Human Molecular Genetics, 2000, Vol. 9, No. 12 1721-1727
© 2000 Oxford University Press
The TSC1 gene product, hamartin, negatively regulates cell proliferation
Obstetrics and Gynecology, University of Vienna, Prenatal Diagnosis and Therapy, Währinger Gürtel 18-20, A-1090 Vienna, Austria and 1MGC Department of Clinical Genetics, Erasmus University, 3015GE Rotterdam, The Netherlands
Received 6 March 2000; Revised and Accepted 19 May 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Tuberous sclerosis is an autosomal dominant hereditary disease caused by mutations in either the TSC1 or the TSC2 tumor suppressor gene. The TSC1 gene on chromosome 9q34 encodes a 130 kDa protein named hamartin, and the TSC2 gene on chromosome 16p13.3 codes for tuberin, a 200 kDa protein. Here we show that expression of hamartin, assayed by immunoblot analyses, is high in G0-arrested cells and hamartin is expressed throughout the entire ongoing cell cycle. An interaction of hamartin and tuberin can be detected in every phase of the cell cycle. Ectopic expression of high levels of hamartin attenuates cellular proliferation. We provide evidence that this effect could depend on a coiled-coil region earlier proposed to be involved in binding of hamartin to tuberin. Further investigations revealed that hamartin affects cell proliferation via deregulation of G1 phase. Our data have a clear impact on understanding the role of hamartin during development of this disease.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Tuberous sclerosis (TSC) is an autosomal dominant condition with an estimated prevalence of ~1 in 6000, with two-thirds being sporadic (1). This disease is characterized by mental retardation, epilepsy and tumours of the skin, retina, heart, kidney and the central nervous system (1–3). In addition to its distinct clinical presentation, two features distinguish TSC from other familial tumour syndromes. First, the tumours that occur in TSC are very rare in the general population, such that several TSC lesions are, by themselves, diagnostic of TSC. Second, TSC tumours, designated hamartomas, rarely progress to malignancy and are therefore probably better described as ‘uncontrolled growths’.
Half of the families affected with TSC are associated with a mutant TSC2 gene, located on chromosome 16, whereas TSC1, which maps to chromosome 9, is implicated in the remainder (4–10). Since identification of the TSC2-encoded protein, designated tuberin, several different functions have been described: (i) it has been reported that tuberin functions as a GTPase-accelerating protein (GAP) for the small molecular weight GTPase Rap1a (11); (ii) transcriptional activation domains in the C-terminus of the TSC2 product have been described (12); (iii) GAP activity of tuberin for Rab5 has been reported (13); and (iv) other data have demonstrated that TSC2 protein can bind and selectively modulate transcription mediated by members of the steroid receptor superfamily of genes (14). Since their first description, as yet none of these activities has been investigated further, especially not with respect to their impact on regulation of the development of the uncontrolled growths (tumours).
From the clinical phenotype one can conclude that the TSC gene products affect cellular proliferation. Accordingly, it was in agreement with the clinical phenotype of TSC patients to find that high levels of ectopic TSC2 inhibit cell proliferation in culture, whereas down-regulation of TSC2 expression has positive effects on proliferation in cell culture (15–18).
The identification of TSC1 and its protein product, hamartin, now allows investigations into the biochemical and biological properties of both hamartin and tuberin. Recently, it was demonstrated that hamartin and tuberin associate physically in vivo and that the interaction is mediated by predicted coiled-coil domains, suggesting that these two proteins function in the same complex (19,20). In this study we show that hamartin protein is expressed throughout the entire ongoing mammalian cell cycle and that its interaction with tuberin can be detected in every phase of the cell cycle. High levels of ectopic hamartin have negative effects on cellular proliferation, which could depend on a coiled-coil region earlier proposed to be involved in hamartin binding to tuberin. The impact of these data on the understanding of the role of hamartin in the development of TSC is discussed.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
We first investigated hamartin protein expression based on western blot analysis in two different inducible Rat1 cell systems, in which the transcription factors E2F-1 or c-Myc can be activated by adding zinc or tamoxifen to the medium, respectively (21,22). We found the expression of hamartin protein to be unaffected (Fig. 1A, compare Rat1E2F+ with Rat1 E2F– and Rat1MycER+ with Rat1MycER–), suggesting that it is independent of the mitogenic as well as of the oncogenic potential of these two transcription factors. Western blot analysis further revealed that hamartin protein is expressed in tuberin-positive and in tuberin-negative rat fibroblasts (Fig. 1A). Although the levels of hamartin protein expression were not the same in these two cell lines, our experiments prove that hamartin protein is expressed at least to a certain extent independently of the expression of its partner tuberin. These data are in agreement with the earlier observation that the tuberin-negative Eker rat tumour-derived cell line ERC18M expresses hamartin (20). We performed serum arrest/restimulation and centrifugal elutriation experiments to analyse cell fractions of different cell cycle phases for hamartin protein expression based on western blot analysis. Immunoblot analyses revealed that hamartin protein is expressed in G0 cells as well as throughout the entire cell cycle in rat and human cells (Fig. 1B and C). The described association of these two proteins suggests that they function in the same complex rather than in separate pathways (19,20), and both loss of functional hamartin and loss of tuberin induce uncontrolled proliferation in patients. Taken together, these data made it interesting to investigate the cell cycle regulation of the interaction of these two proteins. We performed immunoprecipitations with anti-tuberin antibody 5063, raised against amino acids 1387–1784 (11), using protein extracts of the elutriated HeLa cells described above and of serum arrested and restimulated Rat1 cells. Immunoblotting of these precipitates with anti-hamartin antibody 2197 (19) revealed that hamartin bound to tuberin can be detected in every cell cycle phase at similar levels (Fig. 1C).
|
The effects of TSC2 on cell proliferation have been proven in cell culture experiments, but to our knowledge TSC1 has not been studied in comparable experiments. In the first approaches to test the effects of TSC2, this gene was ectopically expressed in rat cells and cell numbers were estimated at time points 0, 1, 2 and 6 days after induction of high tuberin levels (15) or at time points 0, ~80, ~120, ~220 and ~330 days after induction of high tuberin levels (16). These experiments demonstrated that high levels of ectopic TSC2 attenuate proliferation. In this study we used a similar approach to analyse TSC1. We transfected logarithmically growing HeLa cells with an expression vector containing full-length human TSC1 and selected for transfected cells by adding G418 to the medium 24 h after transfection. Cell numbers were estimated at time points 0, 1, 2, 5 and 6 days after induction of selection. Compared with an identical approach using only the empty expression vector, we found TSC1-expressing cells to grow significantly more slowly (Fig. 2). Repetition of this experiment using HeLa cells as well as Rat1 cells always provided results allowing the same conclusion (Figs 2 and 3 and data not shown). We analysed the cyclin-dependent kinase inhibitor p27 under the same experimental conditions. p27 is a potent negative regulator of the mammalian cell cycle involved in regulation of G1 phase (23,24) and, accordingly, we found it to completely arrest proliferation in our experimental system. Compared with p27, TSC1 only attenuated the proliferation of HeLa cells in this experiment, probably caused by different levels of ectopic expression (Fig. 2). In a recent report the amino acid sequence of hamartin was analysed for potential interaction domains. A predicted coiled-coil structure spanning amino acids 719–998 was shown to be necessary for hamartin interaction with tuberin (19) and a potential transmembrane domain at amino acids 127–144 has been described (10). We wanted to investigate whether the interaction of hamartin with tuberin is necessary for the negative effects of hamartin on cell proliferation. We generated two mutant forms of TSC1 and cloned them behind an N-terminal Xpress epitope tag into the pcDNA3 mammalian expression vector. Mutant 127 spanning amino acids 788–1153 does not encode the transmembrane domain but harbours most of the coiled-coil region. Mutant 128 (amino acids 228–572) encodes neither the transmembrane domain nor the coiled-coil region. In transfection experiments performed as described above mutant 127 triggered very similar effects to wild-type TSC1, whereas mutant 128 did not affect the proliferation of HeLa cells (Fig. 3).
|
|
To test which phase of the mammalian cell cycle is affected by high levels of ectopically expressed hamartin, we transfected logarithmically growing HeLa cells with the TSC1 expression vector or with the empty expression vector as a negative control. After 6 days of G418 selection for transfected cells, cells were harvested, fixed and stained with propidium iodide. Cytofluorometric analysis of DNA distributions revealed that high levels of TSC1 trigger an increase in the G1 cell population (Fig. 4A). We further tested for p27 protein expression by western blot analysis 6 days after G418 selection for TSC1-transfected cells. It is known that expression of p27 is high in G1 phase and is down-regulated when cells pass through S phase (reviewed in ref. 24). Accordingly, in a cycling cell population the level of p27 protein is proportional to the number of G1 cells. Our observation that p27 protein expression is increased on TSC1 transfection (Fig. 4B) supports the conclusion that TSC1 affects regulation of G1 phase of the mammalian cell cycle.
|
We next wanted to reproduce these findings using a totally different approach in a different cell. We wished to confirm the effects on G1 phase regulation and on p27 expression. Since these data were obtained by long-term analysis of TSC1 overexpression (up to 6 days), we wanted to investigate short-term effects of high levels of hamartin. We transfected rat fibroblasts with a TSC1 expression vector or with the empty expression vector as a negative control and co-transfected with a green fluorescence protein (GFP) expression vector. Immunoblot analyses 2 days after transfection demonstrated that hamartin was overexpressed and p27 expression was up-regulated (Fig. 5A). While these data suggest that the effects of high levels of TSC1 on p27 are independent of the cell line, they also show that the timing of p27 up-regulation on overexpression of hamartin is different in different cells. In HeLa cells up-regulation of p27 protein is not visible on day 2 after transfection but only later (compare Figs 2 and 4), whereas in rat fibroblasts the effects of TSC1 on p27 are already detectable on day 2 (Fig. 5). Since we do not know the reason for this difference, at the moment we can only speculate that it might be associated with differences in responsiveness to TSC1 effects between transformed (HeLa) and non-transformed (rat fibroblast) cells. In experiment 1 we transfected cell populations harbouring high numbers of G0/G1 cells (74.9 ± 0.1%) and found that TSC1 triggers an increase of 3.4% of cells in G0/G1. In experiments 2 and 3 we transfected logarithmically growing cells (57.9 ± 3.3 and 58.8 ± 1.4% G0/G1 cells) and found an increase in G0/G1 cells of 9.25% on average. Every experiment was performed more than once and the results are presented as means ± SD (Fig. 5B). The set of data obtained by studying logarithmically growing cells (experiments 2 and 3) together with the FACS data in Figure 4A, also representing the effects of TSC1 overexpression on the number of cells in G0/G1 (as means ± SD), prompted us to interpret the changes noted in the proportion of cells in G0/G1 to be a significant effect of TSC1 overexpression. As an expected consequence, in cell populations harbouring already high numbers of G1 cells the potential of hamartin to increase the number of G1 cells was lower (see experiment 1 in Fig. 5B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Inherited tumour syndromes are caused by mutations in genes that have been implicated in a diverse array of cellular processes. Proteins encoded by inherited tumour genes appear to function as transmembrane receptors, cytoplasmic regulatory or structural proteins, transcription factors or regulators of transcription, cell cycle factors or DNA damage repair pathway proteins (25). So far, classification of TSC1 and TSC2 into one of these groups of factors is not possible. One characteristic of inherited tumour genes is that their deregulation can induce quiescent cells to start to proliferate and thereby become susceptible to the transformation process. In fact, the clinical phenotype of TSC, characterized by uncontrolled growths, would suggest that TSC1 and TSC2 act as negative regulators of cell proliferation, so that their functional loss induces proliferation. This function would not be possible if hamartin was not expressed in quiescent cells, which we have shown in this report to be the case. Whereas TSC2 has been proven to negatively regulate proliferation (15,16), experimental proof of negative effects of TSC1 on cellular proliferation has been missing so far. The data presented here demonstrate that high levels of ectopically expressed hamartin attenuate proliferation of mammalian cells. In this context it is very important to note that tumour genes, which mediate effects on cell proliferation, can be divided into different groups: (i) their gene products may be cell cycle regulators (e.g. the cyclin-dependent kinase inhibitor p16 or retinoblastoma protein) and therefore a disease caused by mutations in these genes is based on direct deregulation of the cell cycle machinery; and (ii) the gene products of a wide variety of other inherited tumour genes do not directly regulate the cell cycle machinery although they have effects on cell proliferation; mutations in these genes trigger deregulation of other cellular mechanisms (as just one example, deregulated transport of growth factors), which can also ultimately affect proliferation rates. Now the experimental proof exists that TSC1 (this study) and TSC2 (15–18) affect cell proliferation. However, since we do not know the real biochemical function of these two proteins we cannot clarify whether they are directly involved in control of the mammalian cell cycle machinery or whether deregulated proliferation is merely a late consequence far downstream of a totally different function of these proteins. We have reported that loss of TSC2 triggers inactivation of the cyclin-dependent kinase inhibitor p27 (18). Inactivation of p27 is one characteristic marker for deregulated proliferation (23,24). Since so far we have neither found a direct interaction of tuberin with any cell cycle molecule nor could we prove any other mechanism of how tuberin could directly affect the cell cycle machinery, these data can be interpreted in either of two ways: (i) tuberin could directly affect the cell cycle, which would mean that TSC could be a cell cycle disease; or (ii) the effects of tuberin on cell proliferation and p27 could merely be a late consequence of deregulation of a totally different function of tuberin. The same is true for the observation described in this report that overexpression of TSC1 triggers increased p27 levels, which so far we can only interpret as a marker for decreased proliferation. In a publication described above (18) we reported that one immortalized tuberin-negative cell line has more endogenous p27 in the cytoplasm compared with a tuberin-positive counterpart, although we found both cell lines to express p27 in the nucleus. We speculated that deregulation of p27 localization could be involved in the effects of TSC2 on cell proliferation. Further investigations in our laboratory have led us to believe that neither hamartin nor tuberin affects p27 localization and that the observations described above are rather specific for the immortalized cell line analysed and/or for the method used (T. Soucek, A. Miloloza and M. Hengstschläger, unpublished data). Whereas to our knowledge no data exist placing the TSC gene products in direct control of the mammalian cell cycle, a recent publication demonstrated that gigas, a Drosophila homologue of TSC2, regulates the cell cycle. Clones of gigas mutant cells are enlarged and repeat S phase without entering M phase (26). These results could be interpreted as implying a direct involvement of the TSC gene products in control of the cell cycle machinery, but that the link between TSC1/2 and this machinery has not yet been found in mammalian cells.
Recently it was demonstrated that hamartin and tuberin associate physically in vivo mediated by predicted coiled-coil domains, suggesting that these two proteins function in the same complex (19,20). With the exception of correct localization (19), so far no effect of hamartin or tuberin function has been reported to depend on this interaction. We used mutant forms of hamartin to provide evidence that the negative effects of this protein on cellular proliferation might depend on the predicted coiled-coil region. Since we have further shown that a transmembrane domain described earlier is not essential for the effects of hamartin, these data provide support for the biological importance of an interaction of tuberin and hamartin.
In this report we further provide evidence that the effects of hamartin on cell proliferation are likely mediated via deregulation of G1 phase control. This is in agreement with the effects of tuberin on G1 earlier reported (17). It could be interpreted as additional evidence that these two proteins act together. Still, the question of how they mediate phase-specific deregulation remains elusive. We found hamartin (this study) and tuberin (this study; 17) to be constitutively expressed throughout the ongoing cell cycle. In addition, we here report that the interaction of these two proteins is also constant throughout the ongoing cell cycle.
As recently discussed (1), for a more detailed understanding of the mechanism by which the TSC proteins mediate their effects it will be essential to determine which, if any, of the reported functions (Rap1a GAP, Rab5 GAP, transcriptional control, etc.) is/are necessary for these effects. In future experiments the role of TSC proteins in growth suppression should be further investigated. Can tuberin mediate its effects in hamartin-negative cells and vice versa? Do hamartin and tuberin have additive effects when they are co-overexpressed? Which of these two proteins is the limiting factor? Is any TSC protein stronger than the other, when they are directly compared? Overexpression of TSC gene mutants could further confine the regions within TSC1 and TSC2 which are essential for their growth suppression function(s). A major question is whether the effects of TSC proteins are separable from their tumour suppressor function. This can be experimentally investigated by studying disease-causing naturally occurring mutants of TSC1 and/or TSC2 for their potential to trigger negative effects in proliferation assays.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cells and tissue culture
Rat1E2F cells are stably transfected with human E2F-1 downstream of a mutated metallothionein promoter, which was induced by 24 h of treatment with zinc (21). Rat1MycER cells express a protein containing the hormone-binding domain of the human oestrogen receptor fused to the 3"-end of human Myc, which was activated by 24 h of treatment with 100 nM 4-hydroxytamoxifen (22). TSC2-negative and -positive rat fibroblasts were derived from Eker rat embryos homozygous for the Eker mutant and wild-type TSC2 gene, respectively. HeLa cells (human cervical carcinoma cells) were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum and antibiotics (30 mg/l penicillin, 50 mg/l streptomycin sulphate). All cultures were kept at 37°C and 7% CO2. Serum arrest was performed by cultivation of the cells for 3 days in 0.1% calf serum.
Flow cytometry and centrifugal elutriation
For cytofluorometric analyses, cells were harvested by trypsinization and fixed by rapid submersion in ice-cold 85% ethanol. After at least 1 h fixation at –20°C, cells were pelleted and stained in 1 ml of staining solution (0.25 mg/ml propidium iodide, 0.05 mg/ml RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8). Stained cells were analysed on a Beckton Dickinson FACScan (Beckton Dickinson, San Jose, CA). In the case of GFP co-transfection, GFP-positive cell subpopulations were selectively gated. Separation of logarithmically growing cells into distinct cell cycle phases was accomplished by centrifugal elutriation in a Beckman J2-21M centrifuge with a JE-6B rotor with a standard separation chamber (Beckman, Palo Alto, CA). The rotor was kept at a speed of 2000 r.p.m. (580 g) and temperature of 20°C and medium flow was controlled with a Cole-Parmer Masterflex pump (Cole-Parmer, Chicago, IL). The elutriation medium consisted of 0.9 mM CaCl2, 0.5 mM MgCl2 and 2% calf serum in phosphate-buffered saline. Consecutive fractions of 150–300 ml were collected at increasing flow rates. Cytofluorometric analysis of the cell cycle distribution of each fraction was performed as described above.
Western blot analyses and immunoprecipitations
Protein extracts were prepared in buffer containing 20 mM HEPES, pH 7.9, 0.4 M NaCl, 2.5% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 0.5 mM NaF, 0.5 mM Na3VO4, 0.02 µg/ml leupeptin, 0.02 µg/ml aprotinin, 0.003 µg/ml benzamidinchloride, 0.1 µg/ml trypsin inhibitor and 0.5 mM DTT. Cells were lysed by freezing and thawing, the extracts were centrifuged and the supernatants were stored at –70°C. Protein concentrations were determined using the Bio-Rad (Hercules, CA) protein assay reagent with bovine serum albumin as the standard. Proteins were run on an SDS–polyacrylamide gel and transferred to nitrocellulose. Blots were stained with Ponceau-S to visualize the loaded protein. Immunodetection was performed using the anti-hamartin antibody 2197 or the anti-hamartin antibody 2199 (19), anti-tuberin antibody 5063 (11), anti-p27 antibody (C-19; Santa Cruz Biotechnology, Santa Cruz, CA), anti-cyclin A antibody (C-19; Santa Cruz), anti-cyclin E antibody (C-19; Santa Cruz) or anti-Xpress antibody (Invitrogen, Groningen, The Netherlands). Immunoprecipitations were performed using extracts prepared as described above (27) with the anti-tuberin antibody 5063. Signals were detected using the enhanced chemiluminescence method (Amersham, Little Chalfont, UK).
Transfections
Plasmids used for transfection were: the empty vector pcDNA3; pcDNA3 harbouring full-length TSC1; pcDNA3 harbouring TSC1 cDNA representing amino acids 788–1153 of hamartin cloned behind an Xpress tag (mutant 127); pcDNA3 harbouring TSC1 cDNA representing amino acids 228–572 of hamartin cloned behind an Xpress tag (mutant 128); pcDNA3 harbouring full-length p27; the GFP expression vector described in Kalejta et al. (28). The mutants were generated by EcoRI digestion of full-length TSC1 cDNA, which was described in Van Slegtenhorst et al. (19). Cell transfections were performed using the LipofectAMINE reagent obtained from Life Technologies (Gibco BRL, Lofer, Australia) following the transfection protocol provided by the manufacturer. Selection for transfected cells was started 24 h after transfection. During the first 2 days of selection the G418 concentration was 700 µg/ml medium, thereafter the G418 concentration was set at 1500 µg/ml. Cell counting was performed using a haemocytometer.
|
ACKNOWLEDGEMENTS |
|---|
The authors wish to thank T. Littlewood and G. Evans, M. Eilers, P.D. Adams, J. DeClue, R. Yeung and A.J. Beavis for the generous gift of cell lines and reagents, and T. Soucek and K. Braun for helpful discussions. Work in the laboratory of M.H. has been supported by the Austrian Federal Ministry of Science and Transport (project 4/98). Work in the laboratory of M.N. and D.H. is funded by Noortman b.v. (Maastricht).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +43 1 40400 7847; Fax: +43 1 40400 7848; Email: markus.hengstschlaeger@akh-wien.ac.at
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Young, J. and Povey, S. (1998) The genetic basis of tuberous sclerosis. Mol. Med. Today, July, 313–319.
2 Gomez, M.R., Sampson, J.R. and Whittemore, V.H. (1999) Tuberous Sclerosis Complex, 3rd edn. Oxford University Press, New York, NY.
3 Osborn, J.P., Fryer, A. and Webb, D. (1991) Epidemiology of tuberous sclerosis. Ann. N. Y. Acad. Sci., 615, 125–127.[Medline]
4 Sampson, J.R., Janssen, L.A.J., Sandkuijl, L.A. and the Tuberous Sclerosis Collaborative Group (1992) Linkage investigation of three putative tuberous sclerosis determining loci on chromosome 9q, 11q and 12q. J. Med. Genet., 29, 861–866.
5 Kwiatkowski, D.J., Armour, J., Bale, A.E., Fountain, J.W., Goudie, D., Haines, J.L., Knowles, M.A., Pilz, A., Slaugenhaupt, S. and Povey, S. (1993) Report on the second international workshop on human chromosome 9. Cytogenet. Cell Genet., 64, 94–106.
6 The European Chromosome 16 Tuberous Sclerosis Consortium (1993) Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell, 75, 1305–1315.[Web of Science][Medline]
7 Povey, S., Burley, M.W., Attwood, J., Benham, F., Hunt, D., Jeremiah, S.J., Franklin, D., Gillet, G., Malas, S., Robson, E.B. et al. (1994) Two loci for tuberous sclerosis: one on 9q34 and one on 16p13. Ann. Hum. Genet., 58, 107–127.[Web of Science][Medline]
8 Janssen, B., Sampson, J., van der Est, M., Deelen, W., Verhoef, S., Daniels, I., Hesseling, A., Brook-Carter, P., Nellist, M., Lindhout, D. et al. (1994) Redefined localization of TSC1 by combined analysis of 9q34 and 16p34 data on 14 tuberous sclerosis families. Hum. Genet., 94, 437–440.[Web of Science][Medline]
9 Sampson, J.R. and Harris, P.C. (1994) The molecular genetics of tuberous sclerosis. Hum. Mol. Genet., 3, 1477–1480.[Abstract]
10 The TSC1 Consortium (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277, 805–808.
11 Wiennecke, R., König, A. and DeClue, J. (1995) Identification of tuberin, the tuberous sclerosis-2 product. J. Biol. Chem., 270, 16409–16414.
12 Tsuchiya, H., Orimoto, K., Kobayashi, T. and Hino, O. (1996) Presence of potent transcriptional activation domains in the predisposing tuberous sclerosis (Tsc2) gene product of the Eker rat model. Cancer Res., 56, 429–433.
13 Xiao, G.-H., Shoarinejad, F., Jin, F., Golemis, E.A. and Yeung, R.S. (1997) The tuberous sclerosis 2 gene product, tuberin, functions as a Rab5 GTPase activating protein (GAP) in modulating endocytosis. J. Biol. Chem., 272, 6097–6100.
14 Henry, K.W., Yuan, X., Koszewski, N.J., Onda, H., Kwiatkowski, D.J. and Noonan, D.J. (1998) Tuberous sclerosis gene 2 product modulates transcription mediated by steroid hormone receptor family members. J. Biol. Chem., 273, 20535–20539.
15 Jin, F., Wienecke, R., Xiao, G.-H, Maize, J.C., DeClue, J.E. and Yeung, R.S. (1996) Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (TSC2) gene and its C-terminal region. Proc. Natl Acad. Sci. USA, 93, 9154–9159.
16 Orimoto, K., Tsuchiya, H., Toshiyuki, K., Matsuda, T. and Hino, O. (1996) Suppression of the neoplastic phenotype by replacement of the TSC2 gene in Eker rat renal carcinoma cells. Biochem. Biophys. Res. Commun., 219, 70–75.[Web of Science][Medline]
17 Soucek, T., Pusch, O., Wienecke, R., DeClue, J.E. and Hengstschläger, M. (1997) Role of the tuberous sclerosis gene-2 product in cell cycle control. J. Biol. Chem., 272, 29031–29038.
18 Soucek, T., Yeung, R.S. and Hengstschläger, M. (1998) Inactivation of the cyclin-dependent kinase inhibitor p27 upon loss of the tuberous sclerosis complex gene-2. Proc. Natl Acad. Sci. USA, 95, 15653–15658.
19 Van Slegtenhorst, M., Nellist, M., Nagelkerken, B., Cheadle, J., Snell, R., van den Ouweland, A., Reuser, A., Sampson, J., Halley, D. and van der Sluijs, P. (1998) Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products. Hum. Mol. Genet., 7, 1053–1057.
20 Plank, T.L., Yeung, R.S. and Henske, E.P. (1998) Hamartin, the product of the tuberous sclerosis 1 (TSC1) gene, interacts with tuberin and appears to be localized to cytoplasmic vesicles. Cancer Res., 85, 4766–4770.
21 Qin, X.-Q., Livingston, D.M., Kaelin, W.G. and Adams, P.D. (1994) Deregulated transcription factor E2F-1 expression leads to S-phase entry and p53-mediated apoptosis. Proc. Natl Acad. Sci. USA, 91, 10918–10922.
22 Littlewood, T.D., Hancock, D.C., Danielian, P.S., Parker, M.G. and Evan, G.I. (1995) A modified oestrogen receptor ligand-binding domain as an improved switch for the regulation of heterologous proteins. Nucleic Acids Res., 23, 1686–1690.
23 Weinberg, R.A. (1995) The retinoblastoma protein and cell cycle control. Cell, 81, 323–330.[Web of Science][Medline]
24 Alessandrini, A., Chiaur, D.S. and Pagano, M. (1997) Regulation of the cyclin-dependent kinase inhibitor p27 by degradation and phosphorylation. Leukemia, 11, 342–345.[Web of Science][Medline]
25 Fearon, E.R. (1997) Human cancer syndromes: clues to the origin and nature of cancer. Science, 278, 1043–1050.
26 Ito, N. and Rubin, G.M. (1999) gigas, a drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell, 96, 529–539.[Web of Science][Medline]
27 Dulic, V., Lees, E. and Reed, S.I. (1992) Association of human cyclin E with a periodic G1-S phase protein kinase. Science, 257, 1958–1961.
28 Kalejta, R.F., Shenk, T. and Beavis, A.J. (1997) Use of a membrane-localized green fluorescent protein allows simultaneous identification of transfected cells and cell cycle analysis by flow cytometry. Cytometry, 29, 286–291.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Rosner and M. Hengstschlager Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: rapamycin triggers dephosphorylation and delocalization of the mTORC2 components rictor and sin1 Hum. Mol. Genet., October 1, 2008; 17(19): 2934 - 2948. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zheng, H. Ding, Z. Lu, Y. Li, Y. Pan, T. Ning, and Y. Ke E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6. Genes Cells, March 1, 2008; 13(3): 285 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Goncharova, D. A. Goncharov, A. Chisolm, M. S. Spaits, P. N. Lim, G. Cesarone, I. Khavin, O. Tliba, Y. Amrani, R. A. Panettieri Jr., et al. Interferon {beta} Augments Tuberous Sclerosis Complex 2 (TSC2)-Dependent Inhibition of TSC2-Null ELT3 and Human Lymphangioleiomyomatosis-Derived Cell Proliferation Mol. Pharmacol., March 1, 2008; 73(3): 778 - 788. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Rosner, A. Freilinger, M. Hanneder, N. Fujita, G. Lubec, T. Tsuruo, and M. Hengstschlager p27Kip1 localization depends on the tumor suppressor protein tuberin Hum. Mol. Genet., July 1, 2007; 16(13): 1541 - 1556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. Sandsmark, H. Zhang, B. Hegedus, C. L. Pelletier, J. D. Weber, and D. H. Gutmann Nucleophosmin Mediates Mammalian Target of Rapamycin-Dependent Actin Cytoskeleton Dynamics and Proliferation in Neurofibromin-Deficient Astrocytes Cancer Res., May 15, 2007; 67(10): 4790 - 4799. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Astrinidis, W. Senapedis, and E. P. Henske Hamartin, the tuberous sclerosis complex 1 gene product, interacts with polo-like kinase 1 in a phosphorylation-dependent manner Hum. Mol. Genet., January 15, 2006; 15(2): 287 - 297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. J. Zacharek, Y. Xiong, and S. D. Shumway Negative Regulation of TSC1-TSC2 by Mammalian D-Type Cyclins Cancer Res., December 15, 2005; 65(24): 11354 - 11360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Gan, Z. K. Melkoumian, X. Wu, K.-L. Guan, and J.-L. Guan Identification of FIP200 interaction with the TSC1-TSC2 complex and its role in regulation of cell size control J. Cell Biol., August 1, 2005; 170(3): 379 - 389. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rosner and M. Hengstschlager Tuberin Binds p27 and Negatively Regulates Its Interaction with the SCF Component Skp2 J. Biol. Chem., November 19, 2004; 279(47): 48707 - 48715. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Astrinidis and E. Petri Henske Aberrant Cellular Differentiation and Migration in Renal and Pulmonary Tuberous Sclerosis Complex J Child Neurol, September 1, 2004; 19(9): 710 - 715. [Abstract] [PDF]
|
|
|
|
|
|
D. K. Scheidenhelm and D. H. Gutmann Mouse Models of Tuberous Sclerosis Complex J Child Neurol, September 1, 2004; 19(9): 726 - 733. [Abstract] [PDF]
|
|
|
|
|
|
M. Karbowniczek, T. Cash, M. Cheung, G. P. Robertson, A. Astrinidis, and E. P. Henske Regulation of B-Raf Kinase Activity by Tuberin and Rheb Is Mammalian Target of Rapamycin (mTOR)-independent J. Biol. Chem., July 16, 2004; 279(29): 29930 - 29937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Yu, A. Astrinidis, S. Howard, and E. P. Henske Estradiol and tamoxifen stimulate LAM-associated angiomyolipoma cell growth and activate both genomic and nongenomic signaling pathways Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L694 - L700. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Jozwiak, D. Kwiatkowski, K. Kotulska, M. Larysz-Brysz, J. Lewin-Kowalik, W. Grajkowska, and M. Roszkowski Tuberin and Hamartin Expression Is Reduced in the Majority of Subependymal Giant Cell Astrocytomas in Tuberous Sclerosis Complex Consistent With a Two-Hit Model of Pathogenesis J Child Neurol, February 1, 2004; 19(2): 102 - 106. [Abstract] [PDF]
|
|
|
|
|
|
A. Astrinidis, W. Senapedis, T. R. Coleman, and E. P. Henske Cell Cycle-regulated Phosphorylation of Hamartin, the Product of the Tuberous Sclerosis Complex 1 Gene, by Cyclin-dependent Kinase 1/Cyclin B J. Biol. Chem., December 19, 2003; 278(51): 51372 - 51379. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Karbowniczek, A. Astrinidis, B. R. Balsara, J. R. Testa, J. H. Lium, T. V. Colby, F. X. McCormack, and E. P. Henske Recurrent Lymphangiomyomatosis after Transplantation: Genetic Analyses Reveal a Metastatic Mechanism Am. J. Respir. Crit. Care Med., April 1, 2003; 167(7): 976 - 982. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. C. Mak, K.-I. Takemaru, H. L. Kenerson, R. T. Moon, and R. S. Yeung The Tuberin-Hamartin Complex Negatively Regulates beta -Catenin Signaling Activity J. Biol. Chem., February 14, 2003; 278(8): 5947 - 5951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Tee, D. C. Fingar, B. D. Manning, D. J. Kwiatkowski, L. C. Cantley, and J. Blenis Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling PNAS, October 15, 2002; 99(21): 13571 - 13576. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nellist, M. A. Goedbloed, C. de Winter, B. Verhaaf, A. Jankie, A. J. J. Reuser, A. M. W. van den Ouweland, P. van der Sluijs, and D. J. J. Halley Identification and Characterization of the Interaction between Tuberin and 14-3-3zeta J. Biol. Chem., October 11, 2002; 277(42): 39417 - 39424. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L Khare, A Astrinidis, W Senapedis, P D Adams, and E P. Henske Expression of wild type and mutant TSC2, but not TSC1, causes an increase in the G1 fraction of the cell cycle in HEK293 cells J. Med. Genet., September 1, 2002; 39(9): 676 - 680. [Full Text] [PDF]
|
|
|
|
|
|
A. K. Hodges, S. Li, J. Maynard, L. Parry, R. Braverman, J. P. Cheadle, J. E. DeClue, and J. R. Sampson Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin Hum. Mol. Genet., December 1, 2001; 10(25): 2899 - 2905. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||