Tuberous sclerosis complex gene products,Tuberin and Hamartin,control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb

BACKGROUND: Tuberous Sclerosis Complex (TSC) is a genetic disorder that occurs through the loss of heterozygosity of either TSC1 or TSC2, which encode Hamartin or Tuberin, respectively. Tuberin and Hamartin form a tumor suppressor heterodimer that inhibits the mammalian target of rapamycin (mTOR) nutrient signaling input, but how this occurs is unclear. RESULTS: We show that the small G protein Rheb (Ras homolog enriched in brain) is a molecular target of TSC1/TSC2 that regulates mTOR signaling. Overexpression of Rheb activates 40S ribosomal protein S6 kinase 1 (S6K1) but not p90 ribosomal S6 kinase 1 (RSK1) or Akt. Furthermore, Rheb induces phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and causes 4E-BP1 to dissociate from eIF4E. This dissociation is completely sensitive to rapamycin (an mTOR inhibitor) but not wortmannin (a phosphoinositide 3-kinase [PI3K] inhibitor). Rheb also activates S6K1 during amino acid insufficiency via a rapamycin-sensitive mechanism, suggesting that Rheb participates in nutrient signaling through mTOR. Moreover, Rheb does not activate a S6K1 mutant that is unresponsive to mTOR-mediated signals, confirming that Rheb functions upstream of mTOR. Overexpression of the Tuberin-Hamartin heterodimer inhibits Rheb-mediated S6K1 activation, suggesting that Tuberin functions as a Rheb GTPase activating protein (GAP). Supporting this notion, TSC patient-derived Tuberin GAP domain mutants were unable to inactivate Rheb in vivo. Moreover, in vitro studies reveal that Tuberin, when associated with Hamartin, acts as a Rheb GTPase-activating protein. Finally, we show that membrane localization of Rheb is important for its biological activity because a farnesylation-defective mutant of Rheb stimulated S6K1 activation less efficiently. CONCLUSIONS: We show that Rheb acts as a novel mediator of the nutrient signaling input to mTOR and is the molecular target of TSC1 and TSC2 within mammalian cells.     

Identification of TBC7 having TBC domain as a novel binding protein to TSC1-TSC2 complex

TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.     

Cell cycle-regulated phosphorylation of hamartin, the product of the tuberous sclerosis complex 1 gene, by cyclin-dependent kinase 1/cyclin B

Tuberous sclerosis complex is a tumor suppressor gene syndrome whose manifestations can include seizures, mental retardation, and benign tumors of the brain, skin, heart, and kidneys. Hamartin and tuberin, the products of the TSC1 and TSC2 genes, respectively, form a complex and inhibit signaling by the mammalian target of rapamycin. Here, we demonstrate that endogenous hamartin is threonine-phosphorylated during nocodazole-induced G2/M arrest and during the G2/M phase of a normal cell cycle. In vitro assays showed that cyclin-dependent kinase 1 phosphorylates hamartin at three sites, one of which (Thr417) is in the hamartin-tuberin interaction domain. Tuberin interacts with phosphohamartin, and tuberin expression attenuates the phosphorylation of exogenous hamartin. Hamartin with alanine mutations in the three cyclin-dependent kinase 1 phosphorylation sites increased the inhibition of p70S6 kinase by the hamartin-tuberin complex. These findings support a model in which phosphorylation of hamartin regulates the function of the hamartin-tuberin complex during the G2/M phase of the cell cycle.     

The p53 target Plk2 interacts with TSC proteins impacting mTOR signaling,tumor growth,and chemosensitivity under hypoxic conditions

Tuberous sclerosis complex 1 (TSC1) inhibits mammalian target of rapamycin (mTOR), a central promotor of cell growth and proliferation. The protein product of the TSC1 gene, hamartin (referred to as TSC1) is known to interact with Polo-like kinase 1 (Plk1) in a cell cycle regulated, phosphorylation-dependent manner. We hypothesized that the p53 target gene, Plk2, is a tumor suppressor, mediating its tumor suppressor function through interactions with TSC1 that facilitate TSC1/2 restraint of mTOR under hypoxic stress. We found that human lung tumor cells deficient in Plk2 grew larger than control tumors, and that Plk2 interacts with endogenous TSC1 protein. Additionally, C-terminal Plk2-GST fusion protein bound both TSC1 and TSC2 proteins. TSC1 levels were elevated in response to Adriamycin and cells transiently over-expressing Plk2 demonstrated decreased phosphorylation of the downstream target of mTOR, ribosomal protein p70S6 kinase during hypoxia. Plk2 levels were inversely correlated with cytoplasmic p70S6K phosphorylation. Plk2 levels did not increase in response to DNA damage (Adriamycin, CPT-11) when HCT 116 and H460 cells were exposed to hypoxia. TSC1-deficient mouse embryonic fibroblasts with TSC1 added back demonstrated decreased S6K phosphorylation, which was further decreased when Plk2 was transiently over-expressed. Interestingly, under normoxia, Plk2 deficient tumor cells demonstrated increased apoptosis in response to various chemotherapeutic agents including CPT-11 but increased resistance to apoptotic death after CPT-11 treatment under hypoxia, and tumor xenografts comprised of these Plk2-deficient cells were resistant to CPT-11. Our results point to a novel Plk2-TSC1 interaction with effects on mTOR signaling during hypoxia, and tumor growth that may enable targeting Plk2 signaling in cancer therapy.     

14-3-3beta binds to and negatively regulates the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product,tuberin

TSC2, or tuberin, is the product of the tuberous sclerosis tumor suppressor gene TSC2 and acts downstream of the phosphatidylinositol 3-kinase-Akt signaling pathway to negatively regulate cellular growth. One mechanism underlying its function is to assemble into a heterodimer with the TSC1 gene product TSC1, or hamartin, resulting in a reduction in phosphorylation, and hence activation, of the ribosomal subunit S6 kinase (S6K). We identified a novel interaction between TSC2 and 14-3-3beta. We found that 14-3-3beta does not interfere with TSC1-TSC2 binding and can form a ternary complex with these two proteins. Association between 14-3-3beta and TSC2 requires phosphorylation of TSC2 at a unique residue that is not a known Akt phosphorylation site. The overexpression of 14-3-3beta compromises the ability of the TSC1-TSC2 complex to reduce S6K phosphorylation. The antagonistic activity of 14-3-3beta toward TSC is dependent on the 14-3-3beta-TSC2 interaction, since a mutant of TSC2 that is not recognized by 14-3-3beta is refractory to 14-3-3beta. We suggest that 14-3-3 proteins interact with the TSC1-TSC2 complex and negatively regulate the function of the TSC proteins.     

TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by the formation of hamartomas in a wide range of human tissues. Mutation in either the TSC1 or TSC2 tumour suppressor gene is responsible for both the familial and sporadic forms of this disease. TSC1 and TSC2 proteins form a physical and functional complex in vivo. Here, we show that TSC1-TSC2 inhibits the p70 ribosomal protein S6 kinase 1 (an activator of translation) and activates the eukaryotic initiation factor 4E binding protein 1 (4E-BP1, an inhibitor of translational initiation). These functions of TSC1-TSC2 are mediated by inhibition of the mammalian target of rapamycin (mTOR). Furthermore, TSC2 is directly phosphorylated by Akt, which is involved in stimulating cell growth and is activated by growth stimulating signals, such as insulin. TSC2 is inactivated by Akt-dependent phosphorylation, which destabilizes TSC2 and disrupts its interaction with TSC1. Our data indicate a molecular mechanism for TSC2 in insulin signalling, tumour suppressor functions and in the inhibition of cell growth.     

The tuberous sclerosis genes and regulation of the cyclin-dependent kinase inhibitor p27

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of tumors, named hamartomas, in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation, and a variety of developmental disorders, including autism. This disease is caused by mutations within the tumor suppressor gene TSC1 on chromosome 9q34 encoding hamartin or within TSC2 on chromosome 16p13.3 encoding tuberin. TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells, and loss of heterozygosity has been documented in a wide variety of TSC tumors. Recent data suggest that functional inactivation of TSC proteins might also be involved in the development of other diseases not associated with TSC, such as sporadic bladder cancer, breast cancer, ovarian carcinoma, gall bladder carcinoma, non-small-cell carcinoma of the lung, and Alzheimer's disease. Tuberin and hamartin form a heterodimer, suggesting they might affect the same processes. Tuberin is assumed to be the functional component of the complex and has been implicated in the regulation of different cellular functions. The TSC proteins regulate cell size control due to their involvement in the insulin signalling pathway. Furthermore, they are potent positive regulators of the cyclin-dependent kinase inhibitor p27, a major regulator of the mammalian cell cycle. Here we review the current knowledge on how mutations within the TSC genes could trigger deregulation of stability and localization of the tumor suppressor p27.     

Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase/Akt-dependent and -independent phosphorylation of tuberin

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo.     

TSC2 mediates cellular energy response to control cell growth and survival

Mutations in either the TSC1 or TSC2 tumor suppressor gene are responsible for Tuberous Sclerosis Complex. The gene products of TSC1 and TSC2 form a functional complex and inhibit the phosphorylation of S6K and 4EBP1, two key regulators of translation. Here, we describe that TSC2 is regulated by cellular energy levels and plays an essential role in the cellular energy response pathway. Under energy starvation conditions, the AMP-activated protein kinase (AMPK) phosphorylates TSC2 and enhances its activity. Phosphorylation of TSC2 by AMPK is required for translation regulation and cell size control in response to energy deprivation. Furthermore, TSC2 and its phosphorylation by AMPK protect cells from energy deprivation-induced apoptosis. These observations demonstrate a model where TSC2 functions as a key player in regulation of the common mTOR pathway of protein synthesis, cell growth, and viability in response to cellular energy levels.     

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

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.     

The p38 and MK2 kinase cascade phosphorylates tuberin,the tuberous sclerosis 2 gene product,and enhances its interaction with 14-3-3

Tuberous sclerosis complex (TSC) is a genetic disease caused by mutations in either TSC1 or TSC2 tumor suppressor genes. TSC1 and TSC2 (also known as hamartin and tuberin, respectively) form a functional complex and negatively regulate cell growth by inhibiting protein synthesis. 14-3-3 binds to TSC2 and may inhibit TSC2 function. We have reported previously that phosphorylation of serine 1210 (Ser(1210)) in TSC2 is essential for 14-3-3 binding. Here we show that serum and anisomycin enhance the interaction between TSC2 and 14-3-3 by stimulating phosphorylation of Ser(1210). Activation of p38 MAP kinase (p38) is essential for the stimulating effect of serum and anisomycin although p38 is not directly responsible for the phosphorylation of Ser(1210) in TSC2. Both in vitro and in vivo experiments demonstrate that the p38-activated kinase MK2 (also known as MAPKAPK2) is directly responsible for the phosphorylation of Ser(1210). Our data show that anisomycin stimulates phosphorylation of Ser(1210) of TSC2 via the p38-MK2 kinase cascade. Phosphorylation of TSC2 by MK2 creates a 14-3-3 binding site and thus regulates the cellular function of the TSC2 tumor suppressor protein.     

Rheb binds tuberous sclerosis complex 2 (TSC2) and promotes S6 kinase activation in a rapamycin- and farnesylation-dependent manner

Recently the tuberous sclerosis complex 2 (TSC2) tumor suppressor gene product has been identified as a negative regulator of protein synthesis upstream of the mTOR and ribosomal S6 kinases. Because of the homology of TSC2 with GTPase-activating proteins for Rap1, we examined whether a Ras/Rap-related GTPase might be involved in this process. TSC2 was found to bind to Rheb-GTP in vitro and to reduce Rheb GTP levels in vivo. Over-expression of Rheb but not Rap1 promoted the activation of S6 kinase in a rapamycin-dependent manner, suggesting that Rheb acts upstream of mTOR. The ability of Rheb to induce S6 phosphorylation was also inhibited by a farnesyl transferase inhibitor, suggesting that Rheb may be responsible for the Ras-independent anti-neoplastic properties of this drug.     

Association of focal adhesion kinase with tuberous sclerosis complex 2 in the regulation of s6 kinase activation and cell growth

Tuberous sclerosis complex 1 (TSC1) and TSC2 tumor suppressor proteins have been shown to negatively regulate cell growth through inhibition of the mammalian target of rapamycin (mTOR) pathway. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a critical role in integrin signaling. Here we identify a novel interaction between FAK and TSC2 and show that TSC2 is phosphorylated by FAK. Furthermore, we show that overexpression of FAK kinase dead mutant inhibits the phosphorylation of ribosomal S6 kinase (S6K) and eukaryotic initiation factor 4E-binding protein-1, two key mTOR (mammalian target of rapamycin) downstream targets, and negatively regulates the cell size and that FAK regulation of S6K phosphorylation is through TSC2. Finally, we provide data that FAK plays a positive role in cell adhesion-induced S6K phosphorylation, whereas TSC2 is required for cell suspension-induced S6K inactivation. Together, these results suggest that FAK might regulate S6K activation and cell size through its interaction with and phosphorylation of TSC2 and also provide a previously unappreciated role of TSC2 in the regulation of mTOR signaling by cell adhesion.     

Regulation of TSC2 by 14-3-3 binding

Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1.TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser(1210) in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.     

Identification of FIP200 interaction with the TSC1-TSC2 complex and its role in regulation of cell size control

FIP200 (focal adhesion kinase [FAK] family interacting protein of 200 kD) is a newly identified protein that binds to the kinase domain of FAK and inhibits its kinase activity and associated cellular functions. Here, we identify an interaction between FIP200 and the TSC1-TSC2 complex through FIP200 binding to TSC1. We found that association of FIP200 with the TSC1-TSC2 complex correlated with its ability to increase cell size and up-regulate S6 kinase phosphorylation but was not involved in the regulation of cell cycle progression. Conversely, knockdown of endogenous FIP200 by RNA interference reduced S6 kinase phosphorylation and cell size, which required TSC1 but was independent of FAK. Furthermore, overexpression of FIP200 reduced TSC1-TSC2 complex formation, although knockdown of endogenous FIP200 by RNA interference did not affect TSC1-TSC2 complex formation. Lastly, we showed that FIP200 is important in nutrient stimulation-induced, but not energy- or serum-induced, S6 kinase activation. Together, these results suggest a cellular function of FIP200 in the regulation of cell size by interaction with the TSC1-TSC2 complex.     

Raptor, a binding partner of target of rapamycin

The mammalian target of rapamycin (mTOR) controls cell growth in response to amino acids and growth factors, in part by regulating p70 S6 kinase alpha (p70 alpha) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). Raptor (regulatory associated protein of mTOR) is a 150 kDa mTOR binding protein that is essential for TOR signaling in vivo and also binds 4EBP1 and p70alpha through their respective TOS (TOR signaling) motifs, a short conserved segment previously shown to be required for amino acid- and mTOR-dependent regulation of these substrates in vivo. Raptor appears to serve as an mTOR scaffold protein, the binding of which to the TOS motif of mTOR substrates is necessary for effective mTOR-catalyzed phosphorylation. Further understanding of regulation of the mTOR-raptor complex in response to the nutritional environment would require identification of the interplay between the mTOR-raptor complex and its upstream effectors such as the protein products of tumor suppressor gene tuberous sclerosis complexes 1 and 2, and the Ras-related small G protein Rheb.