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John Blenis - One of the best experts on this subject based on the ideXlab platform.
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Tumor-promoting phorbol esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90 ribosomal S6 kinase
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Philippe P Roux, Rana Anjum, Steven P. Gygi, Bryan A. Ballif, John BlenisAbstract:Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2, which encode Hamartin and tuberin, respectively. Tuberin and Hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/Hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.
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inactivation of the tuberous sclerosis complex 1 and 2 gene products occurs by phosphoinositide 3 kinase akt dependent and independent phosphorylation of tuberin
Journal of Biological Chemistry, 2003Co-Authors: Andrew R. Tee, Rana Anjum, John BlenisAbstract: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.
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Tuberous Sclerosis Complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb
Current Biology, 2003Co-Authors: Andrew R. Tee, Philippe P Roux, Brendan D Manning, Lewis C Cantley, John BlenisAbstract: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.
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tuberous sclerosis complex 1 and 2 gene products function together to inhibit mammalian target of rapamycin mtor mediated downstream signaling
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Andrew R. Tee, Brendan D Manning, Lewis C Cantley, David J Kwiatkowski, Diane C Fingar, John BlenisAbstract:Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products Hamartin and tuberin, respectively. Here, we show that Hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of Hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of Hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, Hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-Hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.
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tuberous sclerosis complex 1 and 2 gene products function together to inhibit mammalian target of rapamycin mtor mediated downstream signaling
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Diane C Fingar, Lewis C Cantley, Brendan D Manning, David J Kwiatkowski, John BlenisAbstract:Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products Hamartin and tuberin, respectively. Here, we show that Hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of Hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of Hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, Hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-Hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.
Elizabeth P Henske - One of the best experts on this subject based on the ideXlab platform.
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Hamartin the tuberous sclerosis complex 1 gene product interacts with polo like kinase 1 in a phosphorylation dependent manner
Human Molecular Genetics, 2006Co-Authors: Aristotelis Astrinidis, William Senapedis, Elizabeth P HenskeAbstract:Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome caused by mutations in TSC1 and TSC2. Hamartin and tuberin, the products of TSC1 and TSC2, respectively, form heterodimers and inhibit the mammalian target of rapamycin. Previously, we have shown that Hamartin is phosphorylated by CDC2/ cyclin B1 during the G 2 /M phase of the cell cycle. Here, we report that Hamartin is localized to the centrosome and that phosphorylated Hamartin and phosphorylated tuberin co-immunoprecipitate with the mitotic kinase Plk1. Plk1 interacts with the N-terminus of Hamartin (amino acids 1-880), which contains two potential Plk1-binding sites (T310 and S332). Phosphorylated Hamartin interacts with Plk1 independent of tuberin with all three proteins present in a complex. A non-phosphorylatable Hamartin mutant with an alanine substitution at residue T310 does not interact with Plk1, whereas a non-phosphorylatable Hamartin mutant at residue S332 in conjunction with alanine mutations at the other CDC2/cyclin B1 sites (T417, S584 and T1047) does not impact Hamartin binding to Plk1. Hamartin negatively regulates the protein levels of Plk1. Finally, Tsc1 -/- mouse embryonic fibroblasts (MEFs) have increased number of centrosomes and increased DNA content, compared to Tsc1 +/+ cells. Both phenotypes are rescued after pre-treatment with the mTOR inhibitor rapamycin. RNAi inhibition of Plk1 in Tsc1 -/- MEFs failed to rescue the increased centrosome number phenotype. These data reveal a novel subcellular localization for Hamartin and a novel interaction partner for the Hamartin/tuberin complex and implicate Hamartin and mTOR in the regulation of centrosome duplication.
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cell cycle regulated phosphorylation of Hamartin the product of the tuberous sclerosis complex 1 gene by cyclin dependent kinase 1 cyclin b
Journal of Biological Chemistry, 2003Co-Authors: Aristotelis Astrinidis, William Senapedis, Thomas R Coleman, Elizabeth P HenskeAbstract: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.
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metastasis of benign tumor cells in tuberous sclerosis complex
Genes Chromosomes and Cancer, 2003Co-Authors: Elizabeth P HenskeAbstract:Lymphangiomyomatosis (LAM) is a life-threatening lung disease affecting almost exclusively young women. Histologically, LAM is characterized by the diffuse, bilateral proliferation of abnormal smooth muscle cells and cystic degeneration of the lung parenchyma. LAM can occur as an isolated disorder (sporadic LAM), or in women with tuberous sclerosis complex (TSC-LAM). Patients with both sporadic LAM and TSC-LAM often have benign renal angiomyolipomas. The smooth muscle cells within the angiomyolipomas are very similar to the smooth muscle cells in pulmonary LAM. Genetic data suggest that pulmonary LAM is the result of a highly unusual disease mechanism: the metastasis of benign cells. If LAM is the result of metastasis, it is remarkable that the metastasis occurs in women, but not in men. In this review, I discuss the genetic data supporting this metastatic model for LAM. The implications of the model for the functions of the TSC1 and TSC2 gene products, Hamartin and tuberin, respectively, will also be considered. Hamartin and tuberin may play functional roles in the suppression of cell migration and/or metastasis, possibly through their regulation of the small GTPase Rho. © 2003 Wiley-Liss, Inc.
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mutation in tsc2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma
The Lancet, 2003Co-Authors: Nisreen Elhashemite, Elizabeth P Henske, Hongbing Zhang, David J KwiatkowskiAbstract:Summary Mutations that inactivate either TSC1 or TSC2 cause tuberous sclerosis. We have used immunoblotting and immunohistochemical analysis to see whether there is phosphorylation of p70 S6 kinase, and the ribosomal S6 protein in angiomyolipomas occurring in tuberous scierosis. Hamartin (encoded by TSC1 ) and S6K was expressed in all samples. Tuberin ( TSC2 ) was weak or absent in angiomyolipomas, but present in healthy kidney, whereas, phosphorylated p70 S6 kinase and p56 were present only in angiomyolipomas. Our results indicate activation of a mammalian target of rapamycin metabolic pathway in tuberous sclerosis lesions, which contributes to their growth. We suggest that treatment with rapamycin and its analogues could benefit such patients.
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the expression of Hamartin the product of the tsc1 gene in normal human tissues and in tsc1 and tsc2 linked angiomyolipomas
Modern Pathology, 1999Co-Authors: Tracey L. Plank, Helen Logginidou, Andres J Kleinszanto, Elizabeth P HenskeAbstract:Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by seizures, mental retardation, and hamartomatous tumors in multiple organs, including subependymal giant cell astrocytomas, cardiac rhabdomyomas, and renal angiomyolipomas. Mutations in two genes are associated with TSC: TSC1, which was cloned in 1997, and TSC2, which was cloned in 1993. We report here the expression of Hamartin, the product of the TSC1 gene, in normal human tissues and in renal angiomyolipomas from TSC1- and TSC2-linked patients. By Western blot analysis, Hamartin is strongly expressed in brain, kidney, and heart, all of which are frequently affected in TSC. By immunohistochemical analysis, the expression pattern of Hamartin in normal human tissues was almost identical to that of tuberin, the product of the TSC2 gene. This is consistent with the recent finding that tuberin and Hamartin interact and with the clinical similarity between TSC1- and TSC2-linked disease. Strong Hamartin expression was seen in cortical neurons, renal tubular epithelial cells, pancreatic islet cells, bronchial epithelial cells, and pulmonary macrophages. Hamartin was also expressed in endocrine tissues, including islet cells of the pancreas, follicular cells of the thyroid, and the zona reticularis of the adrenal cortex. In eight angiomyolipomas from a TSC1-linked patient, no Hamartin expression was detected, whereas tuberin, the product of the TSC2 gene, was expressed. In 19 angiomyolipomas from a TSC2-linked patient, in whose angiomyolipomas loss of tuberin expression had previously been shown, Hamartin expression was present. These data suggest that tuberin and Hamartin immunoreactivity can distinguish tumors with underlying TSC1 mutations from those with TSC2 mutations. This differentiation might have diagnostic implications.
Dicky J. J. Halley - One of the best experts on this subject based on the ideXlab platform.
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Distinct effects of single amino-acid changes to tuberin on the function of the tuberin-Hamartin complex.
European journal of human genetics : EJHG, 2004Co-Authors: Mark Nellist, Miriam Goedbloed, Ans M.w. Van Den Ouweland, Ozgur Sancak, Christan F. Rohé, Diana Van Netten, Karin Mayer, Aimee Tucker-williams, Dicky J. J. HalleyAbstract:Tuberous sclerosis is an autosomal dominant human disorder caused by inactivating mutations to either the TSC1 or TSC2 tumour suppressor gene. Hamartin and tuberin, the TSC1 and TSC2 gene products, interact and the tuberin-Hamartin complex inhibits cell growth by antagonising signal transduction to downstream effectors of the mammalian target of rapamycin (mTOR) through the small GTPase rheb. Previously, we showed that pathogenic tuberin amino-acid substitutions disrupt the tuberin-Hamartin complex. Here, we investigate how these mutations affect the role of tuberin in the control of signal transduction through mTOR. Our data indicate that specific amino-acid substitutions have distinct effects on tuberin function.
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phosphatidylinositol 3 kinase akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin
Journal of Biological Chemistry, 2002Co-Authors: Han C. Dan, Mark Nellist, Raymond S Yeung, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Santo V. Nicosia, W J PledgerAbstract:Normal cellular functions of Hamartin and tuberin, encoded by the TSC1 and TSC2tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the Hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with Hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of Hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27kip1, thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling Hamartin-tuberin function.
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tsc2 missense mutations inhibit tuberin phosphorylation and prevent formation of the tuberin Hamartin complex
Human Molecular Genetics, 2001Co-Authors: Mark Nellist, Arnold J J Reuser, Brenda Verhaaf, Miriam Goedbloed, Ans M.w. Van Den Ouweland, Dicky J. J. HalleyAbstract:Tuberous sclerosis (TSC) is an autosomal dominant disorder characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction and dermatological abnormalities. Inactivating mutations to either of the TSC1 and TSC2 tumour suppressor genes are responsible for the disease. TSC1 and TSC2 encode two large novel proteins called Hamartin and tuberin, respectively. Hamartin and tuberin interact directly with each other and it has been reported that tuberin may act as a chaperone, preventing Hamartin self-aggregation and maintaining the tuberin-Hamartin complex in a soluble form. In this study, the ability of tuberin to act as a chaperone for Hamartin was used to investigate the tuberin-Hamartin interaction in more detail. A domain within tuberin necessary for the chaperone function was identified, and the effects of TSC2 missense mutations on the tuberin-Hamartin interaction were investigated to allow specific residues within the central domain of tuberin that are important for the interaction with Hamartin to be pin-pointed. In addition, the results confirm that phosphorylation may play an important role in the formation of the tuberin-Hamartin complex. Although mutations that prevent tuberin tyrosine phosphorylation also inhibit tuberin-Hamartin binding and the chaperone function, our results indicate that only Hamartin is phosphorylated in the tuberin-Hamartin complex.
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the tuberous sclerosis 1 tsc1 gene product Hamartin suppresses cell growth and augments the expression of the tsc2 product tuberin by inhibiting its ubiquitination
Oncogene, 2000Co-Authors: Giovanna Benvenuto, Julian Roy Sampson, Jeremy Peter Cheadle, Samantha J Brown, Richard Braverman, Shaowei Li, William C Vass, Dicky J. J. Halley, Ralf Wienecke, J E DeclueAbstract:We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product Hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and Hamartin is known to bind tuberin, these results suggested that Hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to Hamartin is not ubiquitinated. Co-expression of tuberin stabilized Hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of Hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize Hamartin. We conclude that Hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin. 1,a,b, 1,a, Samantha J Brown1, Richard Braverman1, William C Vass1, Jeremy P Cheadle2, Dicky JJ Halley3, Julian R Sampson2, Ralf Wienecke4 and Jeffrey E DeClue1
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The tuberous sclerosis-1 ( TSC1 ) gene product Hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination
Oncogene, 2000Co-Authors: Giovanna Benvenuto, Julian Roy Sampson, Jeremy Peter Cheadle, Samantha J Brown, Richard Braverman, William C Vass, Dicky J. J. Halley, Ralf Wienecke, J E DeclueAbstract:We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product Hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and Hamartin is known to bind tuberin, these results suggested that Hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to Hamartin is not ubiquitinated. Co-expression of tuberin stabilized Hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of Hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize Hamartin. We conclude that Hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.
Raymond S Yeung - One of the best experts on this subject based on the ideXlab platform.
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Advances in Brief Hiimartin, the Product of the Tuberous Sclerosis l (TSC1} Gene, Interacts with Tuberin and Appears to Be Localized to Cytoplasmic Vesicles1
2016Co-Authors: Tracey L. Plank, Raymond S Yeung, Elizabeth Petri HenskeAbstract:Tuberous sclerosis is an inherited syndrome associated with mutations in two tumor suppressor genes: TSCI and TSC2. Tuberin, the product of TSC2, appears to be localized to the Golgi apparatus and may have a function in vesicular transport. The function of Hamartin, the product of TSCI, is not known. In this report, we demonstrate an interaction between Hamartin and tuberin, which is detectable at endogenous protein levels. Hamartin is present in a cell line derived from the Eker rat that lacks functional tuberin, indicating that the stability of Hamartin is not depend ent on its interaction with tuberin. Hamartin is localized to the membrane/ particulale (PI(H)) fraction of cultured cells. The I"I(10 localization is unchanged in the Eker cells. Finally, we show that at endogenous expres sion levels, Hamartin has a punctate pattern of immunofluorescence in the cytoplasm. Taken together, the presence of Hamartin in the membrane/ participate fraction and its pattern of cytoplasmic staining suggest that it is localized to cytoplasmic vesicles. If altered vesicular trafficking leads to tumorigenesis in tuberous sclerosis, TSCI and TSC2 may have a novel mechanism of tumor suppression
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The Tuberin-Hamartin Complex Negatively Regulates β-Catenin Signaling Activity
The Journal of biological chemistry, 2003Co-Authors: Baldwin C. Mak, Ken-ichi Takemaru, Heidi L. Kenerson, Randall T. Moon, Raymond S YeungAbstract:Tuberous sclerosis complex (TSC) is characterized by the formation of hamartomas in multiple organs resulting from mutations in the TSC1 or TSC2 gene. Their protein products, Hamartin and tuberin, respectively, form a functional complex that affects cell growth, differentiation, and proliferation. Several lines of evidence, including renal tumors derived from TSC2+/- animals, suggest that the loss or inhibition of tuberin is associated with up-regulation of cyclin D1. As cyclin D1 can be regulated through the canonical Wnt/beta-catenin signaling pathway, we hypothesize that the cell proliferative effects of Hamartin and tuberin are partly mediated through beta-catenin. In this study, total beta-catenin protein levels were found to be elevated in the TSC2-related renal tumors. Ectopic expression of Hamartin and wild-type tuberin, but not mutant tuberin, reduced beta-catenin steady-state levels and its half-life. The TSC1-TSC2 complex also inhibited Wnt-1 stimulated Tcf/LEF luciferase reporter activity. This inhibition was eliminated by constitutively active beta-catenin but not by Disheveled, suggesting that Hamartin and tuberin function at the level of the beta-catenin degradation complex. Indeed, Hamartin and tuberin co-immunoprecipitated with glycogen synthase kinase 3 beta and Axin, components of this complex in a Wnt-1-dependent manner. Our data suggest that Hamartin and tuberin negatively regulate beta-catenin stability and activity by participating in the beta-catenin degradation complex.
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phosphatidylinositol 3 kinase akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin
Journal of Biological Chemistry, 2002Co-Authors: Han C. Dan, Mark Nellist, Raymond S Yeung, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Santo V. Nicosia, W J PledgerAbstract:Normal cellular functions of Hamartin and tuberin, encoded by the TSC1 and TSC2tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the Hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with Hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of Hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27kip1, thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling Hamartin-tuberin function.
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Multicompartmental distribution of the tuberous sclerosis gene products, Hamartin and tuberin.
Archives of biochemistry and biophysics, 2002Co-Authors: Yuji Yamamoto, Baldwin C. Mak, Kathryn A Jones, Atis Muehlenbachs, Raymond S YeungAbstract:Mutations of the TSC1 and TSC2 genes give rise to the clinical disorder of tuberous sclerosis characterized by the development of hamartomas predominantly affecting the central nervous system, kidney, skin, lung, and heart. The function of the gene products, Hamartin and tuberin, is not well understood but we have previously suggested a role in vesicular transport. To define the subcellular compartment(s) involved with these two proteins, biochemical characterization of Hamartin and tuberin was performed in primary tissues and cell lines. Fractionation of cell lysates identified both proteins in the cytosolic, microsomal, and cytoskeletal compartments. In each of these fractions, Hamartin and tuberin formed a stable complex in coimmunoprecipitation analyses. Further, they colocalized extensively in discrete, vesicular structures in the cytoplasm. Within the microsomal compartment, Hamartin and tuberin behaved as peripheral membrane proteins that associate with the cytosolic leaflet of membranous domains. Immunoisolation of tuberin-bound vesicles using magnetic beads showed an enrichment of rap1, rab5, and caveolin-1, all of which have been found in specialized lipid microdomains, caveolae. Our data suggest that Hamartin and tuberin are multicompartmental proteins that partially reside in caveolin-1-enriched structures and potentially affect their signaling.
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tuberous sclerosis as an underlying basis for infantile spasm
International Review of Neurobiology, 2002Co-Authors: Raymond S YeungAbstract:: The study of the molecular pathogenesis of epilepsy in tuberous sclerosis has taken on a new dimension with the identification of the TSC1 and TSC2 genes. While the development of seizures is ultimately related to mutations in one of the two genes, the mechanism underlying the genotype-phenotype relationship remains a puzzle. This chapter, presented arguments in favor of the hypothesis that abnormal cortical excitability originates in and around focal areas of structural malformations (i.e., cortical tubers and dysplasia) and that these "lesions" are the biologic consequences of tuberin and/or Hamartin dysfunction. This model relies on the concept of a multistep process occurring early in cortical development whereby certain progenitor cells in the germinal layer of the ventricular zone destined for the cortex undergo inactivation of the TSC1 or TSC2 locus (Fig. 2). Immature neuroepithelial cells carrying "two-hit" mutations at either locus are believed to proliferate, migrate, and differentiate abnormally, resulting in the formation of "dysplastic" cells that are heterotopic in distribution. The pathology of the classic tuber suggests a clonal expansion of the bizarre-appearing giant cells that display incomplete, multilineage, and often ambiguous phenotype. Further, they infiltrate the six-layered structure of the cortex to form a poorly circumscribed area containing a mixture of cell types to create a highly disorganized region of a neuronal and glial network. Whether arising from the dysplastic "two-hit" target cells themselves or adjacent "innocent" bystander neurons as a result of aberrant cell-cell interaction, abnormal epileptic discharges originate from these structural abnormalities. The mechanism of how TSC1 and TSC2 inactivation causes tuber to develop is not known, but emerging experimental evidence suggests a disruption of the Hamartin-tuberin "haloenzyme" in the regulation of cell size and number via the insulin signaling pathway and a p27/CDK-dependent mechanism. Biochemically, TSC1/TSC2 may associate with cytoskeletal components and vesicular adaptors in regulating sorting and trafficking of newly synthesized and recycling proteins in the post-Golgi compartments. As such, spatial and temporal localization of proteins may be affected in tuberin or Hamartin-deficient neuronal cells where proper synaptic delivery of neurotransmitters plays an important role in normal cerebral function. We are in the earliest stages of understanding the role of TSC genes in epileptogenesis. To test the hypothesis outlined earlier, there is a need to create in vitro and in vivo models, as direct human experimentation is not feasible. To date, there are several rodent models of TSC, both spontaneous and recombinant strains. Unfortunately, none has consistently developed spontaneous cortical tubers, although one example was reported in an otherwise asymptomatic Eker rat (Mizuguchi et al., 2000). If the "two-hit" hypothesis is operational in tubers, as seen in other TSC lesions, it follows that radiation and chemical carcinogens should have a quantitative and qualitative effect on the development of these cerebral malformations. In preliminary experiments, we have found evidence of areas of cortical dysplasia in Eker rats irradiated early in life (Fig. 3). These dysplastic [figure: see text] cells stained positively with NeuN, consistent with the immunophenotype of cells in tubers. Alternatively, one can analyze the in vivo and in vitro characteristics of neuroprogenitor cells that are deficient of Hamartin or tuberin. While homozygous mutants of TSC1 and TSC2 are lethal during midgestation, one of several techniques can be used to derive mutant neuroepithelial cells, including the procurement of -/- cells prior to embryonic deaths and subsequent cortical transplantation into syngeneic animals, development of conditional "knock outs," or chimeric mutants. These approaches, with their unique advantages and disadvantages, will be helpful in gaining insights into the development of cortical tubers and their electrophysiologic consequences.
Lewis C Cantley - One of the best experts on this subject based on the ideXlab platform.
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united at last the tuberous sclerosis complex gene products connect the phosphoinositide 3 kinase akt pathway to mammalian target of rapamycin mtor signalling
Biochemical Society Transactions, 2003Co-Authors: Brendan D Manning, Lewis C CantleyAbstract:The molecular interplay between the phosphoinositide 3-kinase (PI3K) pathway and mammalian target of rapamycin (mTOR) signalling in the control of cell growth and proliferation has been the subject of much interest and debate amongst cell biologists. A recent escalation of research in this area has come from the discovery of the tuberous sclerosis complex gene products, tuberin and Hamartin, as central regulators of mTOR activation. The PI3K effector Akt/protein kinase B has been found to directly phosphorylate tuberin and is thereby thought to activate mTOR through inhibition of the tuberin-Hamartin complex. The many recent studies aimed at defining the molecular nature of this revamped PI3K/Akt/mTOR pathway are reviewed here. The collective data discussed have laid the groundwork for important new insights into the many cancers caused by aberrant PI3K activation and the clinically challenging tuberous sclerosis complex disease and have suggested a possible means of treatment for both.
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Tuberous Sclerosis Complex gene products, Tuberin and Hamartin, control mTOR signaling by acting as a GTPase-activating protein complex toward Rheb
Current Biology, 2003Co-Authors: Andrew R. Tee, Philippe P Roux, Brendan D Manning, Lewis C Cantley, John BlenisAbstract: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.
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tuberous sclerosis complex 1 and 2 gene products function together to inhibit mammalian target of rapamycin mtor mediated downstream signaling
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Andrew R. Tee, Brendan D Manning, Lewis C Cantley, David J Kwiatkowski, Diane C Fingar, John BlenisAbstract:Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products Hamartin and tuberin, respectively. Here, we show that Hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of Hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of Hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, Hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-Hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.
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tuberous sclerosis complex 1 and 2 gene products function together to inhibit mammalian target of rapamycin mtor mediated downstream signaling
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Diane C Fingar, Lewis C Cantley, Brendan D Manning, David J Kwiatkowski, John BlenisAbstract:Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder that occurs upon mutation of either the TSC1 or TSC2 genes, which encode the protein products Hamartin and tuberin, respectively. Here, we show that Hamartin and tuberin function together to inhibit mammalian target of rapamycin (mTOR)-mediated signaling to eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). First, coexpression of Hamartin and tuberin repressed phosphorylation of 4E-BP1, resulting in increased association of 4E-BP1 with eIF4E; importantly, a mutant of TSC2 derived from TSC patients was defective in repressing phosphorylation of 4E-BP1. Second, the activity of S6K1 was repressed by coexpression of Hamartin and tuberin, but the activity of rapamycin-resistant mutants of S6K1 were not affected, implicating mTOR in the TSC-mediated inhibitory effect on S6K1. Third, Hamartin and tuberin blocked the ability of amino acids to activate S6K1 within nutrient-deprived cells, a process that is dependent on mTOR. These findings strongly implicate the tuberin-Hamartin tumor suppressor complex as an inhibitor of mTOR and suggest that the formation of tumors within TSC patients may result from aberrantly high levels of mTOR-mediated signaling to downstream targets.
