1. Rheb Binds and Regulates the mTOR Kinase
Xiaomeng Long, Yenshou Lin, Sara Ortiz-Vega, Kazuyoshi Yonezawa, Joseph Avruch 
Current Biology 
Volume 15, Issue 8, Pages (April 2005)
DOI: /j.cub
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2. Figure 1 Characterization of Wild-Type and Mutant Rheb
(A) The effect of wild-type and mutant Rheb on the phosphorylation in vivo of p70S6K in amino-acid-replete and -deficient medium. The effect of wild-type and mutant Rheb on the phosphorylation in vivo of p70S6K in amino-acid-replete and -deficient medium. HEK293T cells were transfected with pEBG-p70S6K (lanes 1–28) with pCMV5-FLAG vector (lanes 1, 2, 11, 12, 17, 18, 23, and 24) or with pCMV5-FLAG-Rheb wild-type (lanes 3, 4, 13, 14, 25, and 26) or various mutants as indicated above each lane. 40 hr later, some plates were transferred to D-PBS (lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28) and all cells were harvested 2 hr thereafter. Each panel represents a separate experiment. The bar graph is a quantitation of the anti-Thr412-P immunoblot results for the experiments shown with the value of S6K coexpressed with empty vector set to 100. A quantitative summary of the effect of recombinant Rheb on S6K(Thr412P) in replete medium as a % of vector control is: Rheb(wild-type) (0.5 μg/plate, n = 8; not including the experiment in lanes 23 and 25) = 194 ± 49 (= 1 SD)%; Rheb(Asp60Ile) (2.8 μg/plate, n = 3; 3.5 μg, n = 1) 60 ± 16 (= 1 SD)%; Rheb(Ser20Asn), two experiments (0.5 μg/plate) 40.6% and 59.6%.
(B) Guanyl nucleotide binding in vivo by wild-type and mutant Rheb. GST or GST fusions of wild-type or mutant Rheb were transiently expressed in HEK293T cells in triplicate with 0.3 μg of the pEBG vector, except for Ser20Asn (1.0 μg) and Asp60Ile (1.0 or 2.0 μg). 40 hr later, the cells were washed and incubated in phosphate-free DMEM containing 0.2 mCi/ml of [32P] orthophosphate. After 4 hr, the GST and the GST-fusion proteins were extracted and purified by GSH affinity chromatography and washed, and the bound guanyl nucleotides were extracted and separated by thin layer chromatography as described in methods. 32P comigrating with GDP and GTP were quantified by phosphoimager; the total [32P-GDP + GTP] (in arbitrary phosphoimager units) and the percentage of total [32P-GDP + GTP] as GTP are shown, ± one standard deviation. The GST immunoblot shows the relative amounts of purified GST proteins.
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3. Figure 2
Recombinant Rheb Binds Specifically to Endogenous and Recombinant mTOR
(A) Recombinant Rheb binds to endogenous mTOR and raptor. HEK293T cells were transfected with pEBG (lane 1) or pEBG-Rheb wild-type (lane 2). 40 hr later, cells were extracted, and the GST fusion proteins were purified on GSH-Sepharose. The GSH eluates and aliquots of the extract were analyzed by immunoblot for mTOR (top two panels) and raptor (panels 3 and 4 from top). A Coomassie blue stain of the GSH eluate is in the bottom panel.
(B) Coprecipitation of endogenous mTOR and TSC2 by recombinant wild-type or mutant Rheb. HEK293T cells were transfected with pEBG (lanes 1 and 6) or pEBG-Rheb wild-type (lanes 2 and 7) or pEBG-Rheb mutants (Thr38Met, lane 3; Ile39Lys, lane 4; Gln64Leu, lane 5; Ser20Asn, lane 8). The cells were processed as in Figure 2A. The GSH eluates and aliquots of the extract were analyzed by immunoblot for mTOR (top two panels) and TSC2 (fourth and fifth panels from top). A Coomassie blue stain of the GSH eluate is shown in the bottom panel. The bar graph in the third panel from top shows the ratio of endogenous mTOR recovered relative to GST-Rheb with the value for wild-type Rheb set to 100.
(C) mTOR binds preferentially to Rheb as compared to Ras and Rap1b. HEK293T cells were transfected with pcDNA1-HA-mTOR with pEBG (lane 1), pEBG-Rheb (lane 2), Ha-Ras(Gly12Val) (lane 3), or Rap1b(Gly12Val) (lane 4). 40 hr thereafter, some cells were harvested. The GST fusion proteins purified on GSH-Sepharose were eluted and analyzed by anti-HA immunoblot (top) and Coomassie blue stain (bottom).
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4. Figure 3
Recombinant Rheb Binds to the mTOR Catalytic Domain In Vivo and In Vitro
(A) GST-Rheb binding to mTOR fragments during transient expression. HEK293T cells were transfected with plasmids encoding FLAG-tagged mTOR fragments and pEBG vector or pEBG-Rheb. 40 hr later, cells were harvested and lysates subjected to GSH-Sepharose affinity purification. The eluates and cell lysates were analyzed by anti-FLAG immunoblot or by Coomassie blue stain. The recovery of the FLAG-tagged mTOR fragments with GST-Rheb is indicated on the right as + or -. The immunoblot data supporting this summary is shown in Figure S3.
(B) GST-Rheb specifically binds purified mTOR 2148–2300 directly in vitro. The FLAG-tagged mTOR fragments 1967–2191, 1967–2147, 2148–2549, and 2148–2300 were each expressed individually in HEK293T cells and eluted after immunopurification on immobilized anti-FLAG monoclonal antibody. Aliquots of each FLAG-mTOR fragment were incubated in vitro with GSH-Sepharose-immobilized GST (lanes 3, 6, 9, and 12) or GST-Rheb (lanes 2, 5, 8, and 11), both also purified after transient expression and charged in vitro with GMPPNP. After being washed, the polypeptides retained on GSH-Sepharose were analyzed by anti-FLAG immunoblot (top) and Coomassie blue stain (bottom). An aliquot representing 10% of the FLAG-polypeptide loaded with the GST proteins is shown in lanes 1, 4, 7, and 10.
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5. Figure 4
Rheb Binding to Other PIKKs, Conventional Protein Kinases, LST8, and Raptor
(A) GST-Rheb binds to homologous regions of the catalytic domains of the PIKKs, mTOR, ATM, and ATR but not to the catalytic domains of several subfamilies of conventional protein kinases. In the experiment shown in the upper panels, pEBG or pEBG-Rheb was coexpressed with plasmids encoding FLAG-tagged mTOR 2148–2549 (lanes 1 and 2), ATR 2288–2644 (lanes 3 and 4), or ATM 2678–3056 (lanes 5 and 6). A second experiment is shown in the lower panels in which pEBG or pEBG-Rheb was coexpressed with plasmids encoding FLAG-tagged mTOR 2148–2549 (lanes 7 and 8), MST1 1–326 (lanes 9 and 10), Nek6 (lanes 11 and 12), or p70S6KΔCT104 (lanes 13 and 14). The affinity-purified GST fusion proteins were eluted with GSH and examined by anti-FLAG immunoblot and Coomassie blue stain. An anti-FLAG immunoblot of the cell lysates is also shown (middle).
(B) GST-Rheb binds to LST8, the raptor carboxyterminal WD domain, and the TSC2 GAP domain. HEK293T cells were transfected with pEBG (not shown) or pEBG-Rheb and plasmids encoding p70S6K, mTOR complex components, or fragments thereof (as indicated above each lane). 40 hr later, the cells were harvested, and GST-Rheb was processed as in Figure 3A. No FLAG immunoreactivity coprecipitated with GST (not shown).
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6. Figure 5
The Effect of Guanyl Nucleotide on Rheb Binding In Vitro to the c-Raf1 Ras Binding Domain, the mTOR Catalytic Domain, LST8, the Raptor WD Domain, and the TSC2 GAP Domain
GST-Rheb, purified on GSH-Sepharose after transient expression in HEK293 cells, was either stripped of endogenous nucleotide with EDTA and washed in nucleotide-free buffer or charged in vitro with GDP or GMPPNP. Recombinant GST was treated in parallel. Each species of GST or GST-Rheb was incubated with immunoaffinity-purified, soluble FLAG-tagged polypeptides: c-Raf1 1–257 (lanes 2–7), mTOR 2148–2549 (lanes 9–14), LST8 (lanes 16–21), raptor 1009–1335 (lanes 23–28), or TSC2 1531–1807 (lanes 30–35). After being washed, the adsorbed polypeptides were subjected to SDS-PAGE and analyzed by anti-FLAG immunoblot and Coomassie blue stain. Lanes 1, 8, 15, 22, and 29 contain 10% of the amount of FLAG-polypeptide added into the binding reaction.
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7. Figure 6 Regulation of TOR Kinase Activity by Rheb
(A) Coexpression of wild-type but not kinase-dead mTOR with GST-Rheb increases the in vitro Rheb-associated kinase activity toward p70S6K and 4E-BP. Left, GST-Rheb expressed alone (lane 2) or with HA-mTOR (lane 1) was purified on GSH-Sepharose and equal amounts of GST-Rheb polypeptide (second panel from top) were assayed for kinase activity (lowest three panels) toward a recombinant p70S6(355–525) polypeptide (third panel from bottom). Substrate phosphorylation in vitro was estimated by overall incorporation of 32P (second panel from bottom) and by anti-p70S6K(Thr412P) immunoblot (bottom). Right, the kinase activity associated with GST (lane 1) or GST-Rheb (lanes 2 and 3) expressed with wild-type HA-mTOR (lane 2) was compared to GST-Rheb expressed with the kinase-inactive mTOR mutant (Asn2343Lys) (lane 3) in assays equalized for identical amounts of mTOR polypeptide (top). The phosphorylation of p70S6K 355–525 (middle three panels) and 4E-BP1 (lowest three panels) was analyzed by overall incorporation of 32P (second and fourth panels from bottom) and by immunoblot against p70S6K(Thr421P) (fourth panel from bottom) and 4E-BP1(Thr37P, Thr46P), respectively. Each experiment was repeated twice with nearly identical results.
(B) mTOR bound to guanyl nucleotide-deficient Rheb mutants exhibits little or no protein kinase activity. HA-mTOR was expressed with GST (lane 2) or with GST fusions to Rheb wild-type (lane 3), Rheb(Ser20Asn) (lane 4), or Rheb(Asp60Ile) (lane 5). After purification on GSH-Sepharose, the bound proteins were eluted, and aliquots containing equal amounts of HA-mTOR polypeptide (second panel from bottom) were assayed for kinase activity toward p70S6K(355–525) (lanes 1–5) and 4E-BP1 (lanes 6–10). Substrate phosphorylation was estimated by overall 32P incorporation, displayed both by a representative autoradiograph (fifth panel from bottom) and quantitatively by phosphoimager (fourth panel from bottom). The bar graphs display the combined results from three experiments, as a percentage (± 1 SD) of the mTOR kinase activity, set to 100, associated with wild-type Rheb. Representative immunoblots with anti-p70S6K(Thr412P) (third panel from bottom, lanes 1–5) and anti-4E-BP1(Thr37P, Thr46P) (third panel from bottom, lanes 6–10) antibodies are shown.
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8. Figure 7
mTOR Bound to Rheb(Gln64Leu) Has Higher Kinase Activity than mTOR Bound to Wild-Type Rheb
(A) Immunoblots of lysates from cells coexpressing HA-mTOR with GST or with GST fusions to Rheb wild-type, Rheb(Gln64Leu), or Rheb(Asp60Ile).
(B) Immunoblots of the GST proteins and copurifying HA-mTOR polypeptides after purification on GSH-Sepharose. Lanes 1–12 demonstrate the GST polypeptides as 4×, 2×, and 1× aliquots (bottom) and associated HA-mTOR (top).
(C) A repesentative kinase assay of the GST bound HA-mTOR polypeptides. After elution with GSH, aliquots containing equal amounts of HA-mTOR polypeptide, as shown by immunoblot (top) were assayed for kinase activity toward p70S6K(355–525) (third panel from top). Substrate phosphorylation was estimated by immunoblot with anti-p70S6K(Thr412P) (bottom).
(D) A summary of three mTOR kinase assays. The bar graph shows results from two experiments. The results are expressed as in Figure 6B.
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