Pathological mutations in TSC1 and TSC2 disrupt the interaction between hamartin and tuberin.

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.

Loss of tuberin, the tuberous-sclerosis-complex-2 gene product is associated with angiogenesis.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominantly inherited disorder associated with an alteration of the TSC2 tumor suppressor gene which encodes for the protein product tuberin. The disease is characterized by the development of hamartomas, e.g. cutaneous angiofibromas which consist of vascular cells, interstitial cells, and normal components of the skin. The Eker rat model, an animal model of inherited cancer, has been shown to carry a mutation of TSC2. METHODS: Immunohistochemical analyses of human angiofibromas were performed using antibodies directed against tuberin and angiogenic growth factors. Proliferation of human dermal microvascular endothelial cells (HDMEC) was determined after incubation with the supernatants of TSC2 (+/+) and TSC2 (-/-) rat embryonic fibroblasts (REF) that were derived from the Eker strain. RESULTS: Loss of the expression of tuberin was observed in the interstitial cells of 13 of 39 angiofibromas. The expression of tuberin was retained in the vascular cells. In all analyzed angiofibromas, the angiogenic factors bFGF, PD-ECGF, VEGF and angiogenin were detected in the interstitial cells and/or vascular cells. Expression of PDGF-B and TGF-beta1 was weak. Tissue culture supernatants from TSC2 (-/-) REF stimulated the growth of HDMEC significantly more than supernatants from TSC2 (+/+) REF. CONCLUSION: A functional loss of tuberin may stimulate vascular growth.

Epidermal growth factor receptor expression in neurofibromatosis type 1-related tumors and NF1 animal models.

We have found that EGF-R expression is associated with the development of the Schwann cell-derived tumors characteristic of neurofibromatosis type 1 (NF1) and in animal models of this disease. This is surprising, because Schwann cells normally lack EGF-R and respond to ligands other than EGF. Nevertheless, immunoblotting, Northern analysis, and immunohistochemistry revealed that each of 3 malignant peripheral nerve sheath tumor (MPNST) cell lines from NF1 patients expressed the EGF-R, as did 7 of 7 other primary MPNSTs, a non-NF1 MPNST cell line, and the S100(+) cells from each of 9 benign neurofibromas. Furthermore, transformed derivatives of Schwann cells from NF1(-/-) mouse embryos also expressed the EGF-R. All of the cells or cell lines expressing EGF-R responded to EGF by activation of downstream signaling pathways. Thus, EGF-R expression may play an important role in NF1 tumorigenesis and Schwann cell transformation. Consistent with this hypothesis, growth of NF1 MPNST lines and the transformed NF1(-/-) mouse embryo Schwann cells was greatly stimulated by EGF in vitro and could be blocked by agents that antagonize EGF-R function.

The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.

We report here that overexpression of the tuberous sclerosis-1 (TSC1) gene product hamartin results in the inhibition of growth, as well as changes in cell morphology. Growth inhibition was associated with an increase in the endogenous level of the product of the tuberous sclerosis-2 (TSC2) gene, tuberin. As overexpression of tuberin inhibits cell growth, and hamartin is known to bind tuberin, these results suggested that hamartin stabilizes tuberin and this contributes to the inhibition of cell growth. Indeed, transient transfection of TSC1 increased the endogenous level of tuberin, and transient co-transfection of TSC1 with TSC2 resulted in higher tuberin levels. The stabilization was explained by the finding that tuberin is highly ubiquitinated in cells, while the fraction of tuberin that is bound to hamartin is not ubiquitinated. Co-expression of tuberin stabilized hamartin, which is weakly ubiquitinated, in transiently transfected cells. The amino-terminal two-thirds of tuberin was responsible for its ubiquitination and for stabilization of hamartin. A mutant of tuberin from a patient missense mutation of TSC2 was also highly ubiquitinated, and was unable to stabilize hamartin. We conclude that hamartin is a growth inhibitory protein whose biological effect is likely dependent on its interaction with tuberin.

Genetic variants of the tuberous sclerosis 2 tumour suppressor gene in mouse t haplotypes.

The murine t complex on chromosome 17 contains a number of homozygous lethal and semi-lethal mutations that disrupt development of the mouse embryo. We recently characterized an embryonic lethality in the rat that results from a germ-line mutation in the tuberous sclerosis 2 (Tsc-2) tumour suppressor gene (the Eker mutation). Remarkably, mouse embryos homozygous for tw8 mutation display cranial defects reminiscent of those observed in rat embryos homozygous for the Eker mutation. To determine whether the Tsc-2 gene, which is in the t complex, is mutated in tw8 or other t haplotypes, we characterized this gene in a series of t haplotype mice. Four Tsc-2 polymorphisms were identified: three in the coding region and one intronic that appeared to be common to all t haplotypes analysed. No evidence was found to argue that the Tsc-2 gene is altered in tw8 haplotype mice. However, in the tw5 haplotype we found a G to T mutation in Tsc-2 that was present only in this t haplotype. In contrast to other polymorphisms within the Tsc-2 coding region which did not result in amino acid changes in Tsc-2 gene product tuberin, this mutation substituted a phenylalanine for a conserved cysteine in tw5 tuberin. Within the t complex, the Tsc-2 gene and the putative tw5 locus appeared to map to different positions, complicating identification of Tsc-2 as a candidate for the tw5 locus and suggesting that the G to T mutation in the Tsc-2 gene may have arisen independently of the tw5 functional mutation.

Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf.

The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C. L. Farnsworth et al., Nature 376:524-527, 1995; N. P. Fam et al., Mol. Cell. Biol. 17:1396-1406, 1996). Each GRF protein contains a Dbl homology (DH) domain. A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1. In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain. Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction. Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers. Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type. Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive. Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells. In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP. We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.

Tuberous sclerosis-related gene expression in normal and dysplastic brain.

Cortical dysplasia (CD) broadly defines a complex cerebral malformative lesion associated clinically with intractable, pharmacoresistant epilepsy (including infantile spasms), especially in infants and children. In CD, the spectrum of structural brain abnormalities includes (at a minimum) neuronal dyslamination and (in severe cases) neuronal cytomegaly with cytoskeletal alterations and the presence of gemistocyte-like 'balloon cells'. In some CD variants, the neuropathological features are essentially indistinguishable from those of a tuber of tuberous sclerosis (TSC). Two genes associated with the autosomal dominant, multi-system disorder TSC have recently been cloned: TSC2 (on chromosome 16p13.3) encodes the protein tuberin and TSC1 (on 9q34) encodes hamartin. Tuberin has been immunolocalized to neurons and possibly astrocytes in normal brain and CD/TSC tubers, and is widely expressed in normal viscera; loss of heterozygosity and tissue culture studies suggest it functions as a growth suppressor. The TSC1 gene has been cloned within the last year and hamartin as yet has no well-defined cellular function, though its protein product may also function as a growth suppressor. This article focuses on the cellular pathogenesis of CD and TSC brain lesions and how the two may be biologically related. Studies of how TSC1 and TSC2 function in normal and dysplastic cerebral neocortex may provide a paradigm for understanding the neurobiology of other genes that determine epilepsy-associated cerebral malformations (e.g. lissencephaly, double cortex).

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.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription factor E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically growing cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested fibroblasts to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.

Alterations in the rap1 signaling pathway are common in human gliomas.

Several inherited predisposition to cancer syndromes are associated with the development of nervous system tumors. Tuberous sclerosis complex (TSC) is an autosomal dominant disorder in which affected individuals are at risk for developing astrocytomas. One of the genes responsible for this disorder is TSC2, located on chromosome 16p, and encoding a 180 kDa protein (tuberin) that functions in part as a negative regulator of rap1. Previous studies from our laboratory demonstrated that 30% of sporadic astrocytomas have reduced or absent tuberin expression. In addition to loss of tuberin in sporadic astrocytomas, aberrant rap1 mediated signaling may also result from overexpression of rap1. In this study, we test the hypothesis that alterations in the rap1 signaling pathway are frequently observed in certain subsets of gliomas compared to other tumors of the nervous system. Analysis of sporadic astrocytomas and ependymomas demonstrated either increased rap1 or reduced/absent tuberin protein expression in 50-60% of different cohorts of these gliomas, compared to 30-33% of sporadic schwannomas and meningiomas and none of eight oligodendrocyte tumors. These results suggest that alterations in the rap1 signaling pathway are important in the development of certain sporadic human gliomas.

Reduced TSC2 RNA and protein in sporadic astrocytomas and ependymomas.

Individuals affected with tuberous sclerosis complex (TSC) develop several benign and malignant tumors at increased frequency, including astrocytomas. Tuberin, the protein product of the tuberous sclerosis complex-2 (TSC2) tumor suppressor gene, has been shown to directly inhibit cell growth and is expressed at high levels in normal central nervous system neurons and astrocytes. To determine whether TSC2 RNA and protein are reduced in astrocytomas from individuals without tuberous sclerosis, reverse transcriptase-polymerase chain reaction and immunoblotting analyses were performed on 49 adult astrocytomas, 10 pediatric astrocytomas, and 13 ependymomas. Eighteen of 40 (45%) high-grade (World Health Organization [WHO] grade III/IV) astrocytomas and 4 of 8 (50%) adult low-grade (WHO grade II) astrocytomas demonstrated reduced or absent TSC2 expression, including 1 giant cell astrocytoma, whereas none of the 10 pediatric low-grade astrocytomas analyzed showed a reduction in TSC2 expression. Reduced or absent tuberin was observed in 2 of 6 (33%) ependymomas analyzed. These data demonstrate, for the first time, that reduced or absent TSC2 expression may represent one of the critical genetic events associated with the development of sporadic adult, but not pediatric, astrocytomas.

Tuberous sclerosis in a 20-week gestation fetus: immunohistochemical study.

We report an autopsy case of tuberous sclerosis complex (TSC) in a 20-week gestational age female fetus. The brain showed lesions suggestive of early cortical tubers and subependymal hamartomatous nodules. The large cells within these nodular clusters were variably immunoreactive for glial fibrillary acidic protein (GFAP) and vimentin and negative for synaptophysin and neurofilament. Subependymal radial glia expressed both vimentin and GFAP, but subpial radial glia either did not express these markers (in contrast to an age-matched control) or were absent. Tuberin expression was noted in heterotopic neurons in the white matter and brain cells consistent with Cajal Retzius cells in the neocortical molecular layer, very weakly in superficial cortical neurons, neurons in the basal ganglia, Purkinje cells and external granular cells of cerebellum, cranial nerve nuclei neurons, occasional germinal matrix cells, ependymal cells, choroid plexus epithelium, and pituitary gland neuroendocrine cells; it was not seen within the cells of subependymal nodules. The pattern of tuberin immunoreactivity was similar to that which we have observed in older TSC patients. Proliferating cell labeling indexes were comparable in the germinal matrix of the TSC patient and an age-matched control. Abnormal subpial radial glia may be responsible for some of the neuronal migration abnormalities that appear to result in neocortical tubers.

cAMP-dependent protein kinase A is required for Schwann cell growth: interactions between the cAMP and neuregulin/tyrosine kinase pathways.

Schwann cell proliferation is stimulated by contact with neurons or exposure to growth factor ligands for tyrosine kinase receptors, effects of which are potentiated by cAMP. Here we show that treatment of rat Schwann cells with recombinant human glial growth factor 2 (rhGGF2), but not with other mitogenic factors, transiently increases intracellular cyclic AMP (cAMP), with maximal elevation at the G0/G1 boundary. The cAMP-dependent protein kinase (PKA) inhibitor H-89 strongly antagonized GGF- and neuron-induced Schwann cell proliferation, with maximum inhibition observed at G0/G1. H-89 also inhibited Schwann cell proliferation induced by growth factors that did not increase intracellular cAMP. Stimulation of Schwann cells with rhGGF2 resulted in 70-fold activation of MAP kinase; forskolin treatment resulted in a 50% decrease in MAP kinase activity but did not alter Raf-1 phosphorylation on Ser-43. These results demonstrate that the MAP kinase cascade represents an intersection between receptor tyrosine kinase and cAMP signaling pathways in Schwann cells and that PKA plays a critical role in Schwann cell cycle progression.

Expression of the TSC2 product tuberin and its target Rap1 in normal human tissues.

The tuberous sclerosis-2 (TSC2) gene is linked to tuberous sclerosis (TSC), a dominantly inherited genetic syndrome in which inactivation of the normal TSC2 allele is associated with the development of mostly benign tumors and focal dysplasias. TSC2 encodes the protein tuberin, which is a widely expressed 180-kd polypeptide that exhibits specific GTPase activating activity toward Rap1 in vitro and co-localizes with Rap1 in cultured cells. In this study, we have performed immunohistochemical analyses, using affinity-purified anti-tuberin antibodies, to study the distribution of tuberin in a panel of normal human organs that are commonly affected by TSC. Cryosections indicated that tuberin is widely expressed at low levels. More intense staining of tuberin, in the cryosections and in paraffin sections, was observed in the small blood vessels of many organs, including the kidney, skin, and adrenal gland. High levels of tuberin were also detected in cortical neurons and cerebellar Purkinje cells. These findings imply that loss-of-function mutations in TSC2 might lead to the development of highly vascularized tumors, subcortical tubers, and focal atrophy of the cerebellar cortex, which are features commonly associated with TSC. Moreover, Rap1 was also found to be highly expressed in many of the same cells that contained high levels of tuberin, suggesting a functional interaction between tuberin and Rap1 in these tissues.

Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility.

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.

Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.

Localization of tuberous sclerosis 2 mRNA and its protein product tuberin in normal human brain and in cerebral lesions of patients with tuberous sclerosis.

Tuberous sclerosis (TSC), an autosomal dominant disorder, is characterized by malformations, hamartomas and tumors in various organs including the brain. TSC is genetically linked to two loci: TSC1 on chromosome 9q34 and TSC2 on 16p13.3. TSC2 has been cloned, sequenced and encodes a protein (tuberin) which functions as a tumor suppressor. We have analyzed the distribution of TSC2 mRNA and tuberin in the brains of TSC patients and non-affected individuals using both autopsy and biopsy material. High levels of transcript and protein expression were observed in choroid plexus epithelium, ependymal cells, most brainstem and spinal cord motor neurons, Purkinje cells and the external granule cell layer of the cerebellum in both TSC and control cases. Individual balloon cells from TSC patients showed very faint expression while other glia showed no expression of either transcript or tuberin. Neocortical and hippocampal neurons expressed high levels of TSC2 transcript, but only modest levels of tuberin. The internal granule cell layer of the cerebellum expressed abundant transcript but low levels of tuberin. These observations suggest either that tuberin expression is controlled at the level of both transcription and translation or the antibody and in-situ hybridization recognize different splice variants of the TSC2 gene. In TSC patients, dysmorphic cytomegalic neurons expressed high levels of tuberin and transcript, particularly when in an 'ectopic' location. Individual cells within subependymal giant cell astrocytomas (SEGAs) and hamartomas from TSC patients expressed moderate to high levels of TSC2 transcript and tuberin. While the TSC2 transcript is widely expressed primarily within neurons, tuberin is demonstrable primarily within dysplastic/cytomegalic cells of the cortex and subependymal hamartomas/SEGAs. CNS expression of tuberin is unique in that primarily non-dividing cells express it in this location, whereas extra-CNS expression of tuberin is mainly found in actively proliferating cell types such as epithelium.

Co-localization of the TSC2 product tuberin with its target Rap1 in the Golgi apparatus.

Tuberin is the protein product of the tuberous sclerosis-2 (TSC2) gene, which is associated with tuberous sclerosis (TSC), a human genetic syndrome characterized by the development of tumors in a variety of tissues. We have previously shown that tuberin is a widely expressed 180 kDa protein which exhibits specific GTPase activating activity in vitro towards the Ras-related Rap1 protein. In this study we have used affinity-purified antibodies against tuberin to analyse its expression in human and rat tissues and to examine its subcellular localization. Tuberin expression was detected in all adult human tissues tested, with the highest levels found in brain, heart and kidney, organs that are commonly affected in TSC patients. By contrast, in adult rats the highest levels of tuberin were found in brain, liver and testis. Indirect immunofluorescence of tuberin in various cultured cell lines revealed a punctate, mostly perinuclear staining pattern. Double-indirect immunofluorescence analysis with anti-tuberin sera and antisera against known Golgi markers (mannosidase-II and furin) revealed that the staining of tuberin was consistent with its localization in the stacks of the Golgi apparatus. In support of this, treatment of cells with brefeldin A, a drug known to cause disassembly of the Golgi apparatus, abolished the perinuclear staining of tuberin. Moreover, conventional and confocal immunofluorescence demonstrated co-localization of tuberin with Rap1, which has previously been localized to the Golgi apparatus. The co-localization of tuberin and Rap1 in vivo strengthens the likelihood that the in vitro catalytic activity of tuberin toward Rap1 plays a physiologically relevant role in the tumor suppressor function of tuberin.

Suppression of tumorigenicity by the wild-type tuberous sclerosis 2 (Tsc2) gene and its C-terminal region.

The Tsc2 gene, which is mutationally inactivated in the germ line of some families with tuberous sclerosis, encodes a large, membrane-associated GTPase activating protein (GAP) designated tuberin. Studies of the Eker rat model of hereditary cancer strongly support the role of Tsc2 as a tumor suppressor gene. In this study, the biological activity of tuberin was assessed by expressing the wild-type Tsc2 gene in tumor cell lines lacking functional tuberin and also in rat fibroblasts with normal levels of endogenous tuberin. The colony forming efficiency of Eker rat-derived renal carcinoma cells was significantly reduced following reintroduction of wild-type Tsc2. Tumor cells expressing the transfected Tsc2 gene became more anchorage-dependent and lost their ability to form tumors in severe combined immunodeficient mice. At the cellular level, restoration of tuberin expression caused morphological changes characterized by enlargement of the cells and increased contact inhibition. As with the full-length Tsc2 gene, a clone encoding only the C terminus of tuberin (amino acids 1049-1809, including the GAP domain) was capable of reducing both colony formation and in vivo tumorigenicity when transfected into the Eker rat tumor cells. In normal Rat1 fibroblasts, conditional overexpression of tuberin also suppressed colony formation and cell growth in vitro. These results provide direct experimental evidence for the tumor suppressor function of Tsc2 and suggest that the tuberin C terminus plays an important role in this activity.

Schwann cells from neurofibromin deficient mice exhibit activation of p21ras, inhibition of cell proliferation and morphological changes.

Schwann cells are thought to be abnormal in type 1 neurofibromatosis (NF1) and to contribute to the formation of benign and malignant tumors in this disease. To test the role of the NF1 gene product neurofibromin as a Ras-GTPase activating protein in Schwann cells, and to study the effect of the loss of neurofibromin on Schwann cell proliferation, we isolated Schwann cells from mice with targeted disruption of NF1. The properties of these neurofibromin deficient cells were strikingly similar to those of v-ras expressing rat Schwann cells with normal levels of neurofibromin. The similarities included: growth inhibition, noted as a decrease in cell division in response to glial growth factor 2 (GGF2) and of neuronal contact; morphological changes such as the appearance of elaborated processes; and elevated levels of Ras-GTP. Furthermore, Ras-GTP levels in the neurofibromin deficient Schwann cells were consistently elevated in response to GGF2 treatment. In contrast to these results, introduction of v-ras into a Schwannoma cell line (RN22) led to cell transformation. We conclude that neurofibromin functions as a major regulator of Ras-GTP in Schwann cells; however, mutation in NF1 by itself is unlikely to explain the hyperplasia observed in Schwann cell tumors in NF1 disease.

Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity.

Tuberous sclerosis (TSC) is a human genetic syndrome characterized by the development of benign tumors in a variety of tissues, as well as rare malignancies. Two different genetic loci have been implicated in TSC; one of these loci, the tuberous sclerosis-2 gene (TSC2), encodes an open reading frame with a putative protein product of 1784 amino acids. The putative TSC2 product (tuberin) contains a region of limited homology to the catalytic domain of Rap1GAP. We have generated antisera against the N-terminal and C-terminal portions of tuberin, and these antisera specifically recognize a 180-kDa protein in immunoprecipitation and immunoblotting analyses. A wide variety of human cell lines express the 180-kDa tuberin protein, and subcellular fractionation revealed that most tuberin is found in a membrane/particulate (100,000 x g) fraction. Immunoprecipitates of native tuberin contain an activity that specifically stimulates the intrinsic GTPase activity of Rap1a. These results were confirmed in assays with a C-terminal fragment of tuberin, expressed in bacteria or Sf9 cells. Tuberin did not stimulate the GTPase activity of Rap2, Ha-Ras, Rac, or Rho. These results suggest that the loss of tuberin leads to constitutive activation of Rap1 in tumors of patients with tuberous sclerosis.