Recent studies from several groups, including ours, have established that TSC2, which is responsible for approximately 60% of TSC disease, has GAP activity specifically towards Rheb. There is a Rap1-GAP homology domain in the C-terminal region of TSC2 (Fig. ) (16
). The GAP domain is the sole functionally recognizable domain in TSC2. Importantly, frequent point mutations in the TSC2 GAP domain are found in TSC patients, indicating the importance of the GAP domain in TSC2 function (1
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. ). 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
). Based on the three-dimensional structure and binding data, K285 is important to position the catalytic α-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
), the Rheb-C181S mutant also shows a lower GTP level (Fig. ). 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. ). 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
), 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. ), 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.