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. 2009 May 8;284(19):12783-91.
doi: 10.1074/jbc.M809207200. Epub 2009 Mar 19.

Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein

Tatsuhiro Sato  1 , Akio Nakashima, Lea Guo, Fuyuhiko Tamanoi
Affiliations

Specific activation of mTORC1 by Rheb G-protein in vitro involves enhanced recruitment of its substrate protein

Tatsuhiro Sato et al. J Biol Chem. .

Abstract

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.

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Figures

FIGURE 1.
FIGURE 1.
Recombinant Rheb activates mTORC1 but not mTORC2 in vitro. A, a schematic procedure of mTOR in vitro kinase assay is outlined. The cells are first starved for serum and amino acids to shut down mTOR signaling. The cells are then lysed, and mTORC1 or mTORC2 is immunopurified using anti-raptor or -rictor antibody, respectively. In the raptor complex, mTOR and mLST8/GβL are contained together with raptor, whereas the rictor complex has mTOR, mLST8/GβL, rictor and Sin1. Each preparation is mixed with a specific substrate, 4E-BP1 for mTORC1 or Akt for mTORC2, in the kinase buffer supplemented with 0.2 mm ATP and 10 mm MgCl2, and incubated for 2–20 min at 37 °C. The kinase activities are estimated by the phosphorylation level of the substrates. B, in vitro kinase assay of mTOR complexes was performed in the absence or presence of recombinant Rheb loaded with GTPγS at 37 °C for 20 min. The proteins were resolved by SDS-PAGE, and the indicated protein bands were detected by Western blotting. C, time course analysis of mTORC1 activity was performed in the presence (squares) or absence (triangles) of Rheb-GTPγS. Phosphorylation of 4E-BP1 at Thr37/46 was detected by Western blotting. The relative band intensities were measured using Scion Image. The standard deviation was derived from three independent experiments. IP, immunoprecipitation.
FIGURE 2.
FIGURE 2.
Rheb G-protein specifically activates mTORC1. A, the phylogenetic tree of Ras superfamily was generated using ClustalW program. The proteins used in the following experiments were indicated by dots. B, G-proteins indicated were purified from E. coli, loaded with GTPγS, and used for mTORC1 kinase assay in vitro. The proteins were resolved by SDS-PAGE, and phospho-4E-BP1 at Thr37/46 was detected using phospho-specific antibody. C, GTPγS-bound Rheb (GTPγS), GDP-bound Rheb (GDP), and the nucleotide free form of Rheb (Free) were prepared and used for the in vitro mTORC1 kinase assay. The proteins were resolved by SDS-PAGE, and the indicated protein bands were detected by Western blotting. D, in vitro kinase activity of mTORC1 was compared in the absence (Control) or in the presence of recombinant Rheb (Rheb1) or recombinant Rheb2 loaded with GTPγS. Phospho-4E-BP1 at Thr37/46 was detected by Western blotting.
FIGURE 3.
FIGURE 3.
Characterization of Rheb mutants. A, 0.5 μg of each protein was incubated in the buffer containing 0.2 μCi of [35S]GTPγS for 10 min at 37 °C to examine GTP binding. B, wild type (wt) and mutant Rheb proteins were purified from E. coli and were loaded with GTPγS. Each protein was mixed with mTORC1 immunoprecipitates, and mTORC1 activity was evaluated in vitro. The proteins were resolved by SDS-PAGE, and phospho-4E-BP1 at Thr37/46 was detected by Western blotting.
FIGURE 4.
FIGURE 4.
Rheb activation of mTORC1 is independent of FKBP38. A, cells were starved for serum and amino acids and lysed with lysis buffer containing 25 mm NaCl (Lysate). The concentration of sodium chloride was then adjusted as indicated and used for mTORC1 immunoprecipitation (IP). The amount of mTORC1 components and FKBP38 in the lysate or in immunopurified mTORC1 was detected by Western blotting. B, small interference RNA against FKBP38 (FKBP38 siRNA) was transfected in HEK293T cells. Scramble siRNA transfected cells were used as control cells (control siRNA). After culturing for 3 days, the cells were starved for nutrients and lysed with lysis buffer as described under “Experimental Procedures,” and the amount of FKBP38, phospho-S6 at Ser235/236, and total S6 was examined by Western blotting (left panel). From these lysates, mTORC1 was immunopurified, and the in vitro activity was measured in the presence or absence of recombinant Rheb-GTPγS (right panel). The proteins were resolved by SDS-PAGE, and phospho-4E-BP1 at Thr37/46 was analyzed by Western blotting. C, cells were transfected with pCDNA3 (control), FLAG-FKBP38 and/or FLAG-Rheb, respectively. The cells were starved for serum and amino acids (AA –) or restimulated with amino acid mixture after amino acid starvation (AA +). The proteins were resolved by SDS-PAGE and analyzed by Western blotting.
FIGURE 5.
FIGURE 5.
Rheb enhances the binding of 4E-BP1 to mTORC1. A, amino acid-starved cells were collected, and mTORC1 was immunoprecipitated using anti-raptor antibody. The immunoprecipitated mTORC1 was then mixed with 1 μg of recombinant 4E-BP1 in the presence or absence of Rheb, and 4E-BP1 binding assay was performed in the buffer with or without ATP at 37 °C for 20 min. After washing, bound 4E-BP1 was detected by Western blot. B, wild type (wt) or mutant Rheb proteins bound with GTPγS were added to the immunopurified mTORC1, and the amount of 4E-BP1 bound to mTORC1 was measured by Scion Image after Western blot analysis using specific antibody. The standard deviation was derived from three independent experiments. C, amino acid starved cells were lysed in the lysis buffer supplemented with 0.3% CHAPS or with 1% Nonidet P-40. mTOR was immunopurified from each cell lysates using anti-mTOR antibody, and the kinase activity was measured in the presence or absence of Rheb-GTPγS. Proteins were resolved by SDS-PAGE, and phospho-4E-BP1 at Thr37/46 was detected by Western blotting. D, 4E-BP1 bound to mTORC1 was measured in the presence of indicated amount of PRAS40. After washing, the amount of 4E-BP1 or PRAS40 bound to mTORC1 was detected by Western blotting. E, the kinase assay of mTORC1 was performed in vitro. 1 μg of recombinant Rheb-GTPγS and/or described amount of recombinant PRAS40 was added before incubation at 37 °C for 20 min. After reaction, the proteins were resolved by SDS-PAGE, and phospho-4E-BP1 at Thr37/46 was detected by Western blot.
FIGURE 6.
FIGURE 6.
Rheb does not induce autophosphorylation of mTOR. A, HEK293T cells were lysed and mTORC1 was immunoprecipitated using anti-raptor antibody. The in vitro kinase assay of mTORC1 was started in the buffer containing 10 mm Mg2+, 10 mm Mg2+, and 1 μg recombinant Rheb loaded with GTPγS or 10 mm Mn2+ instead of Mg2+. 1 μg of 4E-BP1 or 10 μCi of [γ-32P]ATP was added to examine phosphorylation of 4E-BP1 or autophosphorylation of mTOR. After 20 min reaction at 37 °C, the samples were resolved by SDS-PAGE, and the phosphorylation of mTOR was detected by autoradiography. Other proteins were detected by Western blotting. B, the immunopurified mTORC1 and mTORC2 were isolated as described in Fig. 1A. The kinase assay was performed in the kinase buffer containing 10 mm Mg2+ or 10 mm Mn2+ for 20 min at 37 °C. Phospho-4E-BP1 at Thr37/46 and phospho-Akt at Ser473 were detected by Western blotting. C, HEK293T cells were lysed, and mTORC1 was immunoprecipitated using anti-raptor antibody. The in vitro kinase assay of mTORC1 was started in the kinase buffer containing 1 μg of Rheb-GTPγS and 10 mm Mg2+, 10 mm Mn2+ or the combination of 1 μg recombinant Rheb-GTPγS and 10 mm Mn2+. The kinase reaction was stopped at indicated time points. Sample proteins were resolved by SDS-PAGE, and phosphorylation of 4E-BP1 at Thr37/46 or Thr70 was detected by Western blotting using specific antibodies, respectively.
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