Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling

Abstract

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutation in either TSC1 or TSC2. The TSC1 and TSC2 gene products form a functional complex and inhibit phosphorylation of S6K and 4EBP1. These functions of TSC1/TSC2 are likely mediated by mTOR. Here we report that TSC2 is a GTPase-activating protein (GAP) toward Rheb, a Ras family GTPase. Rheb stimulates phosphorylation of S6K and 4EBP1. This function of Rheb is blocked by rapamycin and dominant-negative mTOR. Rheb stimulates the phosphorylation of mTOR and plays an essential role in regulation of S6K and 4EBP1 in response to nutrients and cellular energy status. Our data demonstrate that Rheb acts downstream of TSC1/TSC2 and upstream of mTOR to regulate cell growth.

Figure 1.

TSC2 has GAP activity toward Rheb. (A) Rheb and TSC2 are an unusual GTPase and GAP. The catalytic active-site arginine (the residue in bold) in RapGAP is not conserved in TSC2. Rheb contains an arginine residue (the residue in bold) at the position corresponding to codon 12 of Ras, which has a glycine. Numbers indicate positions of the last residues. (B) Immunoprecipitated TSC1/TSC2 stimulates GTP hydrolysis of Rheb. Increasing amounts (in microliters) of immunoprecipitated TSC1/TSC2 were incubated with GST-Rheb at room temperature for 20 min. Release of free ³²P-phosphate was measured by radioactive counting. Background activity of a control immunoprecipitation was subtracted. The basal GTPase activity of Rheb was arbitrarily set as 1. (C) Time-dependent GTP hydrolysis of Rheb stimulated by TSC1/TSC2. GTP hydrolysis was determined in the absence (▪) and presence (▵) of TSC1/TSC2. (D) Neither the N-terminal nor the C-terminal region of TSC2 has GAP activity toward Rheb. Two truncated TSC2 constructs (TSC2N, amino acids 1-1007; TSC2C, amino acids 1008-1765) were coexpressed with TSC1 and immunoprecipitated and assayed for GAP activity in vitro. The amount of HA-TSC2 used in this assay is equivalent to 1 μL in B. The expression levels of these proteins were determined by Western blot. (E) TSC2, but not TSC1, has GAP activity. Transfected HEK293 cells were untreated or treated with D-PBS, rapamycin (20 nM), or LY294002 (50 μM) for 30 min as indicated. The relative amount of TSC2 used in the GAP assay was determined by Western blot and is shown below each bar. (F) TSC1/TSC2 decreases the Rheb-GTP levels in vivo. Myc-Rheb was transfected in HEK293 cells and labeled with ³²P-phosphate. Myc-Rheb was immunoprecipitated, and the bound nucleotides were eluted and resolved on a cellulose plate. Cotransfection with TSC1 and TSC2 are indicated. Myc-RacL61 was included as a control. The ratio of GTP/GDP was calculated by the formula GTP counts/3 divided by GDP counts/2, and indicated on top of each lane.

Figure 2.

Rheb stimulates phosphorylation of S6K and 4EBP1. (A) Rheb stimulates phosphorylation of S6K and 4EBP1. HA-S6K and Flag-4EBP1 were transfected into HEK293 cells in the presence or absence of Myc-Rheb (100 ng). Phosphorylation of S6K was determined by phosphospecific antibody, pS6K(T389), whereas phosphorylation of 4EBP1 was determined by mobility shift (upper panels). Phosphorylation of endogenous S6K was also determined (lower panels). (B) Rheb stimulates phosphorylation of S6K in a dose-dependent manner. HA-S6K (15 ng) was cotransfected with 0, 2, 5, 20, 50, and 100 ng of Myc-Rheb. (C) The effector domain of Rheb is required to stimulate S6K phosphorylation. HA-S6K was cotransfected with wild-type Rheb (100 ng), RhebL64 (40 ng), Rheb-5A (200 ng), and RhebN20 (300 ng). The relative expression of Rheb mutants is also shown. (D) Stimulation of S6K phosphorylation by different GTPases. Constitutively active mutants (100 ng for each) of Cdc42, Rap1, Rab5, RhoA, and the wild-type Rheb (40 ng) were used to stimulate phosphorylation of S6K. (E) TSC2, but not TSC1, inhibits S6K phosphorylation. HA-S6K was coexpressed with either Myc-TSC1 or HA-TSC2. Phosphorylation of S6K was determined.

Figure 3.

TSC1/TSC2 inhibits Rheb function in vivo. (A) TSC1/TSC2 inhibits the ability of Rheb to induce S6K phosphorylation. (B) TSC1/TSC2 inhibits the ability of Rheb to induce 4EBP1 phosphorylation.

Figure 4.

Rheb functions upstream of mTOR and is involved in response to various signals. (A) Rheb acts downstream of nutrient signals and upstream of mTOR. HA-S6K was cotransfected with RhebL64 or RasV12 as indicated. Cells were treated with rapamycin or D-PBS for 30 min. Phosphorylation of S6K was determined. (B) Kinase inactive mTOR KD blocks Rheb-induced S6K phosphorylation. (C) Rheb stimulates the phosphorylation of mTOR. Flag-mTOR or GST-Akt was cotransfected with Rheb and immunoprecipitated. (Left) Phosphorylation of mTOR was detected by the anti-phospho mTOR (S2448) antibody. (Right) Phosphorylation of Akt was monitored by the anti-phospho Akt (S473) antibody. (D) Role of Rheb in S6K regulation by various signaling pathways. HA-S6K was transfected alone (left half) or together with RhebL64 (right half). Cells were treated with D-PBS, 1-butanol (0.3%), sorbitol (600 mM), 2-DG (25 mM), or rapamycin (20 nM) for 30 min as indicated.

Figure 5.

A proposed model of Rheb functions downstream of TSC1/TSC2 and upstream of mTOR. TSC2 acts as a GAP to inactivate Rheb by directly stimulating GTP hydrolysis. Rheb stimulates mTOR. Nutrient and cellular energy status signals through Rheb, whereas osmotic stress signals independently of Rheb.