GTP-Binding Protein Rheb
Ras homolog enriched in brain (Rheb) plays a significant role in regulating protein synthesis, cell growth, cell cycle, and autophagy (Aspuria and Tamanoi 2004; Heard et al. 2014). Deregulation of Rheb signaling has been shown to cause diseases ranging from developmental disorders to cancer. Rheb was initially discovered as a protein sharing strong homology with Ras and whose expression was rapidly induced in brain neuronal cell by increased synaptic activity (Yamagata et al. 1994). Since then, Rheb has been shown to be widely expressed and to be conserved in eukaryotes from yeast to human. Higher eukaryotes express two Rheb proteins, Rheb1 and Rheb2 (RhebL1). Two separate genes encode these proteins that share 74% similarity. Their functions appear similar, but their tissue expression profiles differ significantly. Rheb is a member of the Ras superfamily of GTP-binding proteins that act as molecular switches in the cell to activate downstream signaling when GTP is bound and turnoff signaling when GDP is bound. Rheb received significant attention after studies in Drosophila revealed that Rheb activates the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. Recent studies have revealed additional downstream effectors of Rheb, generating excitement over Rheb signaling as a potential therapeutic target. The following sections will detail Rheb structure, control of Rheb activity, Rheb downstream effectors, and the role of Rheb in disease and development.
Rheb Structure, Low GTPase Activity, and High GTP Loading
The overall structure of Rheb determined by X-ray crystallography is similar to that of Ras and Rap proteins (Yu et al. 2005). However, conformation of the switch II region is unique to this subfamily of GTPase. Unlike other GTPases, Gln64 of Rheb (that corresponds to Gln61 in Ras) is buried in a hydrophobic core. Further structural studies on Rheb have revealed Thr38 and Asp65, from the switch I and II regions, respectively, to be the main residues involved in GTP hydrolysis (Mazhab-Jafari et al. 2012). However, these residues are blocked from GTP interaction by the Tyr35 residue, thus keeping Rheb in a highly active state. In fact, analysis of GTP/GDP ratio bound to Rheb in CHOK-1 cells showed that Rheb contains approximately 24% GTP (Im et al. 2002) which is unusually high compared with most Ras superfamily GTPases.
TSC Is a GAP but Exchange Factors Remain Elusive
Most of the Ras superfamily GTPases is downregulated by GAP proteins (GTPase-activating protein) that act to stimulate intrinsic GTPase of these proteins (Colicelli 2004). Tuberous Sclerosis Complex (TSC) has been identified as the GAP protein for Rheb (Garami et al. 2003; Inoki et al. 2003; also reviewed in Heard et al. 2014). TSC GAP consists of three subunits, TSC1, TSC2, and TBC1D7. TSC1 and TSC2 proteins are the product of the TSC1 and TSC2 genes. Mutations of these genes are responsible for tuberous sclerosis, a genetic disorder that is associated with growth of benign tumors. TSC increases Rheb GTPase activity by inserting an asparagine finger (Asn 1643) into the catalytic site of Rheb, interfering with Tyr35 mediated inhibition, and allowing Thr38/Asp65 to facilitate GTP hydrolysis.
Another type of protein called guanine nucleotide exchange factors (GEFs) regulates the Ras superfamily GTPases (Colicelli 2004). This type of protein acts to stimulate exchange reaction, the unloading of GDP and reloading of GTP. Studies in Drosophila suggested that TCTP functions as a GEF for Rheb (Hsu et al. 2007). Further studies are needed to investigate this point. Identification of Rheb GEF is important for understanding the control of Rheb-related diseases.
Farnesylation and Intracellular Localization
Rheb shares another common feature among Ras proteins, a C-terminal –CAAX motif described above. These –CAAX motifs signal posttranslational modification events that include the addition of a lipid moiety in a process called prenylation. The addition of lipid moieties confers the ability of Ras proteins to bind and localize to membrane structures within the cell. For Rheb, this process involves the addition of a farnesyl group by protein farnesyltransferase, after cleavage of the –AAX motif by RceI protein and carboxylmethylation of cysteine by Icmt1. This process is crucial for proper localization of Rheb to activate downstream signaling.
Rheb is localized to endosomal vesicles, and co-localization of Rheb and lysosomal marker LAMP2 has been reported (Menon et al. 2014). This intracellular localization is consistent with the role of Rheb in activating mTORC1 which is localized on the same organelle.
Rheb localization studies have also revealed potential new Rheb signaling pathways (reviewed in Heard et al. 2014). Studies using both imaging techniques and Western analysis of cell fractions have suggested a subset of Rheb protein localizes to other intracellular membranes including peroxisomes and mitochondria. Rheb localization to the mitochondria has suggested its role in mitophagy, important for maintaining cellular homeostasis. In support of this, Rheb has been shown to interact with BNIP3L and LC3II, activating LC3II and mitophagy. It was suggested that another TSC/Rheb/mTORC1 signaling node exists on the peroxisomes, where TSC stimulates Rheb-GTPase activity thus shutting down mTORC1 in response to increased ROS levels in the cell.
Rheb Activates mTORC1
mTORC1 is the most well-studied downstream effector of Rheb (reviewed in Aspuria and Tamanoi 2004). In response to excess glucose, insulin stimulates PI3K signal transduction leading to mTORC1 activation of a variety of pathways including cellular growth and protein synthesis. The mTORC1 is a multiprotein complex consisting of mTOR kinase, Raptor, and mLST. Initial suggestion that Rheb activates TOR signaling downstream of the insulin/PI3K pathway came from the study in Drosophila (Saucedo et al. 2003; Patel et al. 2003). It was shown that overexpression of dRheb increased cell growth and cell cycle progression. Under these conditions PI3K activates AKT, which phosphorylates and inhibits TSC activity, thus keeping Rheb in an active GTP-bound state, resulting in stimulation of mTORC1 signaling. Purified Rheb can activate immunoprecipitated mTORC1 in a GTP and effector domain-dependent manner, suggesting that Rheb interacts directly with the mTORC1 complex (Sato et al. 2009; reviewed in Heard et al. 2014). It is believed that Rheb interacts with mTOR, but rigorous demonstration of this point has not been reported. Studies have shown that Rheb localization to the lysosome is crucial for mTORC1 activation. In response to insulin, AKT inhibitory phosphorylation of TSC causes TSC to come off from lysosomal membrane, thus increasing Rheb-GTP-mediated activation of mTOR (Menon et al. 2014).
Rheb Interacts with Other Proteins
Other interacting proteins of Rheb have been found, including phospholipase D1 (PLDI), beta-secretase 1 (BACE1), phosphodiesterase 4D (PDE4D), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (reviewed in Heard et al. 2014). It is suggested that Rheb is required for serum stimulation of PLD1. PLD1 is responsible for converting phosphatidylcholine to choline and phosphatidic acid, the most abundant phospholipids of the plasmid membrane. Rheb was shown to bind PLD1 in a GTP-dependent manner and stimulate its activity upstream of mTORC1.
Rheb in Disease and Development
Aberrant Rheb signaling has been uncovered in a diverse range of diseases including cancer. The disease Rheb is most prevalently associated with is tuberous sclerosis (TS). Tuberous sclerosis is a disorder characterized by the development of benign harmatomas all over the body, with life-threatening tumors localized in the brain, heart, lungs, and kidney. TS is caused by inactivating mutations in TSC, which leads to overactive Rheb-mTORC1 signaling causing uncontrolled tumor formation and cell growth. Another disease resulting from mutations in TSC, lymphangioleiomyomatosis (LAM), is defined by development of uncontrolled tumor growth localized in the lungs of primarily women. Treatment for these diseases involves sirolimus, an inhibitor of mTORC1 signaling. However, studies have shown that inhibition with sirolimus alone is not enough to rid the patient of disease, suggesting that Rheb-mediated signaling independent of mTORC1 may play a role in these diseases.
Several Rheb mutations have been identified in cancer genome databases. Initial screen using yeast identified a variety of single amino acid changes that activate Rheb (reviewed in Heard et al. 2014). Subsequent large-scale analysis led to the identification of various mutations. One such mutation is at the tyrosine 35 position. It has been shown that mutation of tyrosine 35 to asparagine decreases Rheb-TSC GTPase activity, leading to overactive Rheb signaling (Mazhab-Jafari et al. 2012). A Rheb Y35N point mutation has been found in both renal and endometrial carcinoma and activates mTORC1 signaling (Grabiner et al. 2014). Rheb Y35N has also been shown to competitively bind to AMPKα, blocking a Rheb-WT-mediated activation of AMPK. Decreased AMPK activation resulted in activated MAPK signaling and transformation of NIH3T3 cells in xenograft nude mice (Wang et al. 2017).
Early Drosophila studies revealed the importance of Rheb for embryonic viability (Saucedo et al. 2003; Patel et al. 2003). Mouse models of Rheb have also revealed essential roles for Rheb in development. Mouse studies also uncovered various effects of Rheb knockouts (reviewed in Heard et al. 2014). Rheb1 knockout in mice leads to embryonic death around E12. In these models, the cardiovascular system appears to be most affected, as Rheb1 knockout mice revealed pericardial hemorrhaging and thinning of the ventricular walls at E11.5. Conditional knockout of Rheb in neuronal progenitor cells, germ cells, and cardiac cells result in hypomylenation, male sterility, and hypotrophy, respectively.
Rheb is a member of the Ras family of small GTPases and mediates major signaling pathways that control cell metabolism, growth, and proliferation. Rheb signaling has been identified in a variety of disease settings including defects in development and mutations resulting in cancer progression. Most of what has been discovered about Rheb includes its activation of the mTORC1 signaling pathway downstream of insulin/PI3K axis. However, recent studies uncovering new binding partners of Rheb have revealed potentially new Rheb signaling pathways. These identified and new effectors that remain to be identified require more thorough examination to better understand the role of Rheb in development and disease. The identification of Rheb GEF remains as a major missing piece towards understanding Rheb activity. Our understanding of Rheb signaling has come a long way, but new exciting technologies and studies will continue to unveil Rheb signaling importance and therapeutic potential in the future.
- Colicelli J. Human RAS superfamily proteins and related GTPases. Sci STKE [Internet]. 2004;2004(250):RE13.Google Scholar
- Sato T, Akasu H, Shimono W, Matsu C, Fujiwara Y, Shibagaki Y, et al. Rheb protein binds CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) protein in a GTP- and effector domain-dependent manner and influences its cellular localization and carbamoyl-phosphate synthetase (CPSase) activity. J Biol Chem. 2015;290:1096–105.PubMedCrossRefGoogle Scholar
- Wang Y, Hong X, Wang J, Yin Y, Zhang Y, Zhou Y, et al. Inhibition of MAPK pathway is essential for suppressing Rheb-Y35N driven tumor growth. Oncogene. 2017;36:756–65.Google Scholar