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Markus Hengstschläger - One of the best experts on this subject based on the ideXlab platform.
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Tuberin, p27 and mTOR in different cells.
Amino acids, 2008Co-Authors: S. Burgstaller, Michaela Hanneder, Marsha Rich Rosner, Nicol Siegel, Alessandro Valli, C. Lindengrün, Christiane Fuchs, Markus HengstschlägerAbstract:Mutations in the genes TSC1 or TSC2 cause the autosomal dominantly inherited tumor suppressor syndrome tuberous sclerosis, which is characterized by the development of tumors, named hamartomas, in different organs. The TSC gene products, hamartin and Tuberin, form a complex, of which Tuberin is assumed to be the functional component. Both, hamartin and Tuberin have been implicated in the control of the cell cycle by activating the cyclin-dependent kinase inhibitor p27 and in cell size regulation by inhibiting the mammalian target of rapamycin (mTOR) a regulator of the p70 ribosomal protein S6 kinase (p70S6K) and its target the ribosomal protein S6. The Tuberin/hamartin complex was shown to protect p27 from protein degradation. Within the mTOR signaling pathway Tuberin harbors GTPase activating (GAP) potential toward Rheb, which is a potent regulator of mTOR. In this study, we have analyzed the protein levels of Tuberin, p27, cyclin D1, mTOR and phospho mTOR Ser2448 (activated mTOR), S6 and phospho S6 Ser240/244 (activated S6) and as controls α-tubulin and topoisomerase IIβ, in ten different cells, including primary normal cells, immortalized and transformed cell lines.
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The tuberous sclerosis gene products hamartin and Tuberin are multifunctional proteins with a wide spectrum of interacting partners.
Mutation research, 2008Co-Authors: Margit Rosner, Michaela Hanneder, Nicol Siegel, Alessandro Valli, Markus HengstschlägerAbstract:Mutations in the tumor suppressor genes TSC1 and TSC2, encoding hamartin and Tuberin, respectively, cause the tumor syndrome tuberous sclerosis with similar phenotypes. Until now, over 50 proteins have been demonstrated to interact with hamartin and/or Tuberin. Besides Tuberin, the proteins DOCK7, ezrin/radixin/moesin, FIP200, IKKbeta, Melted, Merlin, NADE(p75NTR), NF-L, Plk1 and TBC7 have been found to interact with hamartin. Whereas Plk1 and TBC7 have been demonstrated not to bind to Tuberin, for all the other hamartin-interacting proteins the question, whether they can also bind to Tuberin, has not been studied. Tuberin interacts with 14-3-3 beta,epsilon,gamma,eta,sigma,tau,zeta, Akt, AMPK, CaM, CRB3/PATJ, cyclin A, cyclins D1, D2, D3, Dsh, ERalpha, Erk, FoxO1, HERC1, HPV16 E6, HSCP-70, HSP70-1, MK2, NEK1, p27KIP1, Pam, PC1, PP2Ac, Rabaptin-5, Rheb, RxRalpha/VDR and SMAD2/3. 14-3-3 beta,epsilon,gamma,eta,sigma,tau,zeta, Akt, Dsh, FoxO1, HERC1, p27KIP1 and PP2Ac are known not to bind to hamartin. For the other Tuberin-interacting proteins this question remains elusive. The proteins axin, Cdk1, cyclin B1, GADD34, GSK3, mTOR and RSK1 have been found to co-immunoprecipitate with both, hamartin and Tuberin. The kinases Cdk1 and IKKbeta phosphorylate hamartin, Erk, Akt, MK2, AMPK and RSK1 phosphorylate Tuberin, and GSK3 phosphorylates both, hamartin and Tuberin. This detailed summary of protein interactions allows new insights into their relevance for the wide variety of different functions of hamartin and Tuberin.
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Ras mediates cell survival by regulating Tuberin
Oncogene, 2007Co-Authors: Angelika Freilinger, Michaela Hanneder, Marsha Rich Rosner, Markus HengstschlägerAbstract:Mutational activation of Ras promotes oncogenesis by controlling cell cycle regulation and cell survival. Ras-mediated activation of both, the PI3K/AKT pathway and the MEK/ERK pathway, can trigger downregulation of the function of Tuberin to block the activities of mTOR and p70S6K. Here we demonstrate that Ras-induced cell survival is accompanied by upregulation of p70S6K activity. Ras harbors the potential to negatively affect Tuberin-induced apoptosis and p70S6K inactivation. These effects of Ras were found to depend on its potential to regulate the MEK/ERK pathway. Experiments using Tuberin-negative fibroblasts revealed that the potential of Ras to counteract apoptosis depends on functional Tuberin. Taken together, we provide evidence that the function of Ras to trigger inactivation of Tuberin plays a major role in the regulation of cell survival upon mutational activation of the oncogene Ras. This is the first description of a functional interaction between the tumor suppressor Tuberin and the oncogene Ras in regulating apoptosis.
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p27Kip1 localization depends on the tumor suppressor protein Tuberin
Human molecular genetics, 2007Co-Authors: Margit Rosner, Angelika Freilinger, Michaela Hanneder, Naoya Fujita, Gert Lubec, Takashi Tsuruo, Markus HengstschlägerAbstract:p27(Kip1) plays an important role in cell cycle regulation by inhibiting cyclin-CDK complex activity in the nucleus. p27(Kip1) is regulated by its concentration as well as by its subcellular localization. Tuberin, encoded by the tuberous sclerosis tumor suppressor gene TSC2, is a potent negative cell cycle regulator. We show herein, that Tuberin induces nuclear p27 localization by inhibiting its 14-3-3-mediated cytoplasmic retention. Tuberin interferes with 14-3-3's counteracting effects on p27-mediated cell cycle arrest. Akt-mediated phosphorylation of p27, but not of Tuberin, negatively regulates Tuberin's potential to trigger p27 nuclear localization. In G0 cells, Tuberin binds p27 triggering downregulation of p27's binding to 14-3-3 and of its cytoplasmic retention. At transition to S phase p27 is phosphorylated by Akt, Tuberin/p27 complex levels are downregulated and binding of p27 to 14-3-3 increases triggering cytoplasmic retention of p27. These findings demonstrate p27 localization during the mammalian cell cycle to be under the control of the tumor suppressor Tuberin.
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Cytoplasmic/nuclear localization of Tuberin in different cell lines.
Amino acids, 2007Co-Authors: Marsha Rich Rosner, Markus HengstschlägerAbstract:Tuberous sclerosis (TSC) is an autosomal dominantly inherited disease affecting 1 in 6000 individuals. The TSC gene products, hamartin and Tuberin, form a complex, of which Tuberin is assumed to be the functional component being involved in a wide variety of different cellular processes. Tuberin has been demonstrated to be localized to both, the cytoplasm and the nucleus. The cytoplasmic/nuclear localization of Tuberin is known to be regulated by the serine/threonine protein kinase Akt. Akt also regulates the cytoplasmic/nuclear localization of the cyclin-dependent kinase inhibitor p27. In this study the localization of these two Akt-regulated proteins was analysed in different cell lines.
Raymond S. Yeung - One of the best experts on this subject based on the ideXlab platform.
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The Loss of Tuberin Promotes Cell Invasion through the β-Catenin Pathway
American journal of respiratory cell and molecular biology, 2009Co-Authors: Elizabeth A. Barnes, Baldwin C. Mak, Heidi L. Kenerson, Raymond S. YeungAbstract:Mutations in the tumor suppressor Tuberin (TSC2) are a common factor in the development of lymphangioleiomyomatosis (LAM). LAM is a cystic lung disease that is characterized by the infiltration of smooth muscle–like cells into the pulmonary parenchyma. The mechanism by which the loss of Tuberin promotes the development of LAM has yet to be elucidated, although several lines of evidence suggest it is due to the metastasis of Tuberin-deficient cells. Here we show that Tuberin-null cells become nonadherent and invasive. These nonadherent cells express cleaved forms of β-catenin. In reporter assays, the β-catenin products are transcriptionally active and promote MMP7 expression. Invasion by the Tuberin-null cells is mediated by MMP7. Examination of LAM tissues shows the expression of cleaved β-catenin products and MMP7 consistent with a model that Tuberin-deficient cells acquire invasive properties through a β-catenin–dependent mechanism, which may underlie the development of LAM.
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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, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Raymond S. Yeung, 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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Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Tuberous Sclerosis Tumor Suppressor Complex by Phosphorylation of Tuberin
The Journal of biological chemistry, 2002Co-Authors: Han C. Dan, Mark Nellist, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Raymond S. Yeung, Santo V. NicosiaAbstract: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.
J E Declue - One of the best experts on this subject based on the ideXlab platform.
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Loss of expression of Tuberin and hamartin in tuberous sclerosis complex-associated but not in sporadic angiofibromas.
Journal of cutaneous pathology, 2003Co-Authors: Ingrid Fackler, J E Declue, Heidi Rust, Heinz Kutzner, Arno Rütten, Steven Kaddu, Christian A. Sander, Matthias Volkenandt, Michael W. JohnsonAbstract:Background: Angiofibromas occur sporadically, and they develop in most patients with tuberous sclerosis complex (TSC), which is associated with alterations of the tumor suppressor genes TSC1 or TSC2. Loss of Tuberin, the protein product of TSC2, has been shown in the interstitial fibroblast compartment of TSC-associated angiofibromas. It is unclear whether there is also a loss of hamartin, the product of TSC1 in TSC-associated and sporadic angiofibromas. Methods: The expression of hamartin and Tuberin was analyzed by immunohistochemistry in 59 TSC-associated and 12 sporadic angiofibromas using affinity-purified antibodies. Results: Loss of expression of both Tuberin and hamartin was detected in 14 angiofibromas, loss of only Tuberin in three, and loss of only hamartin in four TSC-associated angiofibromas; but there was no loss in the sporadic angiofibromas. Only the interstitial cells, but not the vascular cells, showed a loss of expression of Tuberin or hamartin. Conclusions: Loss of Tuberin or hamartin occurred in a minority of the TSC-linked angiofibromas, but not in the sporadic angiofibromas. The absence of both Tuberin and hamartin in some of the tumors suggests that the stability of Tuberin and hamartin, which are believed to form an active complex in vivo, is negatively affected by the absence of either of the partners.
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Reduction of expression of Tuberin, the tuberous-sclerosis-complex-gene-2 product in tuberous sclerosis complex associated connective tissue nevi and sporadic squamous and basal cell carcinomas.
Journal of cutaneous pathology, 2002Co-Authors: Ralf Wienecke, Eckart Klemm, Sarolta Karparti, Neil A. Swanson, Andrew Green, J E DeclueAbstract:Background: Patients affected with tuberous sclerosis complex (TSC) are prone to the development of multiple benign tumors of the skin and other organs. Tuberin, the protein product of the tuberous-sclerosis-complex-2 tumor suppressor gene (TSC2) has been shown to inhibit cell proliferation. In TSC associated kidney tumors and sporadic brain tumors the loss/reduction of Tuberin has been shown. Methods: Specimens of nine squamous cell carcinomas (SCC) and five basal cell carcinomas (BCC) from patients without TSC and six biopsies of connective tissue nevi (CTN) of patients with TSC were obtained. Specimens were analyzed by immunoblotting for the expression of Tuberin. Results: Absent or reduced levels of Tuberin were detected in the dermal parts of three of six shagreen patches, two of five BCC, and four of nine SCC. Conclusions: In tumors/hamartomas of patients with TSC the complete loss of TSC2 and Tuberin is a mechanism which could be shown for CTN, thereby excluding the possibility of haploinsufficiency of TSC2. In a substantial number of cutaneous BCC and SCC the loss or downregulation of Tuberin seems to be epigenetic, as alterations of TSC2 are not known in these tumors. The absence or reduction of Tuberin might contribute to their proliferation.
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Loss of Tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis.
Journal of cutaneous pathology, 2001Co-Authors: Phuong-anh Nguyen-vu, J E Declue, Raymond S. Yeung, Ingrid Fackler, Christian A. Sander, Matthias Volkenandt, Adelheid Rust, Michael J. Flaig, Ralf WieneckeAbstract:Background: Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disorder associated with an alteration of the TSC2 tumor suppressor gene which encodes for the protein product Tuberin. The disease is characterized by the development of hamartomas, e.g. cutaneous angiofibromas which consist of vascular cells, interstitial cells, and normal components of the skin. The Eker rat model, an animal model of inherited cancer, has been shown to carry a mutation of TSC2. Methods: Immunohistochemical analyses of human angiofibromas were performed using antibodies directed against Tuberin and angiogenic growth factors. Proliferation of human dermal microvascular endothelial cells (HDMEC) was determined after incubation with the supernatants of TSC2 (+/+) and TSC2 (−/−) rat embryonic fibroblasts (REF) that were derived from the Eker strain. Results: Loss of the expression of Tuberin was observed in the interstitial cells of 13 of 39 angiofibromas. The expression of Tuberin was retained in the vascular cells. In all analyzed angiofibromas, the angiogenic factors bFGF, PD-ECGF, VEGF and angiogenin were detected in the interstitial cells and/or vascular cells. Expression of PDGF-B and TGF-β1 was weak. Tissue culture supernatants from TSC2 (−/−) REF stimulated the growth of HDMEC significantly more than supernatants from TSC2 (+/+) REF. Conclusion: A functional loss of Tuberin may stimulate vascular growth.
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Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and Tuberin
Human molecular genetics, 2001Co-Authors: Angela Hodges, Jeremy Peter Cheadle, Richard Braverman, J E Declue, Julie Helen Maynard, Lee Parry, Julian Roy SampsonAbstract:Critical functions of hamartin and Tuberin, encoded by the TSC1 and TSC2 genes, are likely to be closely linked. The proteins interact directly with one another and mutations affecting either gene result in the tuberous sclerosis phenotype. However, the regions of hamartin and Tuberin that interact have not been well defined, and the relationship between their interaction and the pathogenesis of tuberous sclerosis has not been explored. To address these issues a series of hamartin and Tuberin constructs were used to assay for interaction in the yeast two-hybrid system. Hamartin (amino acids 302–430) and Tuberin (amino acids 1–418) interacted strongly with one another. A region of Tuberin encoding a putative coiled-coil (amino acids 346–371) was necessary but not sufficient to mediate the interaction with hamartin, as more N-terminal residues were also required. A region of hamartin (amino acids 719–998) predicted to encode coiled-coils was capable of oligermerization but was not important for the interaction with Tuberin. Subtle, non-truncating mutations identified in patients with tuberous sclerosis and located within the putative binding regions of hamartin (N198_F199delinsI;593–595delACT) or Tuberin (G294E and I365del), abolished or dramatically reduced interaction of the proteins as assessed by yeast two-hybrid assays and by co-immunoprecipitation of the full-length proteins from Cos7 cells. In contrast, three non-pathogenic missense polymorphisms of Tuberin (R261W, M286V, R367Q) in the same region as the disease-causing TSC2 mutations did not. These results indicate a requirement for interaction in critical growth suppressing functions of hamartin and Tuberin.
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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
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, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Raymond S. Yeung, 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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Identification and Characterization of the Interaction between Tuberin and 14-3-3ζ
The Journal of biological chemistry, 2002Co-Authors: Mark Nellist, Arnold J J Reuser, Brenda Verhaaf, Miriam Goedbloed, Ans M.w. Van Den Ouweland, Christa De Winter, Anita Jankie, Peter Van Der Sluijs, Dicky J. J. HalleyAbstract:Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called Tuberin. Although it has been shown that hamartin and Tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into Tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with Tuberin. One of the proteins identified was 14-3-3ζ, a member of the 14-3-3 protein family. The interaction between Tuberin and 14-3-3ζ was confirmed in vitro and by co-immunoprecipitation; multiple sites within Tuberin for 14-3-3ζ binding were identified; and it was determined that 14-3-3ζ associated with the Tuberin-hamartin complex. Finally, it was shown that the Tuberin/14-3-3ζ interaction is regulated by Akt-mediated phosphorylation of Tuberin, providing insight into how Tuberin may regulate phosphorylation of S6.
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Phosphatidylinositol 3-Kinase/Akt Pathway Regulates Tuberous Sclerosis Tumor Suppressor Complex by Phosphorylation of Tuberin
The Journal of biological chemistry, 2002Co-Authors: Han C. Dan, Mark Nellist, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Raymond S. Yeung, Santo V. NicosiaAbstract: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.
Mark Nellist - One of the best experts on this subject based on the ideXlab platform.
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Hamartin variants that are frequent in focal dysplasias and cortical tubers have reduced Tuberin binding and aberrant subcellular distribution in vitro
Journal of neuropathology and experimental neurology, 2009Co-Authors: Céline Lugnier, Mark Nellist, Michael Majores, Jana Fassunke, Katharina Pernhorst, Pitt Niehusmann, Matthias Simon, Susanne Schoch, Albert J. BeckerAbstract:Focal cortical dysplasia type IIb is characterized by epilepsy-associated malformations that are often composed of balloon cells and dysplastic neurons. There are many histopathologic similarities between focal cortical dysplasia type IIb and cortical tubers in tuberous sclerosis complex (TSC), an autosomal-dominant phakomatosis caused by mutations in the TSC1 or TSC2 genes that encode hamartin and Tuberin. We previously found that an allelic variant of TSC1 (hamartin) is increased in focal cortical dysplasia type IIb. Here, we investigated the subcellular localization of hamartin and its interaction with Tuberin in vitro. Coimmunoprecipitation assays with Tuberin revealed reduced Tuberin binding of hamartin compared with wild-type hamartin. Tuberin binding was also reduced for 2 TSC1 stop mutants (hamartin and hamartin) that are present in brain lesions of TSC patients. Colocalization assays of hamartin and Tuberin were performed in HEK293T cells, and the subcellular localization of the hamartin variants were studied using immunocytochemistry. There was an impairment of Tuberin binding of hamartin and aberrant nuclear distribution of hamartin in these cells, whereas hamartin and hamartin were, like wild-type Tuberin, localized in the cytoplasm. These data suggest a fundamental functional impairment of hamartin and the 2 TSC1 stop mutants hamartin and hamartin in vitro. Future studies will be needed to characterize the roles of these TSC1 sequence variants in the genesis of dysplastic epileptogenic developmental brain lesions.
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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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Differential localization of hamartin and Tuberin and increased S6 phosphorylation in a tuber.
Neurology, 2004Co-Authors: Floor E. Jansen, Mark Nellist, Robbert G. E. Notenboom, M. A. Goedbloed, D. J. J. Halley, P.n.e. De Graan, O. Van NieuwenhuizenAbstract:In a tuberous sclerosis patient with a mutation in the TSC1 tumor suppressor gene, no second-hit mutation was found in a resected cortical tuber. Tuber giant cells showed predominantly nuclear hamartin, cytosolic Tuberin, and hyperphosphorylation of S6. Differential accumulation of hamartin and Tuberin in separate cellular compartments of giant cells may prevent formation of the hamartin-Tuberin complex, resulting in increased S6 phosphorylation. These data provide an alternative mechanism for tuberogenesis.
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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, Dicky J. J. Halley, Mei Sun, Lin Yang, Richard I. Feldman, Xue Mei Sui, Raymond S. Yeung, 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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Identification and Characterization of the Interaction between Tuberin and 14-3-3ζ
The Journal of biological chemistry, 2002Co-Authors: Mark Nellist, Arnold J J Reuser, Brenda Verhaaf, Miriam Goedbloed, Ans M.w. Van Den Ouweland, Christa De Winter, Anita Jankie, Peter Van Der Sluijs, Dicky J. J. HalleyAbstract:Tuberous sclerosis is caused by mutations to either the TSC1 or TSC2 tumor suppressor gene. The disease is characterized by a broad phenotypic spectrum that includes seizures, mental retardation, renal dysfunction, and dermatological abnormalities. TSC1 encodes a 130-kDa protein called hamartin, and TSC2 encodes a 200-kDa protein called Tuberin. Although it has been shown that hamartin and Tuberin form a complex and mediate phosphoinositide 3-kinase/Akt-dependent phosphorylation of the ribosomal protein S6, it is not yet clear how inactivation of either protein leads to tuberous sclerosis. Therefore, to obtain additional insight into Tuberin and hamartin function, yeast two-hybrid screening experiments were performed to identify proteins that interact with Tuberin. One of the proteins identified was 14-3-3ζ, a member of the 14-3-3 protein family. The interaction between Tuberin and 14-3-3ζ was confirmed in vitro and by co-immunoprecipitation; multiple sites within Tuberin for 14-3-3ζ binding were identified; and it was determined that 14-3-3ζ associated with the Tuberin-hamartin complex. Finally, it was shown that the Tuberin/14-3-3ζ interaction is regulated by Akt-mediated phosphorylation of Tuberin, providing insight into how Tuberin may regulate phosphorylation of S6.
