSC, tuberous sclerosis complex; S6K, S6 kinase; GST, glutathione S-transferase; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; GMPPNP, guanosine 5′-(β,γ-imido)triphosphate. is the founding member of the phosphatidylinositol 3′-OH kinase-related protein (Ser/Thr) kinase (PIKK) family (1Jacinto E. Hall M.N. Nat. Rev. Mol. Cell Biol. 2003; 4: 117-126Crossref PubMed Scopus (511) Google Scholar). The TOR polypeptides in Drosophila and mammalian cells are now known to be major regulators of cell growth in part through their ability to phosphorylate and control the activity of the translational regulators, the p70 S6 kinases (S6Ks), and the 4E-BPs. TOR signaling is effected by two TOR-containing complexes; TOR phosphorylation of these translational regulators is mediated by a rapamycin-sensitive complex of TOR with the polypeptides LST8 and raptor, known as TOR complex 1 (2Martin D.E. Hall M.N. Curr. Opin. Cell Biol. 2005; 2: 158-166Crossref Scopus (440) Google Scholar). LST8 binds to and stimulates the kinase activity of the TOR catalytic domain (3Kim Do-Hung. Sarbassov D.D. Ali S.M. Latek R.R. Guntur K.V.P. Erdjument-Bromage H. Tempst P. Sabatini D.M. Mol. Cell. 2003; 11: 895-904Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar), whereas raptor binds the TOR substrates 4E-BP and p70 S6K and is critical for their effective phosphorylation by the TOR catalytic domain in vivo (4Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). In turn, signaling by TOR is controlled by multiple upstream inputs provided by receptor tyrosine kinases, through their control of PtdIns 3′OH kinase and the protein kinase B (PKB), by energy sufficiency through regulation of the AMP-activated protein kinase, and by amino acid sufficiency, whose effectors in this pathway are as yet unknown (5Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3398) Google Scholar, 6Li Y. Corradetti M.N. Inoki K. Guan K-L. Trends Biochem. Sci. 2003; 29: 32-38Abstract Full Text Full Text PDF Scopus (328) Google Scholar, 7Findlay G.M. Harrington L.S. Lamb R.F. Curr. Opin. Genet. Dev. 2005; 15: 69-76Crossref PubMed Scopus (33) Google Scholar). Genetic evidence from Drosophila, fortified by genetic and biochemical data in mammalian systems, identified the critical regulators situated between PKB and TOR as the tuberous sclerosis complex (TSC), an obligatory heterodimer of the polypeptides Hamartin (TSC1) and Tuberin (TSC2), and Rheb, a Ras-like small GTPase. Rheb is a positive regulator of TOR signaling in vivo; the action of Rheb is opposed by the TSC complex, by virtue of the ability of the TSC complex to act as a Rheb GTPase activator, directly promoting the conversion of Rheb-GTP to Rheb-GDP. The inhibitory action of the TSC complex on Rheb is attenuated by PKB-catalyzed TSC2 phosphorylation, whereas the TSC-Rheb-GTPase activator activity is enhanced by AMP-activated protein kinase-catalyzed TSC2 phosphorylation (5Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3398) Google Scholar, 6Li Y. Corradetti M.N. Inoki K. Guan K-L. Trends Biochem. Sci. 2003; 29: 32-38Abstract Full Text Full Text PDF Scopus (328) Google Scholar, 7Findlay G.M. Harrington L.S. Lamb R.F. Curr. Opin. Genet. Dev. 2005; 15: 69-76Crossref PubMed Scopus (33) Google Scholar). Thus the TSC complex is a major site at which RTKs and energy sufficiency control TOR signaling.As regards the mechanisms by which Rheb acts as a positive regulator of TOR signaling, we recently demonstrated (8Long X. Lin Y. Ortiz-Vega S. Yonezawa K. Avruch J. Curr. Biol. 2005; 15: 702-713Abstract Full Text Full Text PDF PubMed Scopus (730) Google Scholar) that Rheb binds directly to the smaller, amino-terminal lobe of the mTOR catalytic domain, and the kinase activity of the TOR polypeptides bound to Rheb is determined by the state of Rheb nucleotide charging. TOR polypeptides bound to mutant Rhebs that are unable to bind any guanyl nucleotide are essentially devoid of protein kinase activity; conversely, TOR polypeptides bound to RhebQ64L, a mutant that is 90% GTP-bound in vivo (9Li Y. Inoki K. Guan K.L. Mol. Cell. Biol. 2004; 24: 7965-7975Crossref PubMed Scopus (187) Google Scholar), exhibit greater kinase activity than TOR polypeptides bound to wild type Rheb. Thus the mTOR polypeptide itself is a direct target of the Rheb GTPase. These findings do not preclude the operation of other Rheb effectors that may promote mTOR signaling indirectly, e.g. as by increasing intracellular amino acid levels. Notably, we also observed Rheb to be capable of interacting with LST8 and with raptor, independent of its ability to bind to TOR; this finding raised the possibility that, in addition to its ability to promote TOR catalytic activity, Rheb may also play a role in configuring TOR complex 1.The mechanisms and site of action of amino acids in the control of TOR signaling and its relation to the mechanisms of Rheb action are poorly understood. Withdrawal of extracellular amino acids inhibits TOR signaling in vivo (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar), as reflected by the progressive dephosphorylation of specific sites on p70 S6K (especially Thr412, a major site of direct phosphorylation by mTOR; Ref. 11Isotani S. Hara K. Tokunaga C. Inoue H. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 34493-34498Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) and 4E-BP, over a period of 1–2 h. Although this response suggests an inhibition of TOR kinase activity, mTOR immunoprecipitates from amino acid-deprived cells exhibit kinase activity in vitro indistinguishable from that of mTOR isolated from amino acid-replete cells. Moreover, mutations of p70 S6K that eliminate the binding of the S6K polypeptide to raptor (when combined with a deletion of the S6K psuedosubstrate/autoinhibitory domain) render it resistant to dephosphorylation after amino acid depletion (as well as to rapamycin) (10Hara K. Yonezawa K. Weng Q-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 272: 14484-14494Abstract Full Text Full Text PDF Scopus (1101) Google Scholar). Together, these results suggest that amino acid withdrawal may not alter TOR catalytic activity but rather may interfere with the ability of the TOR catalytic domain to phosphorylate raptor-bound substrates. Whatever the mechanism by which amino acid withdrawal inhibits TOR signaling, this inhibition can be overcome by excess active (i.e. GTP-charged) Rheb. Thus, overexpression of recombinant Rheb can restore the phosphorylation of p70 S6K and 4E-BP despite the lack of extracellular amino acids; this effect of Rheb is inhibited by rapamycin (12Saucedo L.J. Gao X. Chiarelli D.A. Li L. Pan D. Edgar B.A. Nat. Cell. Biol. 2003; 5: 566-571Crossref PubMed Scopus (527) Google Scholar, 13Stocker H. Radimerski P. E. Nat. Cell. Biol. 2003; 5: PubMed Scopus Google Scholar, Cell. 2004; Scopus Google of the finding that Rheb can directly with the of TOR complex we the interaction of Rheb with any of these is by amino acid Herein we show that the binding of recombinant Rheb to the mTOR catalytic domain in vivo is inhibited by withdrawal of extracellular amino

MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2-5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 mM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1-MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.