Mutations in the TSC1 or TSC2 genes cause tuberous sclerosis, a benign tumor syndrome in humans. Tsc2 possesses a domain that shares homology with the GTPase-activating protein (GAP) domain of Rap1-GAP2, suggesting that a GTPase might be the physiological target of Tsc2. The small GTPase Rheb (Ras homolog enriched in brain) has been shown to be a direct target of Tsc2 GAP activity both in vivo and in vitro. Point mutations in the GAP domain of Tsc2 disrupt its ability to regulate Rheb without affecting the ability of Tsc2 to form a complex with Tsc1. These studies identify Rheb as a molecular target of the TSC tumor suppressor genes (Zhang, 2003).
TSC1 and TSC2 were initially discovered as tumor suppressor genes mutated in tuberous sclerosis, a human syndrome characterized by the widespread development of benign tumors termed harmatomas. TSC2 encodes a putative GAP protein, whereas TSC1 encodes a novel protein containing two coiled-coil domains. Studies of Drosophila TSC1 and TSC2 homologs have identified a specific function for TSC1-TSC2 in the control of cell growth, with loss of TSC1-TSC2 resulting in increases in cell size. Recent studies further suggest that Tsc1-Tsc2 antagonizes the amino-acid-TOR signalling pathway, which normally couples amino-acid availability to S6 Kinase (S6K) activation, translation initiation and cell growth. Strikingly, loss of Drosophila TSC1-TSC2 results in a TOR-dependent increase of S6K activity that is resistant to amino-acid starvation (Zhang, 2003 and references therein).
Despite these new advances, the biochemical activity of the Tsc1-Tsc2 complex remains unknown. Tsc2 possesses a domain homologous to Rap1-GAP. The GAP homology domain of Tsc2 is important for its function, and mis-sense mutations of this domain have been identified in a high proportion of TSC patients. These observations suggest that an unknown small GTPase might be the direct target of Tsc2. This study set out to determine the target GTPase of Tsc2-GAP using an RNAi-based screen in Drosophila S2 cells. It was reasoned that this putative GTPase should be expressed in S2 cells and that RNAi of this GTPase should result in downregulation of S6K-Thr 398 phosphorylation, a phenotype opposite that caused by Tsc2 RNAi. During the course of the RNAi screen, genetic studies have implicated the small GTPase Rheb as a potential target of Tsc2. In S2 cells, RNAi inhibition of Rheb, but not any of the other 17 GTPases tested so far, abolished S6K-Thr 398 phosphorylation, as predicted for a Tsc2 GAP substrate. Among the 17 GTPases screened were Rab5 and Rap1, two proteins previously implicated as TSC2 GAP substrates from in vitro studies, suggesting that Rab5 and Rap1 are improbable physiological substrates of Tsc2. The highly specific effect of Rheb RNAi on S6K phosphorylation suggests that Rheb might be the physiological substrate of TSC2 GAP activity (Zhang, 2003).
Rheb is an evolutionarily conserved small GTPase found from yeast to mammals. Unlike Ras and most other Ras superfamily GTPases, Rheb has an arginine at the third residue of the G1 box (residue 15 of mammalian Rheb) instead of glycine. Rheb is unique, compared with many small GTPases, in that it exists in a highly activated state in mammalian cells. Studies of mammalian Rheb further implicated the existence of a Rheb-GAP that is normally present at relatively limiting concentrations, since overexpression of Rheb results in a progressive increase in the proportion of Rheb in the active GTP-bound state. Genetic analyses in Drosophila support a model in which Tsc2 functions as a Rheb-GAP. These studies also suggest that similarly to mammalian cells, Tsc2, the putative Rheb-GAP, is normally present in limiting concentrations in Drosophila, because overexpression of wild-type Rheb results in an activated phenotype and overexpression of Tsc2 (together with Tsc1) results in the opposite phenotype (Zhang, 2003).
To test directly whether Rheb is a physiological substrate of Tsc2 GAP activity, it was asked if Tsc2 could regulate Rheb in vivo. Rheb, similar to other small GTPases, cycles between an active GTP-bound form and an inactive GDP-bound form. Thus, the steady state GTP/GDP-loading status of Rheb can be used as a measurement of its in vivo activity. An in vivo labelling procedure was adapted to analyse the steady-state GTP/GDP-binding status of Rheb. Drosophila S2 cells expressing Myc-tagged Rheb were labelled with 32P-orthophosphate. Rheb protein was then purified by immunoprecipitation and Rheb-associated GTP/GDP was analysed by thin-layer chromatography (TLC) on polyethyleneimine (PEI) cellulose plates. In wild-type S2 cells, Rheb binds preferentially to GTP, in agreement with studies of mammalian Rheb. In addition, co-overexpression of Tsc1 and Tsc2 results in a marked decrease (approximately eightfold) in the ratio of GTP to GDP bound on Rheb. Interestingly, overexpression of Tsc2 alone has much weaker effect on GTP:GDP ratio. This observation is consistent with previous studies in Drosophila, which show that co-overexpression of Tsc1 and Tsc2, but not either gene alone, results in growth inhibition. The weaker effect of Tsc2 alone on Rheb GTP loading is caused, at least in part, by the lower level of Tsc2 when expressed alone, as compared with Tsc1 co-expression. Mutual stabilization between Drosophila Tsc1 and Tsc2 has been documented previously (Zhang, 2003).
To demonstrate that the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading was caused by the GAP activity of Tsc2, similar in vivo labelling experiments were performed with Tsc2 variants carrying point mutations in the GAP domain. The mutations Tsc2K1693A and Tsc2N1698K changed residues in the GAP domain that are conserved in Drosophila, human and a probable Schizosaccharomyces pombe Tsc2 homolog. In addition, a mutation analogous to Tsc2K1693A has been shown to abolish Rap1-GAP activity, whereas Tsc2N1698K mimics a disease-causing mutation in human TSC patients. The activity of Tsc2-N, a construct that contains just the amino-terminal half of Tsc2 and thus lacks the carboxy-terminal GAP domain, was also examined. Tsc2-N can associate with Tsc1 normally, but does not interact with Rheb in co-immunoprecipitation assays. Similar to Tsc2-N, neither Tsc2K1693A nor Tsc2N1698K affects the ability of Tsc2 to associate with Tsc1. Despite their ability to associate with Tsc1, these mutants all abolished the effect of Tsc1-Tsc2 overexpression on Rheb GTP loading. Complementary to the results from Tsc1-Tsc2 overexpression, RNAi of Tsc2 increases the ratio of GTP:GDP bound to Rheb. The smaller change in GTP:GDP ratio after Tsc2 RNAi, compared with Tsc1-Tsc2 overexpression, is not surprising given that Rheb is already at a relatively active state in wild-type cells. Taken together, these results provide strong evidence that Rheb is a physiological target of Tsc2 GAP activity (Zhang, 2003).
To test whether Rheb is a direct substrate of Tsc2 GAP in vitro, a fusion protein of glutathione S-transferase (GST) and the Tsc2 GAP domain against GTP-loaded Rheb protein was tested using a nitrocellulose filter assay. alpha-32P-GTP- or gamma-32P-GTP-loaded GST-Rheb was incubated with GST-Tsc2 and the remaining radioactive GTP bound on Rheb was measured at different time intervals. GST-Tsc2 results in a dramatic decrease of Rheb-associated radioactive counts when gamma-32P-GTP, but not alpha-32P-GTP, was used in the assay. Thus, Tsc2 functions as a Rheb GAP in vitro. This GAP activity is highly specific, and no activity was detected, using as a substrate Drosophila Ras1, the closest relative of Rheb among all GTPases. In addition, the K1693A or the N1698K point mutation abrogates the in vitro GAP activity of Tsc2 towards Rheb. These results provide further evidence that Tsc2 functions as a Rheb GAP (Zhang, 2003).
The data presented so far suggest a model in which the tuberous sclerosis tumor suppressor proteins negatively regulate Rheb through the Rheb GAP activity of Tsc2. To further substantiate this model, whether there are any genetic interactions between TSC1-TSC2 and Rheb was tested. Flies homozygous for a null allele of TSC1, TSC129, do not survive beyond the second-instar larval stage. Strikingly, the lethality of TSC1 null animals was partially rescued by removing one of the two copies of Rheb gene from the diploid genome: 61% of TSC129 homozygotes that were also heterozygous for a null allele of Rheb, RhebPDelta1, survived to third-instar larval stage, and 21% of the third-instar survivors continued development and arrested at the pupal stage. Such dose-sensitive interactions are reminiscent of those observed between TSC1-TSC2 and TOR, further supporting the model that Tsc1-Tsc2 negatively regulates Rheb during cell growth (Zhang, 2003).
Finally, how the Tsc-Rheb pathway interacts with the amino acid-TOR-S6K signalling network was investigated. Tsc and Rheb could either function as obligatory components between amino acids and TOR in a linear amino-acid sensing pathway, or in a parallel pathway that converges on TOR. The former (but not the latter) model predicts that the activity of Rheb is dependent on the presence of amino acids. The ratio of GTP:GDP bound to Rheb is not reduced after 5 h of amino-acid starvation. Thus, a model is favored in which TSC and Rheb function in a parallel pathway that converges on TOR. According to this model, loss of Tsc1-Tsc2 or ectopic activation of Rheb results in constitutive activation of TOR, which bypasses the requirement for amino acids and renders S6K activity resistant to amino-acid starvation. How Rheb signals to TOR will be an important question for future investigation (Zhang, 2003).
In summary, the small GTPase Rheb is a direct target of the tuberous sclerosis tumor suppressor proteins. Wild-type Tsc2, but not mutant Tsc2 carrying point mutations in the GAP domain, shows GAP activity towards Rheb both in vitro and in vivo. The importance of Tsc2's GAP activity is further supported by the high proportion of mis-sense mutations localized to the Tsc2 GAP domain among TSC patients. Thus, the Tsc2 tumor suppressor functions as a Rheb-GAP in an analogous way to the neurofibromin (NF1) tumor suppressor as a Ras-GAP. These studies suggest that Rheb represents a novel target for therapeutic intervention in the TSC disease. The identification of a small GTPase as the direct target of the TSC tumor suppressors further implicates the existence of activators of GTPases, such as guanine nucleotide-exchange factors (GEFs), as potential regulators of this disease pathway. Identification of the putative Rheb-GEF represents an important goal for the next phase of TSC research (Zhang, 2003).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.