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Tuberous Sclerosis Causing Mutants of the TSC2 Gene Product A€Ect

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Tuberous Sclerosis Causing Mutants of the TSC2 Gene Product A€Ect Oncogene (2001) 20, 4904 ± 4909 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc Tuberous sclerosis causing mutants of the TSC2 gene product aect proliferation and p27 expression Thomas Soucek1,3, Margit Rosner1, Angelina Miloloza1, Marion Kubista1, Jeremy P Cheadle2, Julian R Sampson2 and Markus HengstschlaÈ ger*,1 1Obstetrics and Gynecology, University of Vienna, Prenatal Diagnosis and Therapy, WaÈhringer GuÈrtel 18-20, A-1090 Vienna, Austria; 2Institute of Medical Genetics, University Hospital of Wales, Health Park, Cardi, Wales, CF14 4XW, UK The autosomal dominant disease tuberous sclerosis hamartomas, of the brain, skin, kidneys, heart and (TSC) is caused by mutations in either TSC1 on other organs. Common clinical features are facial chromosome 9q34, encoding hamartin, or TSC2 on angio®bromas, renal angiomyolipomas, hypopigmented chromosome 16p13.3, encoding tuberin. TSC is char- macules, cardiac rhabdomyomas, and cortical tubers acterized by hamartomas that occur in many organs of and subependymal glial nodules in the brain. The aected patients and these have been considered to likely greatest source of morbidity is the brain tumors, which result from defects in proliferation control. Although the cause seizures in 80 ± 90% of aected individuals, true biochemical functions of the two TSC proteins have mental retardation in about half of aected individuals, not been clari®ed, a series of independent investigations and behavioral abnormalities (mostly autism) in over demonstrated that modulated hamartin or tuberin half of aected individuals (Gomez et al., 1999). Two expression cause deregulation of proliferation/cell cycle TSC genes have been identi®ed, TSC1 on chromosome in human, rodent and Drosophila cells. In support of 9q34 (The TSC1 Consortium, 1997) encoding a tuberin acting as a tumor suppressor, ectopic over- 130 kDa protein named hamartin and TSC2 (The expression of TSC2 has been shown to decrease European Chromosome 16 Tuberous Sclerosis Con- proliferation rates of mammalian cells. Furthermore, sortium, 1993) on chromosome 16p13.3 encoding a overexpression of TSC2 has been demonstrated to 200 kDa protein named tuberin. Published TSC trigger upregulation of the cyclin-dependent kinase mutations include large deletions/rearrangements and inhibitor p27. We report that three dierent naturally missense mutations for TSC2 and insertions, deletions, occurring and TSC causing mutations within the TSC2 nonsense and splicing mutations in both genes (recently gene elliminate neither the anti-proliferative capacity of reviewed in Young and Povey, 1998; Cheadle et al., tuberin nor tuberin's eects on p27 expression. For the 2000). Hamartin and tuberin can associate physically in ®rst time these data provide strong evidence that vivo (Van Slegtenhorst et al., 1998; Plank et al., 1998; deregulation of proliferation and/or upregulation of p27 Nellist et al., 1999). Tuberin has been reported to are not likely to be the primary/only mechanisms of possess GTPase-accelerating protein (GAP) activity for hamartoma development in TSC. These results demand the small molecular weight GTPase Rap1a (Wienecke reassessment of previous hypotheses of the pathogenesis et al., 1995) and for Rab5 aecting the control of of TSC. Oncogene (2001) 20, 4904 ± 4909. endocytosis (Xiao et al., 1997), to be involved in transcriptional regulation (Tsuchiya et al., 1996; Henry Keywords: tuberous sclerosis; TSC2; tuberin; prolifera- et al., 1998), and to aect the control of neuronal tion; p27 dierentiation (Soucek et al., 1998a). Hamartin has been demonstrated to bind ezrin/radixin/moesin pro- tein family members and to activate the Rho GTPase and actin ®bre assembly (Lamb et al., 2000). Tuberous sclerosis (TSC) is an autosomal dominant Furthermore, both tuberin and hamartin are involved phakomatosis with an estimated prevalence of about 1 in the regulation of cellular proliferation, the only in 6000 newborns. It is characterized by the occurence function so far demonstrated for both proteins. of mental retardation, epilepsy, and by the develop- Overexpression and antisense-experiments in mamma- ment of a spectrum of tumor-like growths, named lian cells revealed that high levels of tuberin have negative eects on cell proliferation, whereas down- regulation/loss of tuberin stimulates proliferation (Jin et al., 1996; Orimoto et al., 1996; Soucek et al., 1997, *Correspondence: M HengstschlaÈ ger; 1998b; Pajak et al., 1997; Pasumarthi et al., 2000). E-mail: [email protected] Gigas,aDrosophila homolog of TSC2, was reported to 3 Current address: The Salk Institute for Biological Studies, Cellular be involved in cell cycle control (Ito and Rubin, 1999). Neurobiology La Jolla, CA 92037-1099, USA Received 19 March 2001; revised 26 April 2001; accepted 10 May High ectopic levels of hamartin have also been shown 2001 to negatively regulate cellular proliferation (Miloloza et Tuberous sclerosis causing TSC2 mutants maintain anti-proliferative capacity T Soucek et al 4905 al., 2000; Benvenuto et al., 2000). However, mutations was 0.0078 for the dierences in S phase cells in the TSC genes that are causally involved in the P=0.0117. Surprisingly, overexpression of the three development of TSC in patients have not been dierent mutant clones also triggered eects in investigated in relation to eects of hamartin or logarithmically growing cells (Figure 1B,C). In no case tuberin on cellular proliferation. Answering this did the control cells show an increase in the G1 question is required to clarify the relevance of the fraction or a decline in the S phase fraction relative to functions of the proteins for the development of the overexpression of TSC2 cDNAs (the P values for the disease. dierences in G0/G1 cells compared to the control Tuberin has been proposed to harbor a leucin zipper experiments were 0.0039, 0.0039, 0.0039 for the region at amino acids 75 ± 107, coiled-coil structures at mutants 6 2/25, 6 3/5 and 6 5/2, respectively. For the amino acids 346 ± 371 and 1008 ± 1021, a rap1GAP dierences in S phase cells P(62/25)=0.0273, P(63/ homology domain spanning amino acids 1499 ± 1689 5)=0.0117, P(65/2)=0.0078). Assuming that the main and a rabaptin-binding domain at amino acids 1668 ± targets of overexpressed tuberin are the G0/G1 phase 1726 (The European Chromosome 16 Tuberous and the S phase of the mammalian cell cycle one would Sclerosis Consortium, 1993; Xiao et al., 1997; expect that in cell populations harboring already higher Maheshwar et al., 1997). As mentioned, tuberin possess numbers of G0/G1 cells the potential of tuberin to GAP activity for the GTPase Rap1a (Wienecke et al., increase the number of G0/G1 cells would be lower. 1995). Furthermore, mutation analysis of the rap1GAP Six independent transfections of Rat-1 cells grown related domain in tuberous sclerosis patients led to the under density-restricted growth conditions harboring characterization of disease associated mutations in- about 53% G0/G1 cells demonstrated that TSC2 and cluding missense mutations that were shown to occur the three mutant clones induce weaker eects on the as de novo mutations in sporadic TSC cases (Mahesh- G0/G1-S phase distribution (data not shown). war et al., 1997). Accordingly, we considered missense To exclude the possibility that overexpressed TSC2 mutations within the GAP homologous domain to be mutants mediate their anti-proliferative eects via optimal to address the question whether naturally overactivation of the endogenous wild-type tuberin occurring mutations would disrupt the anti-prolifera- we analysed tuberin-negative ®broblasts. Rat ®bro- tive capacity of tuberin. Three of such reported blasts derived from Eker rat embryos homozygous for mutations, N1643K, N1651S and N1681K, were the Eker mutation (a rearrangement in the rat homolog introduced into full-length wild-type human TSC2 of TSC2; Yeung et al., 1994; Kobayashi et al., 1995) cDNA by site directed mutagenesis, cloned into the were transiently transfected as described above for Rat- mammalian expression vector pSG5 and designated 6 1 cells. As shown by immunoblot analysis these cells do 2/25, 6 3/5 and 6 5/2, respectively. We transiently not express endogenous tuberin. Overexpression levels transfected Rat-1 ®broblasts with wild-type TSC2 in of wild-type TSC2 and of the three mutants were pSG5, with the three missense mutant clones or with comparable (Figure 2A). Investigation of DNA the empty expression vector as a negative control. Two distributions on the ¯ow cytometer revealed that days after transfection immunoblot analyses revealed transient transfection of wild-type TSC2 triggers an that the wild-type and the dierent mutant forms of increase in the proportion of G0/G1 cells from TSC2 were overexpressed to similar levels. For all 41.6+9.4% to 54.9+5.1%, accompanied by a decrease presented ®gures Western blots were chosen for which of S phase cells from 50.6+11.5% to 36.1+3.7%. Very the Ponceau-S staining revealed that the amount of similar eects could be detected after overexpression of protein loaded on each lane was about equal. This the missense TSC2 mutants as concluded from 10 together with analysing and presenting a non-speci®c independent experiments (Figure 2B) (for the dier- band was chosen to check loading (Figure 1A). After ences in G0/G1 phase cells compared to the control co-transfection with the pCMV-EGFP-Spectrin vector, P(wild-type)=0.0029, P(6 2/25)=0.001, P(6 3/5)= that expresses a membrane-associated form of the 0.0029, P(6 5/2)=0.001; for the dierences in the green ¯uorescence protein (GFP) under the control of amount of S phase cells P(wild-type)=0.001, P(6 the CMV promoter, the green emission enables 2/25)=0.001, P(6 3/5)=0.0033, P(6 5/2)=0.0049). transfected from untransfected cells to be separated Again, in no case did the control cells show an on a ¯ow cytometer.
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