Human Molecular Genetics, 2001, Vol. 10, No. 25 2899-2905
© 2001 Oxford University Press
Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin
Institute of Medical Genetics, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK and 1Laboratory of Cellular Oncology, National Institute of Health, Bethesda, MD 20892, USA
Received August 2, 2001; Revised September 24, 2001; Accepted October 8, 2001.
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ABSTRACT |
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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TSC1 and TSC2 are tumour suppressor genes that were originally identified through their involvement in the inherited disorder tuberous sclerosis (TSC) (1,2). They encode the previously unknown proteins hamartin and tuberin. Studies of the hamartomatous growths that characterize TSC have revealed inactivation of either hamartin or tuberin by a classical two-hit mechanism (2–6). Comparable findings have also been reported in lesions, particularly renal adenomas and carcinomas, from naturally occurring and engineered mutant Tsc1 and Tsc2 rodent models (7–12).
Abnormalities of cellular proliferation, differentiation and migration have been indicated by examination of various TSC lesions, suggesting that hamartin and tuberin are required for the normal regulation of these cellular processes (13). In human, rodent and Drosophila cells, overexpression of hamartin and tuberin lengthens the G1 phase of the cell cycle and is associated with an inhibition of cell proliferation and, in Drosophila, reduction in cell size. Conversely, deficiency of hamartin or tuberin in null cells derived from rocky (rcy) (Tsc1 –/–) and gigas (gig) (Tsc2 –/–) Drosophila mutants or the Eker (Tsc2 –/–) rat is associated with shortening of G1 and, in Drosophila, with hypertrophy and normal ploidy (14–19). Giant cells with normal DNA content are also a characteristic feature of central nervous system hamartomas in TSC (20). It is currently unclear whether hamartin and tuberin affect the cell cycle directly or indirectly or exactly how these effects relate to the pathogenesis of TSC. Recent reports suggest that the proteins act downstream of the insulin receptor, PTEN and Akt proteins (19) to influence dS6K, cyclins A, B, D and E (15,18,19,21) and p27 (16).
The C-termini of hamartin (amino acids 719–998), that includes putative coiled-coil domains (2), and of tuberin (amino acids 1049–1809), that includes a GTPase activating protein (GAP)-related domain (1) with reported activity for the Rap1a and Rab5 GTPases (22,23) appear important for growth suppression (14,17). Ezrin binding (amino acids 881–1084) and Rho activation (amino acids 145–510) by hamartin were recently assessed to be important in the assembly of contractile actomyosin filaments with downstream effects on the cytoskeleton and cell-substrate adhesion (24). Such roles could underlie the aberrant cell migration that characterizes cerebral pathology in TSC patients (25), and the apparent spread of abnormal smooth muscle cells from renal cell angiomyolipomas to the lung in TSC-associated lymphangioleiomyomatosis (26).
Whereas distinct functions have been postulated for different regions of hamartin and tuberin, evidence linking these functions to the interaction between the proteins and to the TSC phenotype has been lacking. In contrast, recent evidence suggests that the Drosophila rcy/Tsc1 and gig/Tsc2 phenotypes result from disruption to the hamartin–tuberin complex (18,19). Many co-immunoprecipitation studies have demonstrated a physical interaction between full-length hamartin and full-length tuberin (19,27–32). The complex has been detected in all phases of the cell cycle (17) and appears to stabilize tuberin by preventing its ubiquitination and its subsequent degradation (33). In this study we sought to clarify the areas in hamartin and tuberin that are important for their interaction, and to study the effects of TSC-associated mutations on their binding.
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RESULTS |
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Full-length hamartin and tuberin were shown to co-immunoprecipitate from lysates of transfected Cos7 cells overexpressing both proteins, consistent with previous reports of a direct interaction between the proteins (19,27–32) (Fig. 1). A series of tuberin constructs representing progressive truncations from the C-terminal end of tuberin were assayed for interaction with a large fragment of hamartin encompassing residues 335–1161 by yeast two-hybrid assay (Fig. 2). A construct corresponding to tuberin residues 1–418 gave the largest number of colonies on selection for histidine auxotrophy, and the most intense ß-galactosidase activity, indicating the strongest interaction (Fig. 2A). This region of tuberin includes a weakly predicted coiled-coil structure at amino acids 340–400 that was previously reported to mediate tuberin’s interaction with hamartin (27). However, constructs corresponding to tuberin residues 337–415, 270–415 and a region N-terminal to residue 270 did not interact with hamartin 335–1161, indicating that neither the weakly predicted coiled-coil region nor the more N-terminal region alone could mediate the interaction and that both were required. Larger tuberin constructs that, in addition, contained more C-terminal sequences interacted more weakly than the amino acids 1–418 tuberin construct (Fig. 2A).
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In yeast two-hybrid assays using a range of hamartin constructs, all of those that included part of the region corresponding to residues 171–430 interacted with full-length tuberin and with its N-terminal region (residues 1–418) (Fig. 2B). In assays using the full-range of hamartin and tuberin constructs, the strongest interactions were seen between those encoding residues 335–430 of hamartin and 1–418 of tuberin. An interaction was also seen, although more weakly, with N-terminal constructs of hamartin, suggesting the presence of multiple binding sites within its first 430 residues. In contrast, a series of C-terminal hamartin constructs that included part or all of the predicted coiled-coils region (amino acids 719–998) (2) interacted very weakly or not at all with full-length and truncated tuberin constructs (Fig. 2B).
Next, we investigated the effects on hamartin–tuberin binding of well characterized small in-frame deletions and a missense mutation that have been reported in patients with TSC, and that map to the areas that appeared important for interaction. Introduction of either the missense change G294E (34) or the 3 bp deletion I365del (34) into tuberin abolished the ability of tuberin 1–418 to bind to hamartin in yeast two-hybrid assays (Fig. 2A). In contrast, three non-pathogenic amino acid substitutions, R261W, M286V and R367Q (34–36) that flanked the TSC2 mutations had no or little effect on the interaction with hamartin (Fig. 2A). TSC1 mutations that do not lead to protein truncation are extremely rare and we have been able to verify only one example. We found that this well characterized mutation (N198_F199delinsI;593–595delACT) (37) abolished the interaction of hamartin with tuberin in the yeast two-hybrid assay (Fig. 2B).
Next, we investigated the effects of the TSC associated mutations and the non-pathogenic missense variants on the ability of hamartin and tuberin to co-immunoprecipitate in lysates from Cos7 cells. As we had observed for the wild-type proteins, full-length tuberin that contained each of the three non-pathogenic missense variants co-immunoprecipitated with full-length hamartin (Fig. 3). In contrast, full-length tuberin containing either the G294E or the I365del 3 bp deletion mutation, although stable and overexpressed at the same level as the wild-type protein, failed to co-immunoprecipitate with hamartin (Fig. 3), confirming that these pathological TSC2 mutations disrupt the ability of tuberin to bind to hamartin. The TSC1 3 bp deletion mutation (N198_F199delinsI;593–595delACT) (37), that lies upstream of the predicted minimal binding region for tuberin, was introduced into full-length hamartin. Although the mutant protein was shown to be expressed at a similar level to wild-type there was a marked reduction in its ability to immunoprecipitate tuberin (Fig. 3B).
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DISCUSSION |
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Both TSC1 and TSC2 are considered to act as tumour suppressor genes, since inactivation of the corresponding wild-type allele through allelic loss or intragenic mutation has been reported in a wide variety of hamartomas from patients with TSC (5,38). Studies of lesions from the spontaneous Eker (Tsc2+/–) rat model and from engineered Tsc1 and Tsc2 ‘knockout’ mice (7,9,11,12,39), together with phenotypic rescue of the Eker rat by a Tsc2 transgene (40) support this conclusion. In mammalian cell culture systems, reduction of cellular proliferation by hamartin or tuberin has been associated with lengthening of G0/G1 (15–17). Similar cell cycle effects have been reported in Drosophila, where growth was also restricted as a result of cell size reduction (18,19). These effects in Drosophila were only seen when both proteins were overexpressed together, suggesting an interdependency of hamartin and tuberin for their growth regulatory functions (19). In contrast, an anti-proliferative effect of each of the proteins has been reported on independent overexpression in mammalian cell culture (15–17). Previous studies have even indicated that overexpression of only the C-terminal regions of hamartin (residues 788–1153) or tuberin (1049–1809) can mediate growth suppression in this setting (14,17). We sought to clarify whether at least some TSC disease-related functions of human hamartin and tuberin require a direct interaction between the proteins. Our data, showing that certain non-truncating yet TSC causing mutations of hamartin and tuberin disrupt their binding supports this proposition, whether or not the binding dependent functions are among those already suggested by in vitro studies. Each of the mutations investigated was selected because it was well characterized and mapped to the regions implicated in hamartin–tuberin binding by our yeast two-hybrid experiments. Immunoblotting indicated that all were associated with production of stable mutant forms of hamartin or tuberin. Their dramatic effects on binding would be consistent with disruption of the interacting regions. In contrast, TSC causing missense mutations of tuberin that map outside the interacting region (N1643K, N1651S, N1681K) encode stable proteins that can still bind hamartin (32; unpublished data). We conclude that the binding of hamartin and tuberin is necessary, but not sufficient for their mediation of growth suppression.
Previous work has not assessed in detail the regions of hamartin and tuberin that mediate their interaction. The results of two-hybrid experiments reported here are consistent only in part with the single previous report of hamartin–tuberin binding in the yeast two-hybrid system (27). That report suggested the region encoded by amino acids 346–371 of tuberin was required for interaction with hamartin. Our data indicated that the interaction requires a more extended N-terminal region of tuberin. In contrast to the previous report (27), we did not find that the N-terminal of tuberin interacted with the C-terminal coiled-coils region of hamartin (amino acids 786–1010). We found no interaction or only very weak interaction with a large set of hamartin constructs from this region. Neither does the disruption of tuberin binding that we observed in association with the N-terminal N198_F199delinsI;593–595delACT mutation of hamartin support mediation of the interaction via hamartin’s C-terminal coiled-coils region. Rather, our data indicate that interaction of the proteins occurs primarily through their N-terminal domains.
Although tuberin 1–418 interacted strongly with hamartin, larger tuberin constructs that included the N-terminal binding region interacted much more weakly. This could reflect inappropriate folding of some truncated tuberin fragments or the masking of the binding site in progressively larger fragments. Unmasking of the N-terminal binding site to protein partners is required by the ERM proteins ezrin, radixin and moesin and related family member merlin isoform 1, for activity (41–43). It is also possible that the physiological interaction between hamartin and tuberin is complex and that assays in the yeast nucleus do not fully reflect this. A recent report showed that phosphorylation of tuberin that was dependent on specific C-terminal residues, could regulate its binding to hamartin (32). In light of our data this suggests that critical functions of hamartin and tuberin themselves could be regulated through changes in binding.
The generation of similar or identical phenotypes through mutations at two or more loci (locus heterogeneity) is well established in many mendelian disorders. It may reflect disruption of different steps in a common pathway or equivalent effects resulting from disruption of distinct pathways as well as alterations in the different components of a functional complex. An example of the latter situation is hereditary non-polyposis colorectal cancer, that is associated with mutations in one or other of the hMLH1, hMSH2, hMSH6, hPMS2 or hMLH3 genes. Their products form several multimeric complexes involved in the repair of distinct classes of base pair mismatches (44). However, to our knowledge, non-truncating mutations that prevent complex formation rather than abrogate the functions of the component proteins have not been clearly documented in tumour predisposition syndromes. In contrast, molecular mechanisms analogous to that we propose for some cases of TSC are well known in inherited metabolic disease. For example, certain missense mutations of either the LDL receptor gene or the apoB-100 gene that encodes its ligand both lead to the phenotype of hypercholesterolaemia by impairing binding between the gene products (45,46). The likelihood that some cases of TSC arise through disruption of the hamartin–tuberin interaction may raise opportunities for intervention. Although it is difficult to envisage effective delivery of either protein to all deficient cells, the identification of small molecules that could act as a surrogate for hamartin’s binding to tuberin may be more tractable.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TSC1 and TSC2 constructs
In-frame hamartin and tuberin constructs were made in the yeast two-hybrid activating domain vectors pGAD424 series, pGAD GH (Clontech, Basingstoke, UK) and pAD GAL4-2.1 (Stratagene, Amsterdam, The Netherlands) and in the binding domain vectors pGBT9 (Clontech), pBD-GAL4-Cam (Stratagene), pAS1-CY2 (Clontech) and pYTH9 (a gift from Richard Lamb). For expression and co-immunoprecipitation studies constructs were produced in the mammalian expression vectors pCMV-Tag2 series (for detection with an anti-FLAG antibody) and pCMV-Tag3 series (for detection with an anti-c-Myc antibody) (Stratagene).
The following wild-type constructs were derived from full-length cDNAs (numbers refer to amino acid position). Activating domain yeast two-hybrid constructs TSC1: 23–26, 23–171, 23–271, 23–357, 335–430, 335–483, 335–500, 335–605, 335–673, 335–789, 335–1027, 335–1161, 788–915, 788–960, 788–1010; TSC2: 270–415, 337–415, 1–41, 1–76, 1–163, 1–250, 1–383, 1–418, 1–459, 1–606, 1–934, 1–939; full-length TSC2 1–1807. Binding domain yeast two-hybrid constructs TSC1: 1–235, 335–1161, 788–915, 788–960, 788–1010; TSC2: 270–415, 337–415, 1–41, 1–76, 1–163, 1–250, 1–383, 1–418, 1–459, 1–606, 1–934, 1–1670; full-length TSC2 1–1807. Mammalian expression constructs detectable with an anti-c-Myc antibody: full-length TSC1 1–1164 and full-length TSC2 1–1807. Mammalian expression constructs detectable with an anti-FLAG antibody: full-length TSC1 1–1164 and full-length TSC2 1–1807.
The description of changes (mutations and polymorphisms) at the DNA and protein level are according to the nomenclature described by Antonarakis (47) and den Dunnen and Antonarakis (48). TSC-associated missense changes and small in-frame deletion mutants were created by either cloning RT–PCR products from patient-derived EBV-transformed lymphoblastoid cells into the appropriate vector and screening for the mutant allele by sequencing or by site-directed mutagenesis using the Quik Change kit, according to the manufacturer’s protocol (Stratagene). The TSC-associated mutations investigated were G294E, in exon 9 of TSC2 (34), the TSC2 in-frame deletion mutation I365del in exon 10 of TSC2 (34), and the TSC1 mutation N198_F199delinsI;593–595delACT that has been shown to arise de novo in a sporadic TSC case with subsequent transmission to that individual’s two affected offspring (37). Three amino acid changing polymorphisms that we and others have identified previously (34–36) in the same region as the pathogenic TSC2 mutations were also created by site-directed mutagenesis using the Quik Change kit, according to the manufacturer’s protocol. These were: R261W, 781C
T; M286V, 856AG and R367Q, 1100GA.
Yeast two-hybrid interactions
Yeast host strain CG-1945 was sequentially transformed with 1 µg of each plasmid according to the method specified in the Clontech MATCHMAKER two-hybrid protocol (Clontech). Briefly, for primary transformation of the first construct a single yeast colony was grown in 20 ml of YPD broth (2% peptone, 1% yeast extract, 2% glucose) for 
24 h at 30°C with shaking at 250 r.p.m. (Beckman Innova 4230 incubator), to an OD600 of 1–2. This culture was transferred to 300 ml YPD broth and grown for a further 3 h. After pelleting the cells at 1000 g for 5 min at room temperature, the pellet was resuspended in 50 ml sterile water and re-pelleted. After removing the supernatant, the cells were resuspended in 1.5 ml of 0.1 M lithium acetate/TE buffer (10 mM Tris, 1 mM EDTA pH 7.5). 100 µl of resuspended cells were added to 1 µg of each construct to be transformed, along with 100 µg of salmon sperm carrier DNA (Stratagene). To this was added 600 µl of a mix of 40% polyethylene glycol 3350 (Sigma, Poole, UK), 0.1 M lithium acetate/TE buffer. The mixture was incubated at 30°C with shaking (200 r.p.m.) for 30 minutes. After adding 10% dimethyl sulphoxide (Sigma), the cells were heat-shocked for 15 min at 42°C. The cells were briefly chilled on ice and pelleted for 1 min at 10 000 g and resuspended in 100 µl TE buffer. Cells were plated on minimal media (yeast nitrogen base without amino acids) (Becton Dickinson, Oxford, UK) supplemented with an amino acid dropout mixture (Clontech manual) lacking either the amino acid leucine (-L) for selection of activating domain plasmids or tryptophan (-T) for selection of binding domain plasmids, and 2% glucose. The yeast were grown for 3–5 days at 30°C until single colonies appeared.
For secondary transformation, a single colony from the primary transformation was grown in the appropriate selective media (-L or -T) following the same procedure described above for primary transformation. The cells were resuspended in 200 µl TE buffer at the end of the procedure and 100 µl plated on minimal media lacking leucine and tryptophan (-L,-T) (to indicate transformation efficiency) and minimal media lacking histidine, leucine and tryptophan (-H,-L,-T) (to indicate a positive interaction) and grown for 3–5 days at 30°C. Colonies capable of growth on -H,-L,-T media were tested for LacZ expression by assaying ß-galactosidase activity following colony lifts on to nitrocellulose filters. Filters were immersed in liquid nitrogen for 10 s followed by incubation overnight in 1.8 ml of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 0.1mM MgSO4, pH 7) containing 2.7 µl/ml ß-mercaptoethanol and 0.334 mg/ml 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside in N,N-dimethylformamide (X-gal). Where possible, constructs were made in both activating and binding domain vectors and interactions were assessed using both vector combinations in sequential transformations. All experiments were repeated at least twice. Assessment of interaction was on a four-point scale (+++, ++, + or –) based on the number of colonies capable of growth on selection for histidine auxotrophy (-H), and intensity of blue colour as a measure of ß-galactosidase activity
Co-immunoprecipitation
Full-length hamartin and tuberin were detected using a polyclonal rabbit anti-hamartin antibody raised against human hamartin residues 748–957 (33) and a rabbit anti-tuberin antiserum raised against human tuberin residues 1387–1784 (Tub-C) (22). All hamartin and tuberin fragments in pCMV-Tag2 series or pCMV-Tag3 series expression vectors were detected using a mouse anti-FLAG M2 monoclonal antibody (Sigma, St Louis, MO) or a mouse anti-c-Myc (9E10) monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), respectively.
Cell culture and transfection
Cos7 cells were grown in Dulbecco’s modified Eagle medium (D-MEM) with antibiotics and 10% (v/v) fetal bovine serum. All Cos7 cell co-transfections were carried out with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, using 2 x 106 cells/100 mm plate and 10 µg of DNA (5 µg each of TSC1 and TSC2 constructs).
Cell lysis, immunoprecipitation and immunoblotting
Cells were lysed after 24 h of transfection in C-type lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 1% Triton X-100) with CompleteTM protease inhibitors (Roche Diagnostics, Indianapolis, IN). Lysates were clarified by microcentrifugation at 10 000 g for 10 min at 4°C, and the protein concentration of an aliquot from each lysate was determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Portions of each lysate containing 300 µg protein were subjected to immunoprecipitation with 25 µl of anti-FLAG M2-agarose affinity gel or 25 µl of anti-c-Myc (9E10) monoclonal antibody-conjugated agarose, as appropriate, for 2 h at 4°C. The immunoprecipitates were washed four times with lysis buffer and four times with wash buffer (PBS pH 7.5, 5 mM MgCl2, 1% NP-40), analysed on a 12% polyacrylamide gel, and transferred to a PVDF membrane. For all immunoblotting, the membranes were blocked overnight with milk blocking solution (KPL, Gaithersburg, MD) at 4°C, and then incubated with a 1:1000 dilution of crude antiserum or 1 µg/ml purified antibody in KPL diluent solution for 90 min at room temperature. The membranes were washed three times for 5 min each with KPL wash solution before being incubated with a 1:5000 dilution of HRP-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ). Detection of bound antibodies was carried out using the enhanced chemiluminescence system (Amersham Pharmacia Biotech).
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ACKNOWLEDGEMENTS |
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We thank Nick Fleming for automated sequencing of constructs and Drs Mark Nellist, Dicky Halley, Ans van den Ouweland and Richard Lamb for helpful discussions. This work was supported by the Tuberous Sclerosis Alliance, the Tuberous Sclerosis Association, Tenovus and J. Mendoza.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 29 20744033; Fax: +44 29 20747603; Email: hodgesak@cardiff.ac.uk
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REFERENCES |
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1 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]
2 van Slegtenhorst, M., deHoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., Lindhout, D., van den Ouweland, A., Halley, D., Young, J. et al. (1997) Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277, 805–808.
3 Green, A.J., Johnson, P.H. and Yates, J.R.W. (1994) The tuberous sclerosis gene on chromosome 9q34 acts as a growth suppressor. Hum. Mol. Genet., 3, 1833–1834.
4 Henske, E.P., Scheithauer, B.W., Short, M.P., Wollmann, R., Nahmias, J., Hornigold, N., van Slegtenhorst, M., Welsh, C.T. and Kwiatkowski, D.J. (1996) Allelic loss is frequent in tuberous sclerosis kidney lesions but rare in brain lesions. Am. J. Hum. Genet., 59, 400–406.[Web of Science][Medline]
5 Carbonara, C., Longa, L., Grosso, E., Mazzuco, G., Borrone, C., Garre, M.L., Brisigotti, M., Filippi, G., Scabar, A., Giannotti, A. et al. (1996) Apparent preferential loss of heterozygosity at TSC2 over TSC1 chromosomal region in tuberous sclerosis hamartomas. Genes Chromosome Cancer, 15, 18–25.[Web of Science][Medline]
6 Henske, E.P., Wessner, L.L., Golden, J., Scheithauer, B.W., Vortmeyer, A.O., Zhuang, Z.P., KleinSzanto, A.J.P., Kwiatkowski, D.J. and Yeung, R.S. (1997) Loss of tuberin in both subependymal giant cell astrocytomas and angiomyolipomas supports a two-hit model for the pathogenesis of tuberous sclerosis tumors. Am. J. Pathol., 151, 1639–1647.[Abstract]
7 Kubo, Y., Klimek, F., Kikuchi, Y., Bannasch, P. and Hino, O. (1995) Early detection of Knudson 2-hits in preneoplastic renal-cells of the Eker rat model by the laser microdissection procedure. Cancer Res., 55, 989–990.
8 Kubo, Y., Kikuchi, Y., Mitani, H., Kobayashi, E., Kobayashi, T. and Hino, O. (1995) Allelic loss at the tuberous sclerosis (Tsc2) gene locus in spontaneous uterine leiomyosarcomas and pituitary-adenomas in the Eker rat model. Jpn J. Cancer Res., 86, 828–832.[Web of Science][Medline]
9 Yeung, R.S., Xiao, G.H., Everitt, J.I., Jin, F. and Walker, C.L. (1995) Allelic loss at the tuberous sclerosis 2 locus in spontaneous tumors in the Eker rat. Mol. Carcinogen., 14, 28–36.[Web of Science][Medline]
10 Satake, N., Kobayashi, T., Kobayashi, E., Izumi, K. and Hino, O. (1999) Isolation and characterization of a rat homologue of the human tuberous sclerosis 1 gene (Tsc1) and analysis of its mutations in rat renal carcinomas. Cancer Res., 59, 849–855.
11 Onda, H., Lueck, A., Marks, P.W., Warren, H.B. and Kwiatkowski, D.J. (1999) Tsc2(+/–) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background. J. Clin. Invest., 104, 687–695.[Web of Science][Medline]
12 Kobayashi, T., Minowa, O., Sugitani, Y., Takai, S., Mitani, H., Kobayashi, E., Noda, T. and Hino, O. (2001) A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice. Proc. Natl Acad. Sci. USA, 98, 8762–8767.
13 Gomez, M.R., Sampson, J.R. and Holets-Whittemore, V. (1999) Tuberous Sclerosis Complex, 3rd edn. Oxford University Press, New York.
14 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.
15 Soucek, T., Pusch, O., Wienecke, R., DeClue, J.E. and Hengstschlager, M. (1997) Role of the tuberous sclerosis gene-2 product in cell cycle control—loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase. J. Biol. Chem., 272, 29301–29308.
16 Soucek, T., Yeung, R.S. and Hengstschlager, 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.
17 Miloloza, A., Rosner, M., Nellist, M., Halley, D., Bernaschek, G. and Hengstschlager, M. (2000) The TSC1 gene product, hamartin, negatively regulates cell proliferation. Hum. Mol. Genet., 9, 1721–1727.
18 Tapon, N., Ito, N., Dickson, B.J., Treisman, J.E. and Hariharan, I.K. (2001) The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell, 105, 345–355.[Web of Science][Medline]
19 Potter, C.J., Huang, H. and Xu, T. (2001) Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell, 105, 357–368.[Web of Science][Medline]
20 Shepherd, C.W., Scheithauer, B.W., Gomez, M.R., Altermatt, H.J. and Katzmann, J.A. (1991) Subependymal giant-cell astrocytoma—a clinical, pathological, and flow cytometric study. Neurosurgery, 28, 864–868.[Web of Science][Medline]
21 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]
22 Wienecke, R., Konig, A. and Declue, J.E. (1995) Identification of tuberin, the tuberous sclerosis-2 product—tuberin possesses specific Rap1gap activity. J. Biol. Chem., 270, 16409–16414.
23 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.
24 Lamb, R.F., Roy, C., Diefenbach, T.J., Vinters, H.V., Johnson, M.W., Jay, D.G. and Hall, A. (2000) The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho. Nat. Cell Biol., 2, 281–287.[Web of Science][Medline]
25 Takashima, S., Mizuguchi, M. and Arii, N. (1998) Neuronal migration disorder: expression of gene products in the neocortex. Neuropathology, 18, 427–432.
26 Carsillo, T., Astrinidis, A. and Henske, E.P. (2000) Mutations in the tuberous sclerosis complex gene TSC2 are a cause of sporadic pulmonary lymphangioleiomyomatosis. Proc. Natl Acad. Sci. USA, 97, 6085–6090.
27 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.
28 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., 58, 4766–4770.
29 Nellist, M., van Slegtenhorst, M.A., Goedbloed, M., van den Ouweland, A.M.W., Halley, D.J.J. and van der Sluijs, P. (1999) Characterization of the cytosolic tuberin–hamartin complex—tuberin is a cytosolic chaperone for hamartin. J. Biol. Chem., 274, 35647–35652.
30 Gutmann, D.H., Zhang, Y.J., Hasbani, M.J., Goldberg, M.P., Plank, T.L. and Henske, E.P. (2000) Expression of the tuberous sclerosis complex gene products, hamartin and tuberin, in central nervous system tissues. Acta Neuropathol. (Berl.), 99, 223–230.[Medline]
31 Catania, M.G., Johnson, M.W., Liau, L.M., Kremen, T.J., deVellis, J.S. and Vinters, H.V. (2001) Hamartin expression and interaction with tuberin in tumor cell lines and primary cultures. J. Neurosci. Res., 63, 276–283.[Web of Science][Medline]
32 Aicher, L.D., Campbell, J.S. and Yeung, R.S. (2001) Tuberin phosphorylation regulates its interaction with hamartin: two proteins involved in tuberous sclerosis. J. Biol. Chem., 276, 21017–21021.
33 Benvenuto, G., Li, S.W., Brown, S.J., Braverman, R., Vass, W.C., Cheadle, J.P., Halley, D.J.J., Sampson, J.R., Wienecke, R. and DeClue, J.E. (2000) 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, 19, 6306–6316.[Web of Science][Medline]
34 Jones, A.C., Shyamsundar, M.M., Thomas, M.W., Maynard, J., Idziaszczyk, S., Tomkins, S., Sampson, J.R. and Cheadle, J.P. (1999) Comprehensive mutation analysis of TSC1 and TSC2—and phenotypic correlations in 150 families with tuberous sclerosis. Am. J. Hum. Genet., 64, 1305–1315.[Web of Science][Medline]
35 Niida, Y., Lawrence-Smith, N., Banwell, A., Hammer, E., Lewis, J., Beauchamp, R.L., Sims, K., Ramesh, V.L. and Ozelius, L. (1999) Analysis of both TCS1 and TSC2 for germline mutations in 126 unrelated patients with tuberous sclerosis. Hum. Mutat., 14, 412–422.[Web of Science][Medline]
36 Choy, Y.S., Dabora, S.L., Hall, F., Ramesh, V., Niida, Y., Franz, D., Kasprzyk-Obara, J., Reeve, M.P. and Kwiatkowski, D.J. (1999) Superiority of denaturing high performance liquid chromatography over single-stranded conformation and conformation-sensitive gel electrophoresis for mutation detection in TSC2. Ann. Hum. Genet., 63, 383–391.[Web of Science][Medline]
37 van Slegtenhorst, M., Verhoef, S., Tempelaars, A., Bakker, L., Wang, Q., Wessels, M., Bakker, R., Nellist, M., Lindhout, D., Halley, D. and van den Ouweland, A. (1999) Mutational spectrum of the TSC1 gene in a cohort of 225 tuberous sclerosis complex patients: no evidence for genotype–phenotype correlation. J. Med. Genet., 36, 285–289.
38 Plank, T.L., Logginidou, H., Klein-Szanto, A. and Henske, E.P. (1999) The expression of hamartin, the product of the TSC1 gene, in normal human tissues and in TSC1- and TSC2-linked angiomyolipomas. Mod. Pathol., 12, 539–545.[Web of Science][Medline]
39 Fukuda, T., Kobayashi, T., Momose, S., Yasui, H. and Hino, O. (2000) Distribution of Tsc1 protein detected by immunohistochemistry in various normal rat tissues and the renal carcinomas of Eker rat: detection of limited colocalization with Tsc1 and Tsc2 gene products in vivo. Lab. Invest., 80, 1347–1359.[Web of Science][Medline]
40 Kobayashi, T., Mitani, H., Takahashi, R.I., Hirabayashi, M., Ueda, M., Tamura, H. and Hino, O. (1997) Transgenic rescue from embryonic lethality and renal carcinogenesis in the Eker rat model by introduction of a wild-type Tsc2 gene. Proc. Natl Acad. Sci. USA, 94, 3990–3993.
41 Prekeris, R., Hernandez, R.M., Mayhew, M.W., White, M.K. and Terrian, D.M. (1998) Molecular analysis of the interactions between protein kinase C-
and filamentous actin. J. Biol. Chem., 273, 26790–26798.
42 Bretscher, A. (1999) Regulation of cortical structure by the ezrin–radixin–moesin protein family. Curr. Opin. Cell Biol., 11, 109–116.[Web of Science][Medline]
43 Gusella, J.F., Ramesh, V., MacCollin, M. and Jacoby, L.B. (1999) Merlin: the neurofibromatosis 2 tumor suppressor. Biochim. Biophys. Acta Rev. Cancer, 1423, M29–M36.[Medline]
44 Peltomaki, P. (2001) Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum. Mol. Genet., 10, 735–740.
45 Innerarity, T.L., Weisgraber, K.H., Arnold, K.S., Mahley, R.W., Krauss, R.M., Vega, G.L. and Grundy, S.M. (1987) Familial defective apolipoprotein B-100—low-density lipoproteins with abnormal receptor-binding. Proc. Natl Acad. Sci. USA, 84, 6919–6923.
46 Hobbs, H.H., Russell, D.W., Brown, M.S. and Goldstein, J.L. (1990) The Ldl receptor locus in familial hypercholesterolemia—mutational analysis of a membrane–protein. Annu. Rev. Genet., 24, 133–170.[Web of Science][Medline]
47 Antonarakis, S.E. (1998) Recommendations for a nomenclature system for human gene mutations. Hum. Mutat., 11, 1–3.[Web of Science][Medline]
48 den Dunnen, J.T. and Antonarakis, S.E. (2000) Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum. Mutat., 15, 7–12.[Web of Science][Medline]
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