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Life Sciences Institute,1 Department of Biological Chemistry,2 Institute of Gerontology, University of Michigan, Ann Arbor, Michigan3
Received 18 May 2004/ Accepted 15 June 2004
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TSC2 contains a region of limited homology to the catalytic domain of the Rap1-GTPase-activating protein (GAP) (16). Both genetic and biochemical studies have demonstrated that Rheb (Ras homolog enriched in brain) is a direct target of TSC2. TSC2 functions as a GAP to inhibit Rheb activity (7, 19, 22, 45, 50, 53, 59). In vitro biochemical studies show that TSC2 can stimulate GTP hydrolysis of purified Rheb. Furthermore, Rheb GTP levels are elevated in TSC2-deficient cells (19), and overexpression of TSC1/2 decreases the GTP levels of cotransfected Rheb. These studies strongly indicate that TSC1/2 inhibits the mTOR/S6K/4EBP1 signaling pathway by stimulating the GTP hydrolysis of Rheb and Rheb functions between TSC2 and mTOR (30, 35).
Rheb is a member of the Ras superfamily GTPases and shares the highest homology with Ras and Rap (55). The Rheb gene is highly conserved in eukaryotes from yeast to mammals (41, 54). Genetic studies of fly and fission yeast indicate that Rheb plays an important role in the stimulation of cell growth and regulation of G0/G1 cell cycle progression (32, 41, 45, 50, 56, 59). The growth arrest phenotype caused by Rheb mutation in Schizosaccharomyces pombe can be complemented by human Rheb (56), suggesting the conservation of Rheb function from yeast to human. The precise physiological functions of Rheb were unknown in high eukaryotes until recently. Both genetic studies in Drosophila melanogaster and biochemical studies in mammalian cells have shown that Rheb is involved in signal transduction pathways that regulate cell growth (7, 19, 22, 45, 50, 53, 59). Homozygous inactivation of Rheb is lethal, while mosaic analyses of Rheb mutant cells in Drosophila show that the inactivation of Rheb decreases cell size. In contrast, overexpression of Rheb increases cell size. Genetic epistatic analysis demonstrates that Rheb functions between TSC1-TSC2 and TOR. In cultured cells, overexpression of Rheb potently stimulates the phosphorylation of S6K and 4EBP1, while downregulation by interference RNA decreased the phosphorylation of S6K and 4EBP1. These data establish Rheb as an important regulator between TSC1/2 and mTOR to stimulate cell growth.
Rheb is unique in that it contains an arginine (Arg15) at the position equivalent to Gly12 in Ras (55). This is significant because Gly12 in Ras is highly conserved in the Ras superfamily, and mutation to any other residue except proline results in constitutive activation of Ras (48). As expected, Rheb contains high basal GTP levels. Interestingly, Ras with a mutation in Gly12 cannot be inactivated by Ras-GAP, while wild-type Rheb can still be inactivated by TSC2 GAP activity. These results suggest that the GTP hydrolysis of Rheb stimulated by the GAP activity of TSC2 is fundamentally different from that of Ras and Ras-GAP.
GAPs from different families do not share obvious sequence homology, although members from any one subfamily do (15). Accordingly, they are termed Ras-GAP, Rap-GAP, and so on. The typical model of GAP catalysis has been well established from studies of the Ras/Ras-GAP (p120 GAP) system. Ras-GAP works by stabilizing the existing catalytic machinery of Ras via supplementing an external arginine residue, termed the arginine finger (47). However, this arginine finger is not conserved in the TSC2 GAP domain. Furthermore, it is still an open question whether the GAP activity is absolutely required for the cellular function of TSC2 to inhibit mTOR signaling. Considering the critical role of Rheb and TSC2 in cell growth regulation and tumor development (3, 17), it is of importance to investigate Rheb GTPase activity as well as the mechanism of how the TSC2 GAP stimulates Rheb GTP hydrolysis. To better understand the mechanism of Rheb GTP hydrolysis and to further establish the functional importance of GAP activity in the physiological function of TSC2, we report here the biochemical and functional characterizations of Rheb GTPase and TSC2 GAP activity. Consistent with the recently reported Rap1-GAP structure (13), we show that TSC2 and Rheb employ a novel mechanism to hydrolyze GTP utilizing an "asparagine thumb."
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture, transfection, and immunoprecipitation. HEK293 cells were seeded and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Transfection was performed in serum-free condition with Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. Cells were lysed in lysis buffer (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 1% Triton X-100, 50 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml, 10 µg of aprotinin/ml) and immunoprecipitated with the indicated antibodies and protein G-Sepharose beads. Immunocomplexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting.
Measurement of GTP- and GDP-bound Rheb. HEK293 cells were cultured in six-well plates and cotransfected with various plasmids with the Lipofectamine reagent. Thirty-six hours after transfection, the cells were washed once with phosphate-free DMEM (DMEM without sodium phosphate and sodium pyruvate, catalog number 11971-025; GIBCO) and incubated with 1 ml of phosphate-free DMEM for 90 min. Cells were then incubated with 25 µCi of [32P]phosphate (ICN)/ml for 4 h. After the labeling, cells were lysed with prechilled lysis buffer (0.5% NP-40, 50 mM Tris [pH 7.5], 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride, 10 µg of leupeptin/ml, 10 µg of aprotinin/ml; 200 µl per well of a six-well plate). To avoid lysing the nuclei, the cells were incubated with lysis buffer for just 30 s with gentle shaking. The lysate was then centrifuged at 12,000 x g for 15 min at 4°C. The supernatant (160 µl) was transferred to a fresh tube. Sixteen microliters of NaCl (500 mM) was added to 160 µl of supernatant to inhibit GAP activity in the lysate. To immunoprecipitate Myc-tagged Rheb, 3 µg of anti-Myc antibody and 10 µl of a protein-G Sepharose bead slurry were added to the supernatant and incubated with gentle rocking for 1 h at 4°C. The beads were washed with wash buffer 1 (50 mM Tris [pH 8.0], 500 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5% Triton X-100) three times at 4°C. The beads were then washed with wash buffer 2 (50 mM Tris [pH 8.0], 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.1% Triton X-100) three times at 4°C. The Rheb-bound nucleotides were eluted with 20 µl of elution buffer (2 mM EDTA, 0.2% sodium dodecyl sulfate, 1 mM GDP, 1 mM GTP) at 68°C for 10 min. Ten microliters of eluted nucleotides was then applied onto polyethyleneimine cellulose plates (Baker-flex, 20 cm by 20 cm). After applying the sample, the plate was soaked in methanol and dried with a hair dryer. The bottom portion (below the line where the samples are loaded) of the plate was immersed in methanol again, and the plate was placed in a sealed chromatography chamber that was filled with 0.75 M KH2PO4 (pH 3.4) to a depth of 1 cm. The chamber was closed, and the solvent was allowed to ascend to the top of the plate. The plate was then removed and air dried. GTP and GDP resolved by thin-layer chromatography were visualized and quantified by a phosphoimager.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Consistent with previously reported results, Rheb-R15G still has a high GTP level. The mutation of Gly12 in Ras or its equivalent position in Rap results in resistance to inactivation by its GAP (37). To test the effect of TSC2 GAP activity on these Rheb mutants, in vivo labeling experiments were performed with coexpression of TSC1/2 and the Rheb mutants. We observed that TSC1/2 significantly decreased the GTP levels of the wild type, Rheb-R15V, and Rheb-R15P (Fig. 1B). These data indicate that TSC2 can still stimulate the GTP hydrolysis of Rheb-R15V, while a similar mutation in the oncogenic Ras-G12V is resistant to inactivation by Ras-GAP. Surprisingly, Rheb-R15G was partially resistant to inactivation by TSC1/2 (Fig. 1B). To further test the functional relationship between Rheb-R15G and TSC1/2, we next determined whether Rheb-R15G-induced phosphorylation of S6K is less sensitive to inhibition by TSC1/2 coexpression. Our data showed that Rheb-R15G is reproducibly more resistant to inhibition by TSC1/2 than the wild type as determined by the S6K phosphorylation assay, an indirect functional assay for Rheb (Fig. 1C).
Rheb-Q64L is sensitive to TSC2 GAP. Gln61 of Ras is a catalytic residue important for GTP hydrolysis and is the most frequently mutated residue in human cancer (46). Ras-Q61L has a dramatically increased basal GTP level, and the GTPase activity of Ras-Q61L is resistant to Ras-GAP stimulation (46). We observed that Rheb-Q64L also displays a higher basal GTP level (Fig. 2A). These data are consistent with our previous observation that Rheb-Q64L is more active than wild-type Rheb in stimulating S6K (22). Interestingly, the replacement of Gln64 by leucine in the Rheb GTPase does not abolish the effect of TSC2 GAP. Cotransfection of TSC1/2 significantly increased the GDP level of Rheb-Q64L (Fig. 2A). To further support the above observation, phosphorylation of S6K was determined. We observed that TSC1/2 inhibited the ability of Rheb-Q64L to stimulate S6K phosphorylation (Fig. 2B). These results are completely consistent with the GTP binding data in Fig. 2A. Therefore, TSC2 can still enhance GTP hydrolysis of Rheb-Q64L. Our data suggest that the relationship between Rheb and TSC2 GAP is different from that of Ras and Ras-GAP. TSC2 shares limited sequence homology with Rap-GAP. The three-dimensional structure of Rap1-GAP has recently been reported (13). Our results are completely consistent with the catalytic model proposed for Rap1 and Rap1-GAP in which the glutamine equivalent to Glu61 in Ras is not required for GTP hydrolysis as we observed with Rheb.
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Ras-Q61L/C186S is a cytosol-localized GTP-bound dominant-negative mutant. Ras-Q61L/C186S likely prevents membrane recruitment of Ras targets, such as Raf, by sequestering Raf in the cytoplasm. The membrane localization of Raf is essential for Raf activation. To further investigate the importance of Rheb membrane localization in Rheb signaling, we performed experiments with Rheb-Q64L/C181S, a mutant equivalent to Ras-Q61L/C186S, to test the importance of Rheb membrane association. Our data indicate that Rheb-Q64L/C181S does not inhibit S6K phosphorylation. On the contrary, Rheb-Q64L/C181S activates S6K, albeit less potently than does Rheb-Q64L (Fig. 2C). These observations demonstrate that membrane localization is not essential for Rheb function and further support that unlike Ras targets, activation of Rheb downstream targets does not have to occur at the membrane.
Rheb-S20N, Rheb-D60V, and Rheb-D60K do not act as dominant negatives. Dominant-negative GTPase mutants are important tools used for elucidating the biological function of a particular GTPase. It has been well demonstrated that the mutation of Ser17 to asparagine in Ras family GTPases creates a dominant-negative mutant which can inhibit endogenous Ras activation by sequestering GEFs (9). We attempted to create a dominant-negative Rheb. Rheb-S20N, which is analogous to Ras-S17N, binds less guanine nucleotides than does wild-type Rheb (Fig. 3A), a property similar to that of Ras-S17N. The nucleotides bound in Rheb-S20N represent less than 1% of the wild-type Rheb. We tested the effect of Rheb-S20N on the phosphorylation of S6K, which depends on Rheb activity. However, coexpression of Rheb-S20N had no significant effect on S6K phosphorylation (Fig. 3B to E). These observations can be explained if Rheb-S20N does not function as a dominant negative. An alternative explanation is that Rheb is not required for the basal S6K phosphorylation. We favor the former possibility (see Discussion).
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Replacement of D-PBS by DMEM without serum could activate S6K (Fig. 3E), while the addition of serum alone in PBS did not activate S6K (data not shown). We also examined the effects of Rheb mutants on DMEM-stimulated S6K phosphorylation. Our data show that neither Rheb-D60K nor Rheb-S20N blocked the DMEM-induced S6K phosphorylation (Fig. 3E). Under the same conditions, wild-type Rheb or Rheb-D60V activated S6K. The experiments described above clearly demonstrate that Rheb-D60V and Rheb-D60K do not function as dominant negatives while Rheb-D60V is active to stimulate S6K phosphorylation under several conditions tested. Our data are inconsistent with results described by Tabancay et al., who reported the dominant effect of Rheb-D60K and Rheb-D60V. One possible explanation could be the difference in levels of expression between the two studies. However, it is worth noting that the expression levels of the Rheb mutants in our experiments were high.
TSC1 is not required for TSC2 GAP activity towards Rheb. TSC1 and TSC2 form a stable physical complex, which is important for the functions of these two tumor suppressor gene products (23, 42). The requirement of TSC1 for TSC2 GAP activity is not clear from a review of the literature (7, 19, 22, 53, 59). When same quantity of TSC2 plasmid was used in transfection, coexpression of TSC1 caused a significant increase in the reduction of Rheb GTP levels than did the transfection of TSC2 alone. However, the effect of TSC1 is complex because TSC1 coexpression significantly increased the TSC2 protein level. To avoid this complication, different amounts of TSC2 plasmid were used to achieve a similar level of TSC2 expression in either the presence or absence of TSC1. We observed that expression of TSC2 alone inhibits Rheb GTP levels, similar to that induced by the TSC1 and TSC2 coexpression if the TSC2 expression level is similar (Fig. 4B). These results would suggest that TSC1 is not required for the GAP activity towards Rheb. However, this interpretation is complicated by the fact that transfection of TSC2 in HEK293 cells may elevate endogenous TSC1 protein levels (39). To overcome this problem, we used a TSC2 mutant with a deletion of the N-terminal region, which is required for interaction with TSC1. It has been reported that amino acids (1 to 418) of TSC2 are responsible for the interaction with TSC1 by yeast two-hybrid assay (Fig. 4A) (27). We performed coimmunoprecipitation to confirm whether TSC2(400-C) can interact with TSC1. Here, we present biochemical data that a TSC2(400-C) mutant does not bind endogenous TSC1 in mammalian cells while wild-type TSC2 coimmunoprecipitated with endogenous TSC1 (Fig. 4C), confirming that the N-terminal region of TSC2 is required for interaction with TSC1. The effect of TSC2(400-C) on Rheb GTP levels was examined. We observed that the expression of TSC2(400-C) efficiently decreased the Rheb GTP level (Fig. 4B). Our data strongly indicate that TSC1 is not required for the GAP activity of TSC2.
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The biochemical and structural study of Ras-GAP and Rho-GAP reveal two highly conserved arginines essential for GAP activity (47). However, the first arginine, which functions as the catalytic arginine finger in Ras-GAP, is not conserved in TSC2 homologs. Corresponding to the second conserved arginine in Ras-GAP, there are three positively charged residues of KKR at positions 1595, 1596, and 1597 [KKR(1595/6/7)]. Moreover, there is no additional invariant arginine in the TSC2 domain homologous to Rap-GAP. In contrast, four invariant arginines exist in TSC2 that are C terminal to the GAP domain (Arg1701, Arg1703, Arg1707, and Arg1709) (Fig. 4A). In order to determine potential catalytic residues necessary for TSC2 GAP activity, we mutated all of the above-mentioned residues. These mutants are denoted R1701P, R1703Q, double mutant R1707/1709Q, and triple mutant KKR(1595/6/7)QQQ. Mutation at KKR(1595/6/7), Arg1701, and Arg1703 abolished the GAP activity of TSC2, while mutation at Arg1707 and Arg1709 had no significant effect (Fig. 5A). We also examined the ability of these TSC2 mutants to inhibit S6K phosphorylation. Consistently, mutant R1707/1709Q retains the ability to inhibit S6K phosphorylation, and the mutants KKR(1595/6/7)QQQ, Arg1701, and Arg1703 with abolished GAP activity also lost the ability to inhibit S6K (Fig. 5B and C). Our data indicate the importance of these residues in TSC2 function and GAP activity. Interestingly, there are deletion and point mutations at Arg1701 and Arg1703 identified in TSC patients (12, 25), suggesting that the loss of these two arginines abolishes the GAP activity, thereby causing TSC. The above-mentioned data support a functional importance of GAP activity for TSC2 function.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sequence analyses of Rheb and TSC2 suggest that they comprise a pair of atypical small GTPase and GAP. Rheb has an arginine at the position equivalent to Gly12 of Ras, and the arginine finger in Ras-GAP is not conserved in TSC2. Our studies of the biochemical properties of Rheb mutants further imply that the mechanisms of Rheb GTP hydrolysis and stimulation by TSC2 GAP are different from those of Ras based on the following observations. (i) Rheb-R15 (wild type) is a better substrate than Rheb-R15G for TSC2 GAP activity. Rheb-Q64L is still sensitive to TSC2 GAP activity. These observations are in clear contrast to Ras. Ras-G12 (wild type) is sensitive to Ras-GAP, while Ras-G12R is resistant to Ras-GAP. Furthermore, the Ras-Q61L mutation is resistant to GTP hydrolysis stimulation by Ras-GAP. (ii) The mutation of Arg15 to Gly15 in Rheb does not decrease basal GTP level. Moreover, the mutation of Arg15 to Val15 and Pro15 significantly decreases its GTP-to-GDP ratio. In the Ras protein, Ras-G12V and Ras-G12R have higher basal GTP levels than Ras-G12 (wild type). (iii) Rheb-S20N does not function as a dominant-negative mutant although Rheb-S20N does not bind the guanine nucleotide. Ras-S17N functions as a dominant negative by sequestering the upstream activator GEF. Therefore, Rheb displays many unique properties distinct from those of other Ras family members.
The GAP homologous domain of TSC2 (approximately 150 amino acid residues) shares 30% sequence identity with the GAP domain of Rap1-GAP. During the preparation of this paper, the crystal structure of Rap1-GAP was reported (13). Our results are completely consistent with catalytic mechanisms proposed by Daumke et al. for Rap1-GAP. Similar to TSC2, Rap1-GAP does not use an arginine finger as a catalytic residue to stimulate GTP hydrolysis. Instead, Rap1-GAP utilizes an asparagine, termed the asparagine thumb, as the active site to stimulate GTP hydrolysis. This active-site asparagine in Rap1-GAP corresponds to N1601 in TSC2, which is mutated in human disease and is also required for TSC2 GAP activity (Fig. 5). Therefore, we propose that N1601 is the active-site residue asparagine thumb for TSC2 to stimulate Rheb GTP hydrolysis. Our data showing that TSC2 can stimulate GTP hydrolysis of Rheb-Q64L are also consistent with the data observed for Rap1-GAP and Rap1, in which the Ras Q61-equivalent residue is not required for the stimulation of Rap1 GTP hydrolysis by the Rap1-GAP (4). Therefore, our study demonstrates that the catalytic mechanism of TSC2 and Rheb is similar to Rap1-GAP and Rap but is completely different from Ras-GAP and Ras.
Deletion analysis reveals that TSC2 requires sequences outside the GAP domain (residues 1517 to 1674) for GAP activity. Deletion of the N-terminal 600 residues completely abolishes TSC2 GAP activity, suggesting that the GAP domain of TSC2 is larger than that predicted by sequence homology. Our mutational analysis identified two arginine residues, Arg1701 and Arg1703, important for GAP activity. These two residues are located outside of the predicted GAP domain and are also mutated in TSC patients. These two arginine residues are conserved in Rap1-GAP and have been implicated in substrate binding. In addition, mutation of the conserved KKR(1567/8/9) completely eliminated GAP activity. Daumke et al. reported that the mutation of K285 in Rap1-GAP, which corresponds to K1568 in TSC2, abolished GAP activity (4, 13). Based on the three-dimensional structure and binding data, K285 is important to position the catalytic 
-helix 7 and is important for substrate binding. Our data are consistent with a similar role for K1568 in TSC2.
We observed a tight correlation between GAP activity of TSC2 and its ability to inhibit S6K phosphorylation. Any mutation with a low TSC2 GAP activity concomitantly decreases its function to inhibit mTOR signaling. These data further demonstrate that the GAP activity of TSC2 is important for its physiological function to regulate protein synthesis and cell growth.
There are two types of dominant-negative Ras mutants. Ras-S17N functions as a dominant negative by sequestering upstream GEF. In contrast, Ras-Q61L/C186S, which is not associated with the membrane but can bind Raf, functions as a dominant negative through binding Raf and preventing Raf activation because Raf activation occurs at the membrane (49). We found that neither Rheb-S20N nor Rheb-Q64L/C181S functions as a dominant negative to inhibit S6K phosphorylation. The soluble Rheb-Q61L/C181S, which presumably can still bind to as-yet-unidentified effectors, can also stimulate S6K phosphorylation. Similarly, Rheb-C181S can also stimulate S6K activation, albeit less effectively. Phosphorylation of S6K was used in our study as the functional assay for Rheb. These results indicate that the activation of Rheb targets may not be restricted to the membrane although direct downstream targets of Rheb have not been identified. A less interesting explanation is that S6K phosphorylation does not require Rheb function, while Rheb-S20N and Rheb-Q61L/C181S do function as dominant negatives by inhibiting activation of endogenous Rheb and its downstream effectors, respectively. However, this explanation is unlikely, based on current genetic and biochemical data that S6K phosphorylation is a real physiological readout of Rheb function (30). First, mutation of Rheb decreases S6K phosphorylation. Downregulation of Rheb by interference RNA also inhibits S6K phosphorylation. Second, the ability of nutrients and insulin to stimulate S6K is abolished in cells containing mutant Rheb. Third, overexpression of Rheb stimulates S6K. Fourth, both GTP binding and the effector domain of Rheb are required for Rheb to stimulate S6K phosphorylation. Furthermore, expression of TSC2, a Rheb GAP, decreases S6K phosphorylation. Together, these observations demonstrate an obligatory function of Rheb in S6K phosphorylation which is a bona fide physiological readout of Rheb, similar to ERK phosphorylation as a physiological readout of Ras.
Consistent with the farnesylation-defective mutants of Rheb having compromised ability to stimulate S6K activity (7, 53), the Rheb-C181S mutant also shows a lower GTP level (Fig. 2A). Because a mutation in Cys181 does not change the sequence involved in GTP binding and hydrolysis, the decrease of the GTP-to-GDP ratio is likely due to sensitivity toward either a GEF or a GAP. However, the latter possibility was ruled out because the sensitivity of Rheb-C181S to TSC2 inhibition was similar to that of the wild type (Fig. 2A). It has been shown that prenylation of Ras proteins is required for efficient stimulation by its GEF (43). It is possible that farnesylation of Rheb is important for Rheb stimulation by GEF, although a Rheb GEF has not been identified. Future studies to investigate the existence and identity of Rheb GEF will be of high significance.
The inactivation of TSC1 or TSC2 causes similar phenotypes, suggesting that they may affect the same downstream targets. For instance, genetic studies show that a mutation in either TSC1 or TSC2 elevates S6K activity (27). However, it remains unclear how or whether TSC1 contributes to TSC2's GAP activity. Studies from Zhang et al., Garami et al., and Tee et al. showed that TSC1 is required for TSC2 GAP activity towards Rheb (19, 53, 59), while studies from Castro et al. (7) and our group (22) showed that TSC1 is not required. Here, we further address this issue by testing the GAP activity of various truncated TSC2 mutants. Our data clearly show that TSC2(400-C), which cannot bind TSC1, still has GAP activity (Fig. 4B), suggesting that TSC2 alone is sufficient to promote GTP hydrolysis of Rheb. However, it has been well demonstrated that TSC1 can bind TSC2 and stabilize TSC2 by preventing degradation (2). Furthermore, TSC1 has been implicated in modulating the subcellular localization of TSC2 (39). Therefore, we propose that TSC1 is not required for Rheb-GAP activity per se but likely plays an important role in regulating the physiological function of TSC2 by modulating the protein stability and localization.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Institutes of Health and the Walther Cancer Institute.
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FOOTNOTES |
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