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. 2004 Sep;24(18):7965-75.
doi: 10.1128/MCB.24.18.7965-7975.2004.

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity

Yong Li  1 , Ken Inoki, Kun-Liang Guan
Affiliations

Biochemical and functional characterizations of small GTPase Rheb and TSC2 GAP activity

Yong Li et al. Mol Cell Biol. 2004 Sep.

Abstract

Tuberous sclerosis complex (TSC) is a genetic disease caused by a mutation in either the tsc1 or tsc2 tumor suppressor gene. Recent studies have demonstrated that TSC2 displays GAP (GTPase-activating protein) activity specifically towards the small G protein Rheb and inhibits its ability to stimulate the mTOR signaling pathway. Rheb and TSC2 comprise a unique pair of GTPase and GAP, because Rheb has high basal GTP levels and TSC2 does not have the catalytic arginine finger found in Ras-GAP. To investigate the function of TSC2 and Rheb in mTOR signaling, we analyzed the TSC2-stimulated Rheb GTPase activity. We found that Arg15, a residue equivalent to Gly12 in Ras, is important for Rheb to function as a substrate for TSC2 GAP. In addition, we identified asparagine residues essential for TSC2 GAP activity. We demonstrated a novel catalytic mechanism of the TSC2 GAP and Rheb that TSC2 uses a catalytic "asparagine thumb" instead of the arginine finger found in Ras-GAP. Furthermore, we discovered that farnesylation and membrane localization of Rheb is not essential for Rheb to stimulate S6 kinase (S6K) phosphorylation. Analysis of TSC1 binding defective mutants of TSC2 shows that TSC1 is not required for the TSC2 GAP activity but may function as a regulatory component in the TSC1/TSC2 complex. Our data further demonstrate that GAP activity is essential for the cellular function of TSC2 to inhibit S6K phosphorylation.

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Figures

FIG. 1.
FIG. 1.
Rheb-R15G cannot restore the GTPase activity and is less sensitive to TSC2 GAP activity. (A) Rheb contains an arginine residue (the residue in boldface type) at the position corresponding to codon 12 of Ras, which has a glycine. Numbers indicate positions of the last residues. (B) Mutation of Arg15 affects Rheb GTP binding and sensitivity to TSC2 GAP. Wild-type and mutant Myc-Rheb were transfected into HEK293 cells in the presence or absence of TSC1/2 as indicated and labeled with [32P]phosphate. Myc-Rheb was immunoprecipitated, and the bound nucleotides were eluted and resolved on a cellulose plate. The expression levels of Myc-Rheb and TSC1/2 in a parallel experiment (middle) and quantification of Rheb nucleotide binding (bottom) are shown. The ratio of GTP to GDP was calculated by the following formula: (GTP counts/3)/(GDP counts/2). The values are data from three independent experiments. (C) Rheb-R15G is less sensitive to the TSC1/2 complex. HA-S6K was transfected into HEK293 cells in the presence or absence of Myc-Rheb and TSC1/2. Phosphorylation of S6K was determined by phosphospecific antibody against phospho-S6K(T389). WT, wild type.
FIG. 2.
FIG. 2.
Farnesylation of Rheb is important for GTP loading but not essential to activate downstream signaling. (A) Nucleotide binding of Rheb mutants. Expression levels of Rheb mutants and TSC1/2 are determined by Western blotting (middle). Quantification of Rheb nucleotide binding is also shown (bottom). wt, wild type. (B) TSC1/2 inhibits Rheb-Q64L mutant-induced phosphorylation of S6K. (C) Rheb-Q64L/C181S is less active to stimulate S6K but does not function as a dominant negative.
FIG. 3.
FIG. 3.
Characterizations of potential dominant-negative Rheb mutants. (A) Rheb-S20N is defective in nucleotide binding. Wild-type Rheb (Rheb-wt) and Rheb-S20N were transfected into HEK293 cells. The transfected cells were labeled with [32P]phosphate. The bound nucleotides (top) of immunoprecipitated Rheb (bottom) are shown. (B) Rheb-D60V and Rheb-D60K do not inhibit S6K phosphorylation. HA-S6K was cotransfected with Rheb mutants in HEK293 cells. Thirty minutes before harvesting, cells were stimulated with fresh DMEM with 10% fetal bovine serum. Phosphorylation of S6K was determined by Western blotting. (C) Rheb-D60V, RhebD60K, and Rheb-S20N do not function as dominant negatives. Experiments are similar to those described above (B), except that cells were not stimulated by fresh medium. (D) Rheb-D60V activates S6K. The transfected HEK293 cells were treated with D-PBS (PBS containing 45 mM glucose) for 30 min before harvesting. (E) Rheb-D60V and Rheb-D60K do not block S6K activation by DMEM. The transfected HEK293 cells were treated with D-PBS for 30 min followed by treatment with DMEM (as indicated) without serum for 30 min before harvesting. WT, wild type.
FIG. 4.
FIG. 4.
TSC1 is not required for TSC2 GAP activity. (A) Schematic diagram of TSC2. The amino acids mutated in this study are indicated. GAP denotes the region of homology to the catalytic domain of Rap1-GAP. (B) The TSC1 interaction-defective mutant TSC2(400-C) displays GAP activity. TSC2(400-C), TSC2(600-C), TSC2(800-C), and TSC2(1007-C), TSC2 mutants with deletions of the N-terminal 400, 600, 800, and 1,007 residues, respectively. The bound nucleotides (top), expression levels of the TSC2 (middle), and quantification of the Rheb GTP-to-GDP ratio (bottom) are shown. WB, Western blot. (C) The N-terminal region of TSC2 is required for interaction with TSC1. HA-TSC2 and deletion mutants were transfected into HEK293 cells. Cell lysates were immunoprecipitated (IP) by anti-HA antibody for HA-TSC2 and blotted with anti-HA and anti-TSC1.
FIG. 4.
FIG. 4.
TSC1 is not required for TSC2 GAP activity. (A) Schematic diagram of TSC2. The amino acids mutated in this study are indicated. GAP denotes the region of homology to the catalytic domain of Rap1-GAP. (B) The TSC1 interaction-defective mutant TSC2(400-C) displays GAP activity. TSC2(400-C), TSC2(600-C), TSC2(800-C), and TSC2(1007-C), TSC2 mutants with deletions of the N-terminal 400, 600, 800, and 1,007 residues, respectively. The bound nucleotides (top), expression levels of the TSC2 (middle), and quantification of the Rheb GTP-to-GDP ratio (bottom) are shown. WB, Western blot. (C) The N-terminal region of TSC2 is required for interaction with TSC1. HA-TSC2 and deletion mutants were transfected into HEK293 cells. Cell lysates were immunoprecipitated (IP) by anti-HA antibody for HA-TSC2 and blotted with anti-HA and anti-TSC1.
FIG. 4.
FIG. 4.
TSC1 is not required for TSC2 GAP activity. (A) Schematic diagram of TSC2. The amino acids mutated in this study are indicated. GAP denotes the region of homology to the catalytic domain of Rap1-GAP. (B) The TSC1 interaction-defective mutant TSC2(400-C) displays GAP activity. TSC2(400-C), TSC2(600-C), TSC2(800-C), and TSC2(1007-C), TSC2 mutants with deletions of the N-terminal 400, 600, 800, and 1,007 residues, respectively. The bound nucleotides (top), expression levels of the TSC2 (middle), and quantification of the Rheb GTP-to-GDP ratio (bottom) are shown. WB, Western blot. (C) The N-terminal region of TSC2 is required for interaction with TSC1. HA-TSC2 and deletion mutants were transfected into HEK293 cells. Cell lysates were immunoprecipitated (IP) by anti-HA antibody for HA-TSC2 and blotted with anti-HA and anti-TSC1.
FIG. 5.
FIG. 5.
Identification of arginines essential for TSC2 GAP activity. (A) The GTP-to-GDP ratio of Rheb was measured in the presence of wild-type and mutant TSC2 as indicated. The expression levels of TSC1 and TSC2 (middle) and quantification of GAP activity of TSC2 point mutations (bottom) are shown. (B) Inhibition of S6K by TSC2 arginine mutants. HA-S6K and wild-type (WT) or mutant TSC2 were cotransfected into HEK293 as indicated. Phosphorylation of S6K was determined by phosphospecific antibody against phospho-pS6K(T389). (C) Inhibition of S6K by the TSC2 wild type and mutants. Experiments were similar to those described above (B).
FIG. 6.
FIG. 6.
Identification of GAP activity-essential asparagines corresponding to mutations found in TSC patients. (A) Asparagine residues N1601, N1609, and N1639 are essential for TSC2 GAP activity. The GTP-to-GDP ratio of Rheb was measured in the presence of wild-type and mutant TSC2 as indicated. (B) Alignment of TSC2 mutated residues described above (A). fugu, Fugu rubripes; pombe, S. pombe.
FIG. 6.
FIG. 6.
Identification of GAP activity-essential asparagines corresponding to mutations found in TSC patients. (A) Asparagine residues N1601, N1609, and N1639 are essential for TSC2 GAP activity. The GTP-to-GDP ratio of Rheb was measured in the presence of wild-type and mutant TSC2 as indicated. (B) Alignment of TSC2 mutated residues described above (A). fugu, Fugu rubripes; pombe, S. pombe.
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