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• W1971931044 abstract "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 × 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. 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 × 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. Members of the Ras-related family of small GTP-binding proteins are involved in many different biological functions(1Downward J. Trends Biochem. Sci. 1990; 15: 469-472Abstract Full Text PDF PubMed Scopus (145) Google Scholar, 2Bourne H.R. Sanders D.A. McCormick F. Nature. 1990; 348: 125-132Crossref PubMed Scopus (1844) Google Scholar). These proteins, which bind and hydrolyze GTP, are active when bound to GTP and inactive in the GDP-bound state(3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 4Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). Guanine nucleotide binding by the Ras-related GTPases is regulated by cellular enzymes. Cellular regulatory proteins include both positive regulators, the guanine nucleotide exchange factors and negative regulators, known as GTPase accelerating proteins (GAPs)1( 1The abbreviations used are: GAPGTPase accelerating proteinsNF1neurofibromatosis type 1TSCtuberous sclerosisTub-Ntuberin which encodes amino acids Ala-2 to Ala-306Tub-Ctuberin which encodes amino acids Leu-1387 to Val-1784GSTglutathione S-transferaseDHdbl-homology regionPBSphosphate-buffered salineDTTdithiothreitolPAGEpolyacrylamide gel electrophoresis.) (5Bollag G. McCormick F. Annu. Rev. Cell Biol. 1991; 7: 601-632Crossref PubMed Scopus (289) Google Scholar). The GAP proteins act by stimulating the intrinsic GTPase of the GTP-binding proteins, keeping them in the inactive, GDP-bound state(4Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar). GTPase accelerating proteins neurofibromatosis type 1 tuberous sclerosis tuberin which encodes amino acids Ala-2 to Ala-306 tuberin which encodes amino acids Leu-1387 to Val-1784 glutathione S-transferase dbl-homology region phosphate-buffered saline dithiothreitol polyacrylamide gel electrophoresis. The Ras subfamily of small molecular weight GTPases is known to regulate mitogenic signal transduction pathways linking plasma membrane receptors to the nucleus. Thus Ras proteins are critical in controlling the proliferation of many diverse cell types(4Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1127) Google Scholar, 6Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1198) Google Scholar). The critical role of GAP proteins in regulating the activity of the Ras-like GTPases has been demonstrated by studies of the human genetic disease, neurofibromatosis type 1 (NF1). The product of the NF1 tumor suppressor gene, neurofibromin, acts as a specific GAP for Ras proteins (3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). Loss of neurofibromin expression is associated with the constitutive activation of Ras in cell lines derived from tumors of patients with NF1(7Basu T.N. Gutmann D.H. Fletcher J.A. Glover T.W. Collins F.S. Downward J. Nature. 1992; 356: 713-715Crossref PubMed Scopus (580) Google Scholar, 8DeClue J.E. Papageorge A.G. Fletcher J.A. Diehl S.R. Ratner N. Vass W.C. Lowy D.R. Cell. 1992; 69: 265-273Abstract Full Text PDF PubMed Scopus (517) Google Scholar). Tuberous sclerosis (TSC) presents many striking parallels with NF1, including autosomal dominant inheritance, the appearance of benign tumors and other abnormalities in multiple organ systems, and development of rare malignancies in affected individuals(9Gomez M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar). Unlike NF1, TSC has been linked to two different loci, TSC2 on chromosome 16, and an unidentified gene on chromosome 9 (TSC1)(10Janssen B. Sampson J. van der Est M. Deelen W. Verhoef S. Daniels I. Hesseling A. Brook-Carter P. Nellist M. Lindhout D. Hum. Genet. 1994; 94: 437-440Crossref PubMed Scopus (35) Google Scholar, 11Kwiatkowski D.J. Armour J. Bale A.E. Fountain J.W. Goudie D. Haines J.L. Knowles M.A. Pilz A. Slaugenhaupt S. Povey S. Cytogenet. Cell Genet. 1993; 64: 94-103Crossref Scopus (28) Google Scholar). Loss of heterozygosity at the TSC2 locus has been demonstrated in tumors of human TSC patients, strongly suggesting that this gene functions as a tumor suppressor(12Green A.J. Smith M. Yates J.R.W. Nature Genet. 1994; 6: 193-196Crossref PubMed Scopus (330) Google Scholar). This implication has been substantiated by studies of the Eker rat strain, in which susceptibility to bilateral renal cell carcinoma is caused by a germline mutation in the rat TSC2 gene, and tumor development is associated with somatic loss of heterozygosity at TSC2(13Yeung R.S. Xiao G.-H. Jin F. Lee W.-C. Testa J.R. Knudson A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (273) Google Scholar, 14Kobayashi T. Hirayama Y. Kobayashi E. Kubo Y. Hino O. Nat. Genet. 1995; 9: 70-74Crossref PubMed Scopus (293) Google Scholar). The 5.5-kilobase transcript of TSC2 is widely expressed, consistent with the multiple organs affected in TSC, including the brain, heart, skin, kidneys, lungs, and others(9Gomez M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar, 13Yeung R.S. Xiao G.-H. Jin F. Lee W.-C. Testa J.R. Knudson A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (273) Google Scholar, 15Cell The European Chromosome 16 Tuberous Sclerosis Consortium. 1993; 75: 1305-1313Google Scholar). The predicted TSC2 product, designated tuberin, comprises 1784 amino acids. A 58-amino acid region near the C terminus has limited homology (19 identical amino acids) with a portion of the catalytic domain of Rap1GAP(15Cell The European Chromosome 16 Tuberous Sclerosis Consortium. 1993; 75: 1305-1313Google Scholar). Rap1GAP acts as a negative regulator of the Rap1a and Rap1b GTPases, which share greater than 50% amino acid identity with Ras(16Pizon V. Lerosey I. Chardin P. Tavitian A. Nucleic Acids Res. 1988; 16: 7719Crossref PubMed Scopus (100) Google Scholar, 17Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). Although originally identified as an antagonist of Ras transformation (see “Discussion”), Rap1 (also referred to as K-rev1 and smg p21) has been shown to induce DNA synthesis and morphological changes when microinjected into Swiss 3T3 fibroblast cells(18Yoshida Y. Kawata M. Miura Y. Musha T. Sasaki T. Kikuchi A. Takai Y. Mol. Cell. Biol. 1992; 12: 3407-3414Crossref PubMed Scopus (102) Google Scholar). These results demonstrated that like Ras, the Rap1 proteins may act as positive mitogenic signaling molecules. The homology between tuberin and Rap1GAP suggests a possible molecular similarity between neurofibromin, a regulatory GAP for Ras, and tuberin, a potential regulator of Rap1. Investigation of this possibility has awaited the identification and characterization of the TSC2 product. To address these questions, we have raised antisera to bacterial fusion proteins encoding the N-terminal and C-terminal regions of tuberin. Immunoprecipitation and immunoblotting with these antisera have identified tuberin as a widely expressed 180-kDa molecule, with associated GAP activity that is specific for Rap1. These findings have potential implications for the tumor suppressor function of TSC2. Two cDNA fragments encoding regions of the human TSC2 gene were obtained by polymerase chain reaction amplification from a fetal brain cDNA library (Clontech)(19Tung J.-S. Daugherty B.L. O'Neill L. Law S.W. Han J. Mark G.E. Erlich H.A. PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York1989: 99-104Crossref Google Scholar). One encodes amino acids Ala-2 to Ala-306 of tuberin (Tub-N), and the other encodes amino acids Leu-1387 to Val-1784 (Tub-C)(15Cell The European Chromosome 16 Tuberous Sclerosis Consortium. 1993; 75: 1305-1313Google Scholar). The DNA fragments were cloned into the vector pGEX-4T2 (Pharmacia Biotech Inc.) using BamHI and SalI, for expression as glutathione S-transferase (GST) fusion proteins. The DNA sequences of the fragments were verified by direct DNA sequencing (U. S. Biochemical Corp.). For control purposes, a region of the mouse CDC25mM/GRF gene, comprising the dbl-homology (DH) region (amino acids Arg-211 to Leu 512), was also cloned into pGEX-4T2(20Cen H. Papageorge A.G. Zippel R. Lowy D.R. Zhang K. EMBO J. 1992; 11: 4007-4015Crossref PubMed Scopus (97) Google Scholar). For expression as a GST fusion protein in Sf9 cells, the fragment encoding Tub-C was subcloned into the baculovirus transfer vector pAcG2T (Pharmingen) using BamHI and EcoRI. All cell lines were obtained from the American Type Tissue Collection, and were cultured according to the conditions supplied. HB101 Escherichia coli cells bearing the pGEX-4T2 plasmids encoding GST-Tub-N, GST-Tub-C, or GST-DH were grown to an optical density of 1.0 (600 nm) and induced with 50 μM isopropyl-β-D-thiogalactopyranoside for 2 h at 28°C. The cells were pelleted, resuspended in 1/50 volume of phosphate-buffered saline (PBS), 0.1% Nonidet P-40, and 1 mM dithiothreitol (DTT), and lysed by sonication. Sonicated lysates were centrifuged at 15,000 × g to pellet insoluble material. Aliquots of the supernatants were used immediately for experiments measuring GAP activity. To estimate the concentration of the GST fusion proteins in the lysates, glutathione-coupled Sepharose beads (Pharmacia) were added to an aliquot of the supernatant, incubated for 2 h at 4°C, washed four times with PBS, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) together with purified carbonic anhydrase (Sigma) as a standard. The gels were stained with Coomassie Brilliant Blue-G (BDH). To isolate fusion proteins present in inclusion bodies, the bacteria were induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 3 h at 37°C. Bacteria were lysed by sonication in PBS, 1% Nonidet P-40, and 1 mM DTT. Unlysed cells were removed by low-speed centrifugation, and the inclusion bodies were pelleted by 15,000 × g for 15 min, washed twice with PBS, 1% Nonidet P-40, 1 mM DTT, and 2 M urea, and repelleted. Concentration and purity were estimated by SDS-PAGE. The purity of GST-Tub-N and GST-Tub-C in these preparations was 80-90%. Acetone powder preparations of HB101 cells expressing GST-Tub-N, GST-Tub-C, and GST-DH were prepared as described(21Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988Google Scholar). 300-400 μg of purified GST-Tub-N and GST-Tub-C (in the form of inclusion bodies) were used to immunize New Zealand White rabbits with Freund's adjuvant. Booster injections were carried out 3, 5, and 9 weeks later. For use in Western blotting, immunoglobulin G (IgG) was isolated from the sera of rabbits immunized with Tub-C by binding to protein A, and conjugated with digoxigenin-3-O-methylcarbonyl-ϵ-aminocaproic acid-N-hydroxysuccinimidine ester according to the manufacturer's instructions (Boehringer Mannheim). For analysis by combined immunoprecipitation/Western blotting, 107 cells per sample lane (in a volume of 300 μl) were used. Cell lysis and immunoprecipitation were performed as described (22DeClue J.E. Cohen B.D. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9914-9918Crossref PubMed Scopus (179) Google Scholar), using 10 μl of antiserum (a 1:30 dilution). The samples were subjected to SDS-PAGE on 6% gels, and proteins were electroblotted onto an Immobilon-P membrane (Millipore). For blocking experiments, antisera were incubated with acetone powder suspensions (0.1% w/v) of bacterial proteins for 2 h at 4°C, then clarified by centrifugation before use. Following electroblotting, the membrane was blocked overnight with Tris-buffered saline, 0.1% Nonidet P-40, and 3% bovine serum albumin. After blocking, the membrane was incubated with 15 μg/ml digoxigenin-labeled anti-Tub-C IgG (for Fig. 2a, 2c, 3, and 4). Immunoblotted proteins were detected by incubation with sheep anti-digoxigenin Fab--alkaline phosphatase conjugate, and chemiluminescence substrate Lumi-Phos530 (Boehringer Mannheim). The immunoblot shown in Fig. 2b employed different nonconjugated antisera at a dilution of 1:2,000, as indicated. This blot was developed with an anti-rabbit peroxidase-based enhanced chemiluminescence detection kit (Kirkegaard and Perry). To estimate the molecular weight of tuberin, both prestained molecular weight standards (BRL) and unstained standards (Pharmacia) were employed. Sf9 cells were co-transfected with the baculovirus transfer vector pAcG2T-GST-Tub-C and BaculoGold baculovirus DNA (Pharmingen), and recombinant baculoviruses encoding the GST-Tub-C protein were isolated and enriched according to the manufacturer's protocol. Sf9 cells (3 × 107) were infected with virus (encoding either GST-Tub-C, or bovine papilloma virus L1 protein as a control), and were harvested after 50 h. Cells were washed with GAP assay buffer, and lysed by sonication in 1 ml of GAP assay buffer. Lysates were clarified by centrifugation, and the concentration of GST-Tub-C was estimated as for bacterial lysates. 1.4 × 108 K-562 cells were pelleted, washed with PBS, and split in two. One portion of the cells was lysed with 2 ml of lysis buffer, while the other aliquot was used to prepare the 100,000 × g supernatant (S100) and particulate/membrane (P100) fractions, as described previously(22DeClue J.E. Cohen B.D. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9914-9918Crossref PubMed Scopus (179) Google Scholar). Equal portions of the S100, P100, and the lysis-buffer lysates were subjected to immunoprecipitation and immunoblotting as described above. For each GAP assay reaction with immune complexes of tuberin, the anti-Tub-C serum was used to immunoprecipitate tuberin from lysates of 4 × 106 K-562, prepared in 120 μl of lysis buffer. Immunoprecipitates were bound to protein A-Sepharose beads, and the immune complexes were washed three times with lysis buffer, then three times with GAP assay buffer (20 mM Tris·HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, 1 mM DTT, and 40 μg/ml bovine serum albumin). Then the beads were resuspended in GAP assay buffer in one-fourth of the original volume. GAP assays were carried out essentially as described(23Zhang K. Papageorge A.P. Martin P. Vass W.C. Olah Z. Polakis P. McCormick F. Lowy D.R. Science. 1991; 254: 1630-1634Crossref PubMed Scopus (30) Google Scholar). Briefly, 0.5 μM GTP-binding proteins were loaded with 5 μM [γ-32P]GTP in loading buffer (0.1 M sodium phosphate, 5 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, and 50 μg/ml sodium deoxycholate), in a volume of 10 μl, then diluted into 600 μl of cold GAP assay buffer with 10 μM unlabeled GTP. 50 μl of this mixture was then added to the different tuberin or GST-Tub-C preparations. Assays included 30 μl of native tuberin bound to protein A-Sepharose beads (isolated from 4 × 106 K-562 cells), 10 μl of crude (200 nM GST-Tub-C) bacterial lysate, or (1 μM GST-Tub-C) Sf9 cell lysate. Reactions were carried out in duplicate at 32°C, and 10-μl portions of each reaction were removed at the start or after the indicated times. Remaining p21-bound [γ-32P]GTP was determined by nitrocellulose filter binding. H6-tagged Ha-Ras and Rap1a, purified from Sf9 cells, were provided by Dr. Albert Reynolds; bacterial Rho and Rac were provided by Dr. Alan Hall. Rap2 was expressed as described in HB101 cells containing a rap2 expression plasmid, provided by Dr. Jean de Gunzburg(24Lerosey I. Chardin P. de Gunzburg J. Tavitian A. J. Biol. Chem. 1991; 266: 4315-4321Abstract Full Text PDF PubMed Google Scholar). To allow for the specific detection of tuberin, antisera were raised against an N-terminal and a C-terminal region of the predicted protein product (designated anti-Tub-N and anti-Tub-C, respectively) (Fig. 1). Extracts of the K-562 erythroleukemia cell line were subjected to immunoprecipitation with anti-Tub-N, unrelated or preimmune sera. After SDS-PAGE analysis, immunoprecipitates were transferred to a filter, and the filter was incubated with anti-Tub-C serum (Fig. 2a). We observed the presence of a specific band of 180 kDa in the anti-Tub-N immunoprecipitates. Next, extracts of the G-401 Wilms tumor cell line were subjected to immunoprecipitation with either anti-Tub-C or anti-Tub-N sera, and the immunoprecipitates were blotted. The blot was cut into strips, which were incubated with preimmune, anti-Tub-C, or unrelated serum (Fig. 2b). Again, a specific band of 180 kDa was detected in the anti-Tub-N and anti-Tub-C immunoprecipitates, but only when the blot was probed with anti-Tub-C serum (the anti-Tub-N serum did not bind to tuberin in immunoblot analyses). The observed migration of this band is close to the predicted molecular mass of tuberin (198 kDa)(15Cell The European Chromosome 16 Tuberous Sclerosis Consortium. 1993; 75: 1305-1313Google Scholar). Furthermore, the combined immunoprecipitation/immunoblot analysis provides a high degree of certainty that the observed protein is in fact tuberin, since antisera against different regions of tuberin were employed for immunoprecipitation and for immunoblotting. As additional proof of specificity, a significant reduction in the intensity of the 180-kDa band was observed following preincubation of the anti-Tub-N serum with an acetone powder extract prepared from E. coli expressing GST-Tub-N, but not by preincubation with acetone powder preparations of E. coli expressing GST-Tub-C or GST-DH (Fig. 2c). Immunoprecipitation of extracts from [35S]methionine-labeled K-562 and G-401 cells with these antisera again revealed a 180-kDa band, which could be specifically blocked by preadsorption with acetone powder preparations from E. coli expressing the cognate fusion protein, but not by preadsorption with control acetone powder preparations (data not shown). Immunoblot analyses performed without prior immunoprecipitation also revealed a 180-kDa protein in the cell lysates. However, the background when either procedure was employed singly was higher than in the combined immunoprecipitation/immunoblot analyses, which led us to use the latter procedure in most of the subsequent experiments. To determine the subcellular localization of tuberin, K-562 cells were lysed by Dounce homogenization in the absence of detergent. The postnuclear supernatant was centrifuged at 100,000 × g to yield membrane/particulate (P100) and nonparticulate (S100) fractions. Control lysates from an equal amount of cells were prepared with detergent, and each of the fractions was analyzed by immunoprecipitation (with anti-Tub-N, preimmune, or anti-Tub-C) followed by Western blotting (with anti-Tub-C). A substantial majority of the tuberin was recovered in the P100 fraction (Fig. 3), raising the possibility that tuberin may associate with the cell plasma membrane, or with intracellular membranes or organelles. The wide variety of organ systems affected in TSC patients suggests that the 180-kDa tuberin protein is expressed in many different cell types. Indeed, expression of the TSC2 mRNA in diverse cell types has been demonstrated by Northern blot analysis. We tested a panel of 13 different human cell lines for tuberin expression (Fig. 4). These lines represent a variety of cell types (see legend to Fig. 4). Cells were lysed, and portions of each lysate containing an equal amount of protein were subjected to combined immunoprecipitation/immunoblot analysis. With the exception of HL-60 and U-937 cells, tuberin was expressed in all of the cell lines tested. Interestingly, each of the tuberin-negative cell lines represents a different maturation level of the myelocytic/monocytic lineage; abnormalities in this lineage have not been linked to TSC patients(9Gomez M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar, 25Lübbert M. Herrmann F. Koeffler H.P. Blood. 1991; 77: 909-924Crossref PubMed Google Scholar). Small differences in the migration rate of tuberin were observed in some of the cell lines (e.g. SW-13 and HT-29 cells). These differences may reflect post-translational modification of tuberin, or the products of alternatively-spliced variants of the TSC2 mRNA. Splicing variants have been demonstrated for the TSC2 homolog in rats.2( 2R. S. Yeung, personal communication.) The region of tuberin bearing homology to Rap1GAP (58 amino acids) is substantially shorter than the Rap1GAP catalytic domain (331 residues), and is also shorter than the homology seen between different Ras-specific GAP proteins (about 350 residues)(3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, 26Rubinfeld B. Crosier W.J. Albert I. Conroy L. Clark R. McCormick F. Polakis P. Mol. Cell. Biol. 1992; 12: 4634-4642Crossref PubMed Scopus (49) Google Scholar). However, if tuberin does indeed function as a GAP for Rap1, then immunoprecipitates of tuberin should contain this type of activity. Therefore, native tuberin was isolated by immunoprecipitation from K-562 cell lysates, and the immunoprecipitates were incubated with [γ-32P]GTP-bound Rap1a to assay potential GAP activity (Fig. 5a). We observed significant levels of GAP activity toward Rap1a in the tuberin immunoprecipitates, but not in immunoprecipitates in which a control antiserum (anti-papillomavirus structural protein L2) was used. As a positive control, we analyzed the GAP activity toward Rap1a present in non-immunoprecipitated lysates of K-562 cells. Three μl of K-562 lysate contained more GAP activity toward Rap1a than tuberin immunocomplexes isolated from 120 μl of K-562 lysate. These differences may be due to incomplete isolation of tuberin from the lysates, although the reaction kinetics of Sepharose bead-bound tuberin are undoubtedly different from those of soluble proteins present in the K-562 lysate. In addition, the K-562 cells may express other sources of Rap1-specific GAP activity, such as one of the previously characterized Rap1GAP activities(23Zhang K. Papageorge A.P. Martin P. Vass W.C. Olah Z. Polakis P. McCormick F. Lowy D.R. Science. 1991; 254: 1630-1634Crossref PubMed Scopus (30) Google Scholar, 27Polakis P.G. Rubinfeld B. Evans T. McCormick F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 239-243Crossref PubMed Scopus (74) Google Scholar). The results described above strongly suggest that tuberin itself possesses GAP activity for Rap1, although we could not exclude the possibility that this activity is due to a coimmunoprecipitating Rap1GAP. To distinguish between these possibilities, we tested the ability of the GST-Tub-C fusion protein, which contains the last 398 amino acids (and includes the putative catalytic domain) of tuberin, to stimulate the GTPase activity of Rap1a. The GST-Tub-C protein, expressed in both E. coli and Sf9 cells, was tested for GAP activity toward Rap1a. When lysates of bacteria expressing GST-Tub-C were tested, a dose dependent, albeit weak, GAP activity was observed, while E. coli lysates expressing a control GST-fusion protein lacked detectable GAP activity (Fig. 5b). In addition, lysates of Sf9 cells expressing GST-Tub-C contained enhanced GAP activity toward Rap1a, when compared with lysates of Sf9 cells expressing a control protein (Fig. 5c). Insect cells express endogenous Rap1GAP, which probably accounts for the activity observed in the control cell lysates(28Hariharan I.K. Carthew R.W. Rubin G.M. Cell. 1991; 67: 717-722Abstract Full Text PDF PubMed Scopus (104) Google Scholar). To determine the specificity of tuberin GAP activity, we incubated immunoprecipitates of native tuberin with [γ-32P]GTP-bound recombinant Rap1a, Rap2, Ras, or Rho proteins. The GAP activity of tuberin toward all four of the GTPase proteins was measured in the same experiment. Consistent with the results of Fig. 5a, immunoprecipitates with anti-tuberin antisera contained significant levels of GAP activity toward Rap1a, compared to control immunoprecipitates (Fig. 6a). By contrast, the intrinsic GTPase activity of Ras, Rap2, and Rho was not stimulated by incubation with anti-tuberin immunoprecipitates (Fig. 6, b, c, and d). In additional experiments, the GTPase activity of Rac was not enhanced by tuberin (data not shown). Here we have described the identification of the TSC2 gene product, tuberin, and its initial biochemical characterization. In particular, the discovery of tuberin-associated GAP activity toward Rap1a suggests that this activity represents a primary biochemical function of tuberin. Full-length native tuberin, isolated by immunoprecipitation, displayed significantly more GAP activity than a recombinant C-terminal fragment of tuberin, suggesting that this activity may be positively regulated by post-translational modification of tuberin in mammalian cells. Alternatively, full GAP activity may require a larger region of the C terminus than that which was tested (amino acids 1387-1784). In either case, it is clear that tuberin does interact physically with Rap1a, and the primary site of this interaction resides within the tuberin C terminus. Additional evidence that the C-terminal region of tuberin is critical for its biological function comes from the study of tumor induction in the Eker rat strain, which is caused by an insertion resulting in premature termination of tuberin upstream from the catalytic domain(13Yeung R.S. Xiao G.-H. Jin F. Lee W.-C. Testa J.R. Knudson A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (273) Google Scholar, 14Kobayashi T. Hirayama Y. Kobayashi E. Kubo Y. Hino O. Nat. Genet. 1995; 9: 70-74Crossref PubMed Scopus (293) Google Scholar). These results do not, however, determine whether Rap1a represents the sole or even the primary target for the GAP activity of tuberin, as more than 50 small molecular weight GTP-binding proteins are known to be present inside mammalian cells(3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). Rap1b, which represents the other isoform of Rap1, is likely to be sensitive to tuberin GAP activity, as Rap1b is highly homologous to Rap1a and no biochemical differences between the isoforms have been demonstrated(16Pizon V. Lerosey I. Chardin P. Tavitian A. Nucleic Acids Res. 1988; 16: 7719Crossref PubMed Scopus (100) Google Scholar, 29Pizon P. Chardin P. Lerosey I. Olofsson B. Touchot N. Tavitian A. Oncogene. 1988; 3: 201-204PubMed Google Scholar). The finding that Ras, Rho, Rac, and especially Rap2 (which shares 60% homology with Rap1) are resistant to the GAP activity of tuberin strongly suggests that Rap1 is the primary target for this activity. The vast majority of tuberin was found in the particulate/membrane fraction of cells, consistent with the possibility that tuberin might co-localize with Rap1a, which is associated with intracellular vesicles (30Pizon V. Desjardins M. Bucci C. Parton R.G. Zerial M. J. Cell Sci. 1994; 107: 1661-1670PubMed Google Scholar). If Rap1 does serve as the primary target for the GAP activity of tuberin, the role of this protein in the development of tumors in TSC patients may be through one of several different mechanisms. Evidence that Rap1 mediates a positive growth signal has come from studies in which it was shown to induce DNA synthesis and morphological changes, when micro-injected into Swiss 3T3 cells(18Yoshida Y. Kawata M. Miura Y. Musha T. Sasaki T. Kikuchi A. Takai Y. Mol. Cell. Biol. 1992; 12: 3407-3414Crossref PubMed Scopus (102) Google Scholar). In another study, a gain-of-function mutant in the Drosophila melanogaster homologue of rap1 was shown to result in a roughened eye phenotype. Revertants of this mutant displayed significantly lower expression of the rap1 gene. Loss-of-function mutants of rap1 were found to be lethal, suggesting a positive role for the rap1 product in cell proliferation or cell viability (28Hariharan I.K. Carthew R.W. Rubin G.M. Cell. 1991; 67: 717-722Abstract Full Text PDF PubMed Scopus (104) Google Scholar). In light of these findings suggesting a positive signaling role for Rap1, the presumed lack of functional tuberin in tumors of patients with TSC and in renal cell tumors of the Eker rat may reflect aberrant regulation of Rap1, resulting in its constitutive activation. While tuberin is clearly not the sole source of Rap1GAP activity in K-562 cells, its activity could be critical in certain cell types, particularly in cells that are predisposed to tumor formation in TSC patients. In this scenario, tuberin would be functionally analogous to the NF1 product neurofibromin, a RasGAP critical for regulation of Ras in tumor cells of NF1 patients(7Basu T.N. Gutmann D.H. Fletcher J.A. Glover T.W. Collins F.S. Downward J. Nature. 1992; 356: 713-715Crossref PubMed Scopus (580) Google Scholar, 8DeClue J.E. Papageorge A.G. Fletcher J.A. Diehl S.R. Ratner N. Vass W.C. Lowy D.R. Cell. 1992; 69: 265-273Abstract Full Text PDF PubMed Scopus (517) Google Scholar). Recent studies have demonstrated that while neurofibromin is critical as a regulator of GTP·Ras in Schwann cells, it is not required as a regulator in other cell types (e.g. NIH 3T3)(31Johnson M.R. DeClue J.E. Felzmann S. Vass W.C. Xu G. White R. Lowy D.R. Mol. Cell. Biol. 1994; 14: 641-645Crossref PubMed Scopus (90) Google Scholar, 32Kim H. Kombrinck K. Marchioni M. Ratner N. DeClue J. Oncogene. 1995; (in press)Google Scholar). In spite of this fact, overexpression of NF1 does inhibit the growth of NIH 3T3(31Johnson M.R. DeClue J.E. Felzmann S. Vass W.C. Xu G. White R. Lowy D.R. Mol. Cell. Biol. 1994; 14: 641-645Crossref PubMed Scopus (90) Google Scholar). One explanation for these findings is that neurofibromin binds to Ras, preventing Ras from interacting with its mitogenic effectors. Such a model could also apply to tuberin and Rap1a: loss of tuberin expression in cells would free up Rap1, thereby allowing it to transmit a positive signal through its effectors. An alternative model for the function of tuberin and Rap1 is suggested by studies which have raised the possibility that Rap1 has a negative effect on cell growth. These studies originated with the demonstration that rap1a can antagonize ras-induced transformation. rap1a was identified as a cDNA that, when overexpressed, could cause reversion of the transformed phenotype in NIH 3T3 bearing an oncogenic K-ras gene(17Kitayama H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). In addition, expression of constitutively active rap1 (valine 12) was shown to inhibit the activation of MAP kinase following simulation with epidermal growth factor and lysophosphatidic acid, although with less efficiency than a dominant-inhibitory mutant of ras (rasN17)(33Cook S.J. Rubinfeld B. Albert I. McCormick F. EMBO J. 1993; 12: 3475-3485Crossref PubMed Scopus (335) Google Scholar). Further studies have demonstrated that Rap1 is capable of binding to all of the putative mitogenic effectors for mammalian Ras: RasGAP, Raf, and phosphatidylinositol-3′ kinase(34Frech M. John J. Pizon V. Chardin P. Tavitian A. Clark R. McCormick F. Wittinghofer A. Science. 1990; 249: 169-171Crossref PubMed Scopus (187) Google Scholar, 35Zhang X.F. Settleman J. Kyriakis J.M. Takeuchi-Suzuki E. Elledge S.J. Marshall M.S. Bruder J.T. Rapp U.R. Avruch J. Nature. 1993; 364: 308-313Crossref PubMed Scopus (689) Google Scholar).3( 3J. Downward, personal communication.) These results have been interpreted as supporting a model whereby Rap1 antagonizes Ras through competitive binding of Rap1 to Ras effector molecules. An alternative (although not mutually exclusive) possibility is that Rap1 proteins participate in the transduction of growth-inhibitory signals, thereby overcoming the mitogenic signals originating from Ras(36Rubinfeld B. Munemitsu S. Clark R. Conroy L. Watt K. Crosier W.J. McCormick F. Polakis P. Cell. 1991; 65: 1033-1042Abstract Full Text PDF PubMed Scopus (209) Google Scholar). In accordance with this scenario, tuberin might function as an effector protein (as well as a GAP) for Rap1, and the loss of tuberin expression would prevent transmission of the growth-inhibitory signals originating from Rap1. While Rap1 is likely to be a primary target for the GAP activity of tuberin, we cannot exclude the possible involvement of other small molecular weight GTP-binding proteins. Recently, Spa-1, a protein with homology to Rap1GAP and tuberin, was shown to have GAP activity toward both Rap1 and the nuclear-localized, Ras-related GTPase Ran(37Hattori M. Tsukamoto N. Nur-E-Kamal M.S.A. Rubinfeld B. Iwai K. Kubota H. Maruta H. Minato N. Mol. Cell. Biol. 1995; 15: 552-560Crossref PubMed Scopus (66) Google Scholar). Also, given the fact that Rap1 binds to RasGAP with high affinity, it is possible that tuberin suppresses cell growth by binding to Ras, without stimulating its GTPase. In future studies, we will pursue the possibility that the tumor suppressor function of tuberin is related to its ability to regulate, or interact with, other members of the Ras superfamily in addition to Rap1. Cell lines derived from tumors of the Eker rat should greatly facilitate these investigations of the tumor suppressor function of tuberin. We thank Dr. Jean de Gunzburg, Dr. Alan Hall, and Dr. Albert Reynolds for providing p21 proteins and/or expression plasmids, respectively. We also thank Lisa Leonardsen and Alex Papageorge for advice and Doug Lowy for continual support and interest." @default.
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