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Mark Nellist - One of the best experts on this subject based on the ideXlab platform.

  • structural basis of the interaction between tuberous sclerosis complex 1 TSC1 and tre2 bub2 cdc16 domain family member 7 tbc1d7
    Journal of Biological Chemistry, 2016
    Co-Authors: Jiayue Qin, Marianne Hoogeveenwesterveld, Zhizhi Wang, Guobo Shen, Weimin Gong, Mark Nellist
    Abstract:

    Mutations in TSC1 or TSC2 cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterized by the occurrence of benign tumors in various vital organs and tissues. TSC1 and TSC2, the TSC1 and TSC2 gene products, form the TSC protein complex that senses specific cellular growth conditions to control mTORC1 signaling. TBC1D7 is the third subunit of the TSC complex, and helps to stabilize the TSC1-TSC2 complex through its direct interaction with TSC1. Homozygous inactivation of TBC1D7 causes intellectual disability and megaencephaly. Here we report the crystal structure of a TSC1-TBC1D7 complex and biochemical characterization of the TSC1-TBC1D7 interaction. TBC1D7 interacts with the C-terminal region of the predicted coiled-coil domain of TSC1. The TSC1-TBC1D7 interface is largely hydrophobic, involving the α4 helix of TBC1D7. Each TBC1D7 molecule interacts simultaneously with two parallel TSC1 helices from two TSC1 molecules, suggesting that TBC1D7 may stabilize the TSC complex by tethering the C-terminal ends of two TSC1 coiled-coils.

  • targeted next generation sequencing reveals previously unidentified TSC1 and tsc2 mutations
    BMC Medical Genetics, 2015
    Co-Authors: Mark Nellist, Rutger W W Brouwer, Monique Van Veghelplandsoen, Caroline Withagenhermans, Lida Prinsbakker, Alan Mrsic, Mike M P Van Den Berg, Christel E M Kockx, Marianne Hoogeveenwesterveld, Anna E Koopmans
    Abstract:

    Background Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene.

  • Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex.
    PLOS ONE, 2014
    Co-Authors: Arthur Jorge Santiago Lima, Anneke Maat-kievit, Akio Nakashima, Ans M W Van Den Ouweland, Dicky J. J. Halley, Ushio Kikkawa, Marianne Hoogeveen-westerveld, Mark Nellist
    Abstract:

    The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction.

  • The TSC1-TSC2 complex consists of multiple TSC1 and TSC2 subunits
    BMC Biochemistry, 2012
    Co-Authors: Marianne Hoogeveen-westerveld, Leontine Van Unen, André T. Hoogeveen, Ans M W Van Den Ouweland, Dicky J. J. Halley, Mark Nellist
    Abstract:

    Background Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the activity of the target of rapamycin (TOR) complex 1 (TORC1) and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation.

  • Functional assessment of variants in the TSC1 and TSC2 genes identified in individuals with Tuberous Sclerosis Complex
    Human Mutation, 2011
    Co-Authors: Mark Nellist, Kay Metcalfe, Sue Povey, Rosemary Ekong, Melika Mozaffari, Marjolein Wentink, Marianne Hoogeveen-westerveld, Diana Van Den Heuvel, Johan T. Den Dunnen, Stephanie Vallee
    Abstract:

    The effects of missense changes and small in-frame deletions and insertions on protein function are not easy to predict and the identification of such variants in individuals at risk of a genetic disease can complicate genetic counselling. One option is to perform functional tests to assess whether the variants affect protein function. We have used this strategy to characterise variants identified in the TSC1 and TSC2 genes in individuals with, or suspected of having, Tuberous Sclerosis Complex (TSC). Here we present an overview of our functional studies on 45 TSC1 and 107 TSC2 variants. Using a standardised protocol we classified 16 TSC1 variants and 70 TSC2 variants as pathogenic. In addition we identified 8 putative splice site mutations (5 TSC1 and 3 TSC2). The remaining 24 TSC1 and 34 TSC2 variants were classified as probably neutral.

David J Kwiatkowski - One of the best experts on this subject based on the ideXlab platform.

  • TSC1 involvement in bladder cancer diverse effects and therapeutic implications
    The Journal of Pathology, 2013
    Co-Authors: Yanan Guo, Yvonne Chekaluk, Jianming Zhang, Nathanael S Gray, David J Kwiatkowski
    Abstract:

    TSC1 is often mutated in bladder cancer. However the importance of this event in disease pathogenesis and its implications for therapy are uncertain. We used genomic sequencing to examine the involvement of TSC1 in bladder cancer, and signalling pathway analysis and small-molecule screening to identify targeted therapeutic strategies in TSC1 mutant bladder cancer cell lines. TSC1 loss of heterozygosity was seen in 54% of bladder cancers. Two (4.9%) of these 41 bladder cancers had TSC1 mutations by exon-based sequencing. Analysis of 27 bladder cancer cell lines demonstrated inactivating TSC1 mutations in three: RT-4, HCV29, 97-1. Interestingly, only RT-4 showed classic feedback inhibition of AKT, and was highly sensitive to treatment with mTOR inhibitors rapamycin and Torin1. 97-1 cells showed constitutive EGFR activation, and were highly sensitive to combined treatment with the mTOR inhibitor Torin1 and EGFR inhibitors Lapatinib or Afatinib. A BRAF missense mutation G469V was found in HCV29 cells, and AKT activation was dependent on BRAF, but independent of ERK. A kinase inhibitor screen of HCV29 cells showed strong growth inhibition by the Hsp90 inhibitor NVP-AUY922, and we then found synergistic inhibitory effects of NVP-AUY922 combined with either Torin1 or rapamycin on cell survival for both HCV29 and 97-1 cells. In aggregate, these findings indicate that there are highly variable mutation profiles and signalling pathway activation in TSC1-mutant bladder cancer. Furthermore, combined Hsp90/mTOR inhibition is a promising therapeutic approach for TSC1 mutant bladder cancer.

  • tbc1d7 is a third subunit of the TSC1 tsc2 complex upstream of mtorc1
    Molecular Cell, 2012
    Co-Authors: Christian C Dibble, Winfried Elis, Justin Klekota, Peter Finan, Suchithra Menon, John M Asara, David J Kwiatkowski, Leon O Murphy
    Abstract:

    The tuberous sclerosis complex (TSC) tumor suppressors form the TSC1-TSC2 complex, which limits cell growth in response to poor growth conditions. Through its GTPase-activating protein (GAP) activity toward Rheb, this complex inhibits the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1), a key promoter of cell growth. Here, we identify and biochemically characterize TBC1D7 as a stably associated and ubiquitous third core subunit of the TSC1-TSC2 complex. We demonstrate that the TSC1-TSC2-TBC1D7 (TSC-TBC) complex is the functional complex that senses specific cellular growth conditions and possesses Rheb-GAP activity. Sequencing analyses of samples from TSC patients suggest that TBC1D7 is unlikely to represent TSC3. TBC1D7 knockdown decreases the association of TSC1 and TSC2 leading to decreased Rheb-GAP activity, without effects on the localization of TSC2 to the lysosome. Like the other TSC-TBC components, TBC1D7 knockdown results in increased mTORC1 signaling, delayed induction of autophagy, and enhanced cell growth under poor growth conditions.

  • identification of a region required for TSC1 stability by functional analysis of TSC1 missense mutations found in individuals with tuberous sclerosis complex
    BMC Medical Genetics, 2009
    Co-Authors: Melika Mozaffari, Sue Povey, Rosemary Ekong, Marianne Hoogeveenwesterveld, Dicky J. J. Halley, Johan Den T Dunnen, David J Kwiatkowski, Julian R Sampson, Ans M W Van Den Ouweland, Mark Nellist
    Abstract:

    Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Recently it has been shown that missense mutations to the TSC1 gene can cause TSC. Methods: We have used in vitro biochemical assays to investigate the effects on TSC1 function of TSC1 missense variants submitted to the Leiden Open Variation Database. Results: We identified specific substitutions between amino acids 50 and 190 in the N-terminal region of TSC1 that result in reduced steady state levels of the protein and lead to increased mTOR signalling. Conclusion: Our results suggest that amino acid residues within the N-terminal region of TSC1 are important for TSC1 function and for maintaining the activity of the TSC1-TSC2 complex.

  • A hypomorphic allele of Tsc2 highlights the role of TSC1/TSC2 in signaling to AKT and models mild human TSC2 alleles
    Human Molecular Genetics, 2009
    Co-Authors: Kristen Pollizzi, Izabela Malinowska-kolodziej, Cheryl A. Doughty, June Goto, Charles Betz, Jian Ma, David J Kwiatkowski
    Abstract:

    Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome in which hamartomas develop in multiple organ systems. Knockout and conditional alleles of TSC1 and Tsc2 have been previously reported. Here, we describe the generation of a novel hypomorphic allele of Tsc2 (del3), in which exon 3, encoding 37 amino acids near the N terminus of tuberin, is deleted. Embryos homozygous for the del3 allele survive until E13.5, 2 days longer than Tsc2 null embryos. Embryos die from underdevelopment of the liver, deficient hematopoiesis, aberrant vascular development and hemorrhage. Mice that are heterozygous for the del3 allele have a markedly reduced kidney tumor burden in comparison with conventional Tsc2 +/― mice. Murine embryo fibroblast (MEF) cultures that are homozygous for the del3 allele express mutant tuberin at low levels, and show enhanced activation of mTORC1, similar to Tsc2 null MEFs. Furthermore, the mutant cells show prominent reduction in the activation of AKT. Similar findings were made in the analysis of homozygous del3 embryo lysates. Tsc2-del3 demonstrates GTPase activating protein activity comparable to that of wild-type Tsc2 in a functional assay. These findings indicate that the del3 allele is a hypomorphic allele of Tsc2 with partial function due to reduced expression, and highlight the consistency of AKT downregulation when TSC1/Tsc2 function is reduced. Tsc2-del3 mice also serve as a model for hypomorphic TSC2 missense mutations reported in TSC patients.

  • mtorc1 dependent and independent regulation of stem cell renewal differentiation and mobilization
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Boyi Gan, David J Kwiatkowski, Ergun Sahin, Shan Jiang, Abel Sanchezaguilera, Kenneth L Scott, Lynda Chin, David A Williams, Ronald A Depinho
    Abstract:

    The Tuberous Sclerosis Complex component, TSC1, functions as a tumor suppressor via its regulation of diverse cellular processes, particularly cell growth. TSC1 exists in a complex with TSC2 and functions primarily as a key negative regulator of mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis, although the TSC1/TSC2 complex also shows mTORC1-independent outputs to other pathways. Here, we explored the role of TSC1 in various aspects of stem cell biology and dissected the extent to which TSC1 functions are executed via mTORC1-dependent versus mTORC1-independent pathways. Using hematopoietic stem cells (HSCs) as a model system, we demonstrate that somatic deletion of TSC1 produces striking stem cell and derivative effector cell phenotypes characterized by increased HSC cell cycling, mobilization, marked progressive depletion, defective long-term repopulating potential, and hematopoietic lineage developmental aberrations. On the mechanistic level, we further establish that TSC1 regulation of HSC quiescence and long-term repopulating potential and hematopoietic lineage development is mediated through mTORC1 signaling. In contrast, TSC1 regulation of HSC mobilization is effected in an mTORC1-independent manner, and gene profiling and functional analyses reveals the actin-bundling protein FSCN1 as a key TSC1/TSC2 target in the regulation of HSC mobilization. Thus, TSC1 is a critical regulator of HSC self-renewal, mobilization, and multilineage development and executes these actions via both mTORC1-dependent and -independent pathways.

Dicky J. J. Halley - One of the best experts on this subject based on the ideXlab platform.

  • Identification of regions critical for the integrity of the TSC1-TSC2-TBC1D7 complex.
    PLOS ONE, 2014
    Co-Authors: Arthur Jorge Santiago Lima, Anneke Maat-kievit, Akio Nakashima, Ans M W Van Den Ouweland, Dicky J. J. Halley, Ushio Kikkawa, Marianne Hoogeveen-westerveld, Mark Nellist
    Abstract:

    The TSC1-TSC2-TBC1D7 complex is an important negative regulator of the mechanistic target of rapamycin complex 1 that controls cell growth in response to environmental cues. Inactivating TSC1 and TSC2 mutations cause tuberous sclerosis complex (TSC), an autosomal dominant disorder characterised by the occurrence of benign tumours in various organs and tissues, notably the brain, skin and kidneys. TBC1D7 mutations have not been reported in TSC patients but homozygous inactivation of TBC1D7 causes megaencephaly and intellectual disability. Here, using an exon-specific deletion strategy, we demonstrate that some regions of TSC1 are not necessary for the core function of the TSC1-TSC2 complex. Furthermore, we show that the TBC1D7 binding site is encoded by TSC1 exon 22 and identify amino acid residues involved in the TSC1-TBC1D7 interaction.

  • The TSC1-TSC2 complex consists of multiple TSC1 and TSC2 subunits
    BMC Biochemistry, 2012
    Co-Authors: Marianne Hoogeveen-westerveld, Leontine Van Unen, André T. Hoogeveen, Ans M W Van Den Ouweland, Dicky J. J. Halley, Mark Nellist
    Abstract:

    Background Mutations to the TSC1 and TSC2 genes cause the disease tuberous sclerosis complex. The TSC1 and TSC2 gene products form a protein complex that integrates multiple metabolic signals to regulate the activity of the target of rapamycin (TOR) complex 1 (TORC1) and thereby control cell growth. Here we investigate the quaternary structure of the TSC1-TSC2 complex by gel filtration and coimmunoprecipitation.

  • identification of a region required for TSC1 stability by functional analysis of TSC1 missense mutations found in individuals with tuberous sclerosis complex
    BMC Medical Genetics, 2009
    Co-Authors: Melika Mozaffari, Sue Povey, Rosemary Ekong, Marianne Hoogeveenwesterveld, Dicky J. J. Halley, Johan Den T Dunnen, David J Kwiatkowski, Julian R Sampson, Ans M W Van Den Ouweland, Mark Nellist
    Abstract:

    Background: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene on chromosome 9q34, or the TSC2 gene on chromosome 16p13.3. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). Recently it has been shown that missense mutations to the TSC1 gene can cause TSC. Methods: We have used in vitro biochemical assays to investigate the effects on TSC1 function of TSC1 missense variants submitted to the Leiden Open Variation Database. Results: We identified specific substitutions between amino acids 50 and 190 in the N-terminal region of TSC1 that result in reduced steady state levels of the protein and lead to increased mTOR signalling. Conclusion: Our results suggest that amino acid residues within the N-terminal region of TSC1 are important for TSC1 function and for maintaining the activity of the TSC1-TSC2 complex.

  • A reliable cell-based assay for testing unclassified TSC2 gene variants
    European Journal of Human Genetics, 2008
    Co-Authors: Ricardo Coevoets, Sermin Arican, Erik J. Simons, Ans M W Van Den Ouweland, Dicky J. J. Halley, Marianne Hoogeveen-westerveld, Mark Nellist
    Abstract:

    Tuberous sclerosis complex (TSC) is characterised by seizures, mental retardation and the development of hamartomas in a variety of organs and tissues. The disease is caused by mutations in either the TSC1 gene or the TSC2 gene. The TSC1 and TSC2 gene products, TSC1 and TSC2, form a protein complex that inhibits signal transduction to the downstream effectors of the mammalian target of rapamycin (mTOR). We have developed a straightforward, semiautomated in-cell western (ICW) assay to investigate the effects of amino acid changes on the TSC1–TSC2-dependent inhibition of mTOR activity. Using this assay, we have characterised 20 TSC2 variants identified in individuals with TSC or suspected of having the disease. In 12 cases, we concluded that the identified variant was pathogenic. The ICW is a rapid, reproducible assay, which can be applied to the characterisation of the effects of novel TSC2 variants on the activity of the TSC1–TSC2 complex.

  • overlapping neurologic and cognitive phenotypes in patients with TSC1 or tsc2 mutations
    Neurology, 2008
    Co-Authors: F E Jansen, O Braams, P Anbeek, A Jennekensschinkel, Ans M W Van Den Ouweland, Ale Algra, Dicky J. J. Halley, Bernard A. Zonnenberg, Koen L. Vincken, Alexander C. Van Huffelen
    Abstract:

    Objective: The purpose of this study was to systematically analyze the associations between different TSC1 and TSC2 mutations and the neurologic and cognitive phenotype in patients with tuberous sclerosis complex (TSC). Methods: Mutation analysis was performed in 58 patients with TSC. Epilepsy variables, including EEG, were classified. A cognition index was determined based on a comprehensive neuropsychological assessment. On three-dimensional fluid-attenuated inversion recovery MR images, an automated tuber segmentation program detected and calculated the number of tubers and the proportion of total brain volume occupied by tubers (tuber/brain proportion [TBP]). Results: As a group, patients with a TSC2 mutation had earlier age at seizure onset, lower cognition index, more tubers, and a greater TBP than those with a TSC1 mutation, but the ranges overlapped considerably. Familial cases were older at seizure onset and had a higher cognition index than nonfamilial cases. Patients with a mutation deleting or directly inactivating the tuberin GTPase activating protein (GAP) domain had more tubers and a greater TBP than those with an intact GAP domain. Patients with a truncating TSC1 or TSC2 mutation differed from those with nontruncating mutations in seizure types only. Conclusions: Although patients with a TSC1 mutation are more likely to have a less severe neurologic and cognitive phenotype than those with a TSC2 mutation, the considerable overlap between both aspects of the phenotype implies that prediction of the neurologic and cognitive phenotypes in individuals with tuberous sclerosis complex should not be based on their particular TSC1 or TSC2 mutation.

Petrus J De Vries - One of the best experts on this subject based on the ideXlab platform.

  • intellectual ability in tuberous sclerosis complex correlates with predicted effects of mutations on TSC1 and tsc2 proteins
    Journal of Medical Genetics, 2015
    Co-Authors: Ho Tin Wong, Christopher J. Howe, Julian R Sampson, Deborahl Mccartney, Julia Lewis, Petrus J De Vries
    Abstract:

    Background Tuberous sclerosis complex is a multisystem genetic disease, caused by mutation in the TSC1 or TSC2 genes, associated with many features, including intellectual disability (ID). We examined psychometric profiles of patients with TSC1 or TSC2 mutations and tested whether different mutation types were associated with different degrees of intellectual ability. Methods One hundred subjects with known TSC1 / TSC2 mutations were assessed using a range of IQ or developmental quotient (DQ) measures. Effects of mutations on TSC1/TSC2 proteins were inferred from sequence data and published biochemical studies. Results Most individuals with TSC1 mutations fell on a normal distribution identical to the general population, with ∼10% showing profound ID. Of individuals with TSC2 mutations, 34% showed profound ID, and the remainder a pattern of IQ/DQ more variable and shifted to the left than in TSC1 or the general population. Truncating TSC1 mutations were all predicted to be subject to nonsense-mediated mRNA decay. Mutations predicted to result in unstable protein were associated with less severe effects on IQ/DQ. There was a statistically significant negative correlation between length of predicted aberrant C-terminal tails arising from frameshift mutations in TSC1 and IQ/DQ; for TSC2 a positive but not statistically significant correlation was observed. Conclusion We propose a model where (i) IQ/DQ correlates inversely with predicted levels and/or deleterious biochemical effects of mutant TSC1 or TSC2 protein, and (ii) longer aberrant C-terminal tails arising from frameshift mutations are more detrimental for TSC1 and less for TSC2. Predictions of the model require replication and biochemical testing.

  • evolution of the TSC1 tsc2 tor signaling pathway
    Science Signaling, 2010
    Co-Authors: Jaco Serfontein, Ellen R R Nisbet, Christopher J. Howe, Petrus J De Vries
    Abstract:

    The TSC1/TSC2-TOR signaling pathway [the signaling pathway that includes the heterodimeric TSC1 (tuberous sclerosis 1 protein)–TSC2 (tuberous sclerosis 2 protein) complex and TOR (target of rapamycin)] regulates various cellular processes, including protein synthesis, in response to growth factors and nutrient availability. Homologs of some pathway components have been reported from animals, fungi, plants, and protozoa. These observations led to the perception that the whole pathway is evolutionarily conserved throughout eukaryotes. Using complete genome sequences, we show that, contrary to this view, the pathway was built up from a simpler one, present in the ancestral eukaryote, coupling cell growth to energy supplies. Additional elements, such as TSC1 and TSC2, were “bolted on” in particular eukaryotic lineages. Our results also suggest that unikonts [Opisthokonta (including animals and fungi) and Amoebozoa] form a monophyletic group with the Excavata and Chromalveolata. A previous proposal, that the root of the eukaryotic “tree of life” lies between the unikonts and other organisms, should therefore be reevaluated.

  • Evolution of the TSC1/TSC2-TOR Signaling Pathway
    Science Signaling, 2010
    Co-Authors: Jaco Serfontein, R. Ellen R. Nisbet, Christopher J. Howe, Petrus J De Vries
    Abstract:

    The TSC1/TSC2-TOR signaling pathway [the signaling pathway that includes the heterodimeric TSC1 (tuberous sclerosis 1 protein)-TSC2 (tuberous sclerosis 2 protein) complex and TOR (target of rapamycin)] regulates various cellular processes, including protein synthesis, in response to growth factors and nutrient availability. Homologs of some pathway components have been reported from animals, fungi, plants, and protozoa. These observations led to the perception that the whole pathway is evolutionarily conserved throughout eukaryotes. Using complete genome sequences, we show that, contrary to this view, the pathway was built up from a simpler one, present in the ancestral eukaryote, coupling cell growth to energy supplies. Additional elements, such as TSC1 and TSC2, were "bolted on" in particular eukaryotic lineages. Our results also suggest that unikonts [Opisthokonta (including animals and fungi) and Amoebozoa] form a monophyletic group with the Excavata and Chromalveolata. A previous proposal, that the root of the eukaryotic "tree of life" lies between the unikonts and other organisms, should therefore be reevaluated.

Ken Inoki - One of the best experts on this subject based on the ideXlab platform.

  • TSC1 tsc2 and rheb have different effects on torc1 and torc2 activity
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Qian Yang, Ken Inoki, Kunliang Guan
    Abstract:

    Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (Rheb) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of Rheb in TORC1 activation remains to be determined. However, the function of Rheb in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRheb has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRheb on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that Rheb does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of Rheb, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and Rheb have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.

  • TSC1/TSC2 and Rheb have different effects on TORC1 and TORC2 activity
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Qian Yang, Ken Inoki, Kunliang Guan
    Abstract:

    Target of rapamycin (TOR) plays a central role in cell growth regulation by integrating signals from growth factors, nutrients, and cellular energy levels. TOR forms two distinct physical and functional complexes, termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). TORC1, which is sensitive to rapamycin, regulates translation and cell growth, whereas TORC2, which is insensitive to rapamycin, regulates cell morphology and cell growth. The Ras homology enriched in brain (Rheb) small GTPase is known to be a key upstream activator of TORC1, although the mechanism of Rheb in TORC1 activation remains to be determined. However, the function of Rheb in the TORC2 regulation has not been elucidated. By measuring Akt and S6K phosphorylation as a functional assay for TORC1 and -2, here, we report that dRheb has an inhibitory effect on dTORC2 activity in Drosophila S2 cells. This negative effect of dRheb on dTORC2 is possibly due to a feedback mechanism involving dTORC1 and dS6K. We also observed that Rheb does not activate TORC2 in human embryonic kidney 293 cells, although it potently stimulates TORC1. Furthermore, tuberous sclerosis complex 1 (TSC1) and TSC2, which are negative regulators of Rheb, have negative and positive effects on TORC1 and -2, respectively. Our observations suggest that TSC1/2 and Rheb have different effects on the activity of TORC1 and -2, further supporting the complexity of TOR regulation.

  • TSC1 stabilizes tsc2 by inhibiting the interaction between tsc2 and the herc1 ubiquitin ligase
    Journal of Biological Chemistry, 2006
    Co-Authors: Huira Chongkopera, Ken Inoki, Francesc R Garciagonzalo, Yong Li, Jose Luis Rosa
    Abstract:

    Abstract Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.

  • tsc2 filling the gap in the mtor signaling pathway
    Trends in Biochemical Sciences, 2004
    Co-Authors: Yong Li, Michael N Corradetti, Ken Inoki
    Abstract:

    Abstract The tumor-suppressor proteins TSC1 and TSC2 are associated with an autosomal dominant disorder known as tuberous sclerosis complex (TSC). TSC1 and TSC2 function as a heterodimer to inhibit cell growth and proliferation. Another protein, mTOR (mammalian target of rapamycin), is regarded as a central controller of cell growth in response to growth factors, cellular energy and nutrient levels. Recent breakthroughs in TSC research link the TSC1/2 heterodimer protein to the mTOR signaling network. It has recently been shown that TSC2 has GTPase-activating protein (GAP) activity towards the Ras family small GTPase Rheb (Ras homolog enriched in brain), and TSC1/2 antagonizes the mTOR signaling pathway via stimulation of GTP hydrolysis of Rheb. Thus, TSC1/2 and Rheb have pivotal roles in mediating growth factors, nutrient and energy sensing signals to mTOR-dependent targets. These discoveries lend new insight into TSC pathogenesis.

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