7248

TSC1

LAM|TSC;tuberous sclerosis 1;TSC1;tuberous sclerosis 1

hamartin|tuberous sclerosis 1 protein|tumor suppressor

9q34

protein-coding

Count: 4; Pubmed_search,TAG,Generif,UniProt

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between beta1 and beta2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.

Akt signal transduction induces coordinated increases in glycolysis and apoptosis resistance in a broad spectrum of cancers. Downstream of Akt, the FoxO transcription factors regulate apoptosis via Bim, but the contributions of FoxOs in regulating Akt-induced glycolysis are not well described. We find that FoxO3a knockdown is sufficient to induce apoptosis resistance in conjunction with elevated glycolysis. Glycolysis in FoxO3a-deficient cells was associated with increased S6K1 phosphorylation and was sensitive to rapamycin, an inhibitor of the mTORC1 pathway that has been linked to glycolysis regulation. We show that mTORC1-dependent glycolysis is increased in FoxO3a knockdown cells due to decreased expression of the TSC1 tumor suppressor that opposes mTORC1 activation. FoxO3a binds to and transactivates the TSC1 promoter, indicating a key role for FoxO3a in regulating TSC1 expression. Together, these data demonstrate that FoxO3a regulates glycolysis downstream of Akt through transcriptional control of Tsc1.

PURPOSE Perivascular epithelioid cell tumors (PEComas) represent a family of mesenchymal neoplasms, mechanistically linked through activation of the mTOR signaling pathway. There is no known effective therapy for PEComa, and the molecular pathophysiology of aberrant mTOR signaling provided us with a scientific rationale to target this pathway therapeutically. On this mechanistic basis, we treated three consecutive patients with metastatic PEComa with an oral mTOR inhibitor, sirolimus. PATIENTS AND METHODS Patients with advanced PEComa were treated with sirolimus and consented to retrospective collection of data from their medical records and analysis of archival tumor specimens. Tumor response was determined by computed tomography scans obtained at the clinical discretion of the treating physicians. Tumors were assessed for immunohistochemical evidence of mTORC1 activation and genetic evidence of alterations in TSC1 and TSC2. Results Radiographic responses to sirolimus were observed in all patients. PEComas demonstrated loss of TSC2 protein expression and evidence of baseline mTORC1 activation. Homozygous loss of TSC1 was identified in one PEComa. CONCLUSION Inhibition of mTORC1, pathologically activated by loss of the TSC1/TSC2 tumor suppressor complex, is a rational mechanistic target for therapy in PEComas. The clinical activity of sirolimus in PEComa additionally strengthens the pathobiologic similarities linking PEComas to other neoplasms related to the tuberous sclerosis complex.

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.

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either the TSC1 or TSC2 genes. TSC1 and TSC2 proteins form a functionally interdependent dimeric complex. Phosphorylation of either TSC subunit by different kinases regulates the function of TSC and represents a major mechanism to integrate various signals into a centralized cell growth pathway. The majority of disease-associated mutations targeting either TSC1 or TSC2 results in a substantial decrease in protein level, suggesting that protein turnover also plays a critical role in TSC regulation. Here we report that TSC2 protein binds to FBW5, a DDB1-binding WD40 (DWD) protein, and is recruited by FBW5 to the DDB1-CUL4-ROC1 E3 ubiquitin ligase. Overexpression of FBW5 or CUL4A promotes TSC2 protein degradation, and this is abrogated by the coexpression of TSC1. Conversely, depletion of FBW5, DDB1, or CUL4A/B stabilizes TSC2. Ddb1 or Cul4 mutations in Drosophila result in Gigas/TSC2 protein accumulation and cause growth defects that can be partially rescued by Gigas/Tsc2 reduction. These results indicate that FBW5-DDB1-CUL4-ROC1 is an E3 ubiquitin ligase regulating TSC2 protein stability and TSC complex turnover.

Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by the formation of hamartomatous lesions in many organs, including brain, heart or kidneys. It has been found that TSC is caused by the mutation in one of the two tumor suppressor genes: TSC1 or TSC2, encoding hamartin and tuberin, respectively. According to Knudson's two-hit model of tumorigenesis, second-hit mutation and resulting loss of heterozygosity (LOH) of a tumor suppressor gene is necessary for tumor formation. In fact, LOH is commonly found in several types of hamartomas formed in the process of tuberous sclerosis, but, interestingly, not in brain lesions, containing characteristic giant cells. In this paper, we review literature covering origination of giant cells and present several hypotheses explaining why in spite of the presence of hamartin and tuberin, brain lesions form in TSC patients.

The TSC1 and TSC2 proteins, which function as a TSC1/TSC2 tumor suppressor complex, are associated with lymphangioleiomyomatosis (LAM), a genetic disorder characterized by the abnormal growth of smooth muscle-like cells in the lungs. The precise molecular mechanisms that modulate LAM cell growth remain unknown. We demonstrate that TSC2 regulates LAM cell growth. Cells dissociated from LAM nodules from the lungs of five different patients with LAM have constitutively activated S6K1, hyperphosphorylated ribosomal protein S6, activated Erk, and increased DNA synthesis compared with normal cells from the same patients. These effects were augmented by PDGF stimulation. Akt activity was unchanged in LAM cells. Rapamycin, a specific S6K1 inhibitor, abolished increased LAM cell growth. The full-length TSC2 was necessary for inhibition of S6 hyperphosphorylation and DNA synthesis in LAM cells, as demonstrated by co-microinjection of the C-terminus, which contains the GTPase activating protein homology domain, and the N-terminus, which binds TSC1. Our data demonstrate that increased LAM cell growth is associated with constitutive S6K1 activation, which is extinguishable by TSC2 expression. Loss of TSC2 GAP activity or disruption of the TSC1/TSC2 complex dysregulates S6K1 activation, which leads to abnormal cell proliferation associated with LAM disease.

Dysregulated signaling by the checkpoint kinase TOR (target of rapamycin) has been linked to numerous human cancers. The tuberous sclerosis tumor suppressors TSC1 and TSC2 form a protein complex that integrates and transmits cellular growth factor and stress signals to negatively regulate TOR activity. Several recent reports have identified the stress response gene REDD1 as an essential regulator of TOR activity through the TSC1/2 complex in both Drosophila and mammalian cells. REDD1 is induced in response both to hypoxia and energy stress, and cells that lack REDD1 exhibit highly defective TOR regulation in response to either of these stress signals. While the precise mechanism of REDD1 function remains to be determined, the finding that REDD1-dependent TOR regulation contributes to cell growth/cell size control in flies and mammals suggests that abnormalities of REDD1-mediated signaling might disrupt energy homeostasis and/or promote tumorigenesis.

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.

TSC1 and TSC2 are responsible for the tumor suppressor gene syndrome tuberous sclerosis (TSC). Mammalian TSC genes have been shown to be involved in cell cycle regulation. Recently, in Drosophila, these data have been confirmed and TSC genes have further been demonstrated to affect cell size control. Here we provide supporting data for the fact that the latter function is conserved in mammals. Human TSC1 and TSC2 trigger mammalian cell size reduction and a dominant-negative TSC2 mutant induces increased size. These effects occur in all cell cycle phases, are dependent on the activity of the phosphoinositide-3-kinase and are abolished by co-overexpression of a dominant-negative Akt mutant. Two independent naturally occurring and disease-causing mutations within the TSC2 gene eliminate tuberin's capacity to affect cell size control, emphasizing the relevance of this function for the development of the disease. The same mutations have earlier been shown not to affect tuberin's antiproliferative capacity. That the consequences of modulated TSC gene expression on cell proliferation and on cell size can be assigned to separable functions is further supported by two findings: A mutation within the TSC1 gene, earlier shown to still harbor anti-proliferative effects, was found to eliminate the cell size regulating functions. An important mammalian cell size regulator, c-Myc, was found to inhibit tuberin's antiproliferative capacity, but to have no effects on tuberin-dependent cell size control. To obtain further mechanistical insights, microarray screens for genes involved in TSC1- or TSC2-mediated cell size effects were performed. Antisense experiments revealed that the so observed regulation of the FK506-binding protein, FKBP38, plays a role in TSC gene-dependent cell size regulation. These data provide new insights into mammalian cell size regulation and allow a better understanding of the function of human TSC genes.

PURPOSE: Because loss of chromosome 9 is known to be the most common finding in human bladder tumors, we studied the mutation of the tumor suppressor gene TSC1 (chromosome 9q34) in bladder tumors. Since another tumor suppressor gene, TSC2 (chromosome 16p13.3), is reported to interact with TSC1 in the pathway that modulates tumor suppression, we assessed loss of heterozygosity (LOH) at 16p13.3. Furthermore, we also examined the expression of p27 because the TSC1 product is reported to influence the level of p27. MATERIALS AND METHODS: Microsatellite markers were used to evaluate LOH at 9q34 or 16p13.3. mutations of TSC1 were screened by single strand conformation polymorphism analysis and verified by direct sequencing. The expression of p27 was examined by reverse transcriptase-polymerase chain reaction and immunohistochemical examination. RESULTS: We identified LOH at 9q34 in 12 of 37 bladder tumors (32.4%) but no LOH at 16p13.3 was observed. Furthermore, on single strand conformational polymorphism analysis we identified tumor specific mutations of TSC1 in 4 cases, of which all had LOH at 9q34, demonstrating the 2-hit mutations of TSC1. The expression of p27 was suppressed in all 4 cases with the 2-hit mutations of TSC1. Unexpectedly p27 suppression was detected at the transcription level, although its mechanism is unknown. CONCLUSIONS: Our data suggest that the TSC1 mutation possibly has a causative role in the initiation or progression of some bladder tumors and this process is possibly related to the functional loss of p27.

Tuberous sclerosis complex, an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. The proteins encoded by TSC1 and TSC2, hamartin and tuberin, respectively, associate with each other forming a tight complex. Here we show that hamartin binds the neurofilament light chain and it is possible to recover the hamartin-tuberin complex over the neurofilament light chain rod domain spanning amino acids 93-156 by affinity precipitation. Homologous rod domains in other intermediate filaments such as neurofilament medium chain, alpha-internexin, vimentin, and desmin are not able to bind hamartin. In cultured cortical neurons, hamartin and tuberin co-localize with neurofilament light chain preferentially in the proximal to central growth cone region. Interestingly, in the distal part of the growth cone hamartin overlaps with the ezrin-radixin-moesin family of actin binding proteins, and we have validated the interaction of hamartin with moesin. These results demonstrate that hamartin may anchor neuronal intermediate filaments to the actin cytoskeleton, which may be critical for some of the CNS functions of the hamartin-tuberin complex, and abolishing this through mutations in TSC1 or TSC2 may lead to certain neurological manifestations associated with the disease.

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2 tumor 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 p27(kip1), 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.

The mechanisms that regulate T cell quiescence are poorly understood. We report that the tumor suppressor Tsc1 established a quiescence program in naive T cells by controlling cell size, cell cycle entry and responses to stimulation of the T cell antigen receptor. Abrogation of quiescence predisposed Tsc1-deficient T cells to apoptosis that resulted in loss of conventional T cells and invariant natural killer T cells. Loss of Tsc1 function dampened in vivo immune responses to bacterial infection. Tsc1-deficient T cells had more activity of the serine-threonine kinase complex mTORC1 but less mTORC2 activity, and activation of mTORC1 was essential for the disruption of immune homeostasis. Therefore, Tsc1-dependent control of mTOR is crucial in actively maintaining the quiescence of naive T cells to facilitate adaptive immune function.

The phosphoinositide 3-kinase (PI3K) pathway regulates mammalian cell growth, survival, and motility and plays a major pathogenetic role in human prostate cancer (PCa). However, the oncogenic contributions downstream of the PI3K pathway made by mammalian target of rapamycin complex 1 (mTORC1)-mediated cell growth signal transduction in PCa have yet to be elucidated in detail. Here, we engineered constitutive mTORC1 activation in prostate epithelium by a conditional genetic deletion of tuberous sclerosis complex 1 (Tsc1), a potent negative regulator of mTORC1 signaling. Epithelial inactivation was not immediately tumorigenic, but Tsc1-deficient mice developed prostatic intraepithelial neoplasia (mPIN) in lateral and anterior prostates by 6 months of age, with increasing disease penetrance over time. Lateral prostate lesions in 16- to 22-month-old mutant mice progressed to two types of more advanced lesions, adenomatous gland forming lesion (Type 1) and atypical glands embedded in massively expanded reactive stroma (Type 2). Both Type 1 and Type 2 lesions contained multiple foci of microinvasive carcinoma. Epithelial neoplastic and atypical stromal lesions persisted despite 4 weeks of RAD001 chemotherapy. Rapalogue resistance was not due to AKT or extracellular signal-regulated kinase 1/2 activation. expression of the homeobox gene Nkx3.1 was lost in Tsc1-deficient mPIN, and it cooperated with TSC1 loss in mPIN initiation in doubly mutant Tsc1:Nkx3.1 prostatic epithelial knockout mice. Thus, TSC1 inactivation distal to PI3K and AKT activation is sufficient to activate a molecular signaling cascade producing prostatic neoplasia and focal carcinogenesis.CI - (c)2010 AACR.

Mammalian target of rapamycin (mTOR) is a central regulator of protein synthesis whose activity is modulated by a variety of signals. Energy depletion and hypoxia result in mTOR inhibition. While energy depletion inhibits mTOR through a process involving the activation of AMP-activated protein kinase (AMPK) by LKB1 and subsequent phosphorylation of TSC2, the mechanism of mTOR inhibition by hypoxia is not known. Here we show that mTOR inhibition by hypoxia requires the TSC1/TSC2 tumor suppressor complex and the hypoxia-inducible gene REDD1/RTP801. Disruption of the TSC1/TSC2 complex through loss of TSC1 or TSC2 blocks the effects of hypoxia on mTOR, as measured by changes in the mTOR targets S6K and 4E-BP1, and results in abnormal accumulation of Hypoxia-inducible factor (HIF). In contrast to energy depletion, mTOR inhibition by hypoxia does not require AMPK or LKB1. Down-regulation of mTOR activity by hypoxia requires de novo mRNA synthesis and correlates with increased expression of the hypoxia-inducible REDD1 gene. Disruption of REDD1 abrogates the hypoxia-induced inhibition of mTOR, and REDD1 overexpression is sufficient to down-regulate S6K phosphorylation in a TSC1/TSC2-dependent manner. Inhibition of mTOR function by hypoxia is likely to be important for tumor suppression as TSC2-deficient cells maintain abnormally high levels of cell proliferation under hypoxia.

Insulin-like growth factors elicit many responses through activation of phosphoinositide 3-OH kinase (PI3K). The tuberous sclerosis complex (TSC1-2) suppresses cell growth by negatively regulating a protein kinase, p70S6K (S6K1), which generally requires PI3K signals for its activation. Here, we show that TSC1-2 is required for insulin signaling to PI3K. TSC1-2 maintains insulin signaling to PI3K by restraining the activity of S6K, which when activated inactivates insulin receptor substrate (IRS) function, via repression of IRS-1 gene expression and via direct phosphorylation of IRS-1. Our results argue that the low malignant potential of tumors arising from TSC1-2 dysfunction may be explained by the failure of TSC mutant cells to activate PI3K and its downstream effectors.CI - Copyright The Rockerfeller University Press