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 × 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 To fusion proteins in the bacteria were induced with 1 mM isopropyl-β-D-thiogalactopyranoside for h at were lysed by in PBS, Nonidet P-40, and 1 mM cells were by and the were by 15,000 × g for washed with PBS, Nonidet P-40, 1 mM and 2 and and were by The of and in these was of HB101 cells GST-Tub-N, GST-Tub-C, and GST-DH were as E. D. A Scholar). of purified and the of were used to New with were and 9 For in was from the of with Tub-C by binding to protein and with according to the For by cells a volume of were Cell and immunoprecipitation were as J.E. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google using of The were subjected to on and proteins were an membrane For antisera were incubated with of bacterial proteins for 2 h at 4°C, by the membrane was with 0.1% Nonidet P-40, and the membrane was incubated with and proteins were by with and The shown in different antisera at a of as This was with an and To estimate the molecular weight of tuberin, both molecular weight and (Pharmacia) were Sf9 cells were with the baculovirus transfer vector and baculovirus DNA and encoding the protein were and according to the Sf9 cells × were with GST-Tub-C, or protein as a and were 50 were washed with GAP and lysed by in 1 of GAP were by and the concentration of was as for bacterial × cells were pelleted, washed with PBS, and in One portion of the cells was lysed with 2 of the other aliquot was used to the × g and as J.E. Lowy D.R. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). portions of the and the lysates were subjected to immunoprecipitation and immunoblotting as For GAP reaction with of tuberin, the was used to tuberin from lysates of × in of Immunoprecipitates were bound to protein and the were washed times with times with GAP mM mM mM 1 mM and the beads were resuspended in GAP in of the GAP assays were as K. Papageorge P. Vass W.C. P. McCormick F. Lowy D.R. 1991; PubMed Scopus Google Scholar). μM GTP-binding proteins were with μM in mM mM mM and 50 in a volume of into of GAP with μM 50 of this was added to the different tuberin or of native tuberin bound to protein beads from × of bacterial or μM Sf9 cell were in at and portions of reaction were at the or the was by and purified from Sf9 cells, were by bacterial and were by was expressed as in HB101 cells a expression by I. Chardin P. J. Tavitian A. J. Biol. 1991; Full Text PDF PubMed Google Scholar). To for the specific of tuberin, antisera were raised against an N-terminal and a C-terminal region of the predicted protein product and of the cell were subjected to immunoprecipitation with or were to a and the was incubated with We the of a specific of in the of the tumor cell were subjected to immunoprecipitation with or and the were The was into which were incubated with or a specific of was in the and when the was with did not bind to tuberin in The of this is to the predicted molecular of tuberin The European Chromosome 16 Tuberous Sclerosis Consortium. 1993; 75: 1305-1313Google Scholar). the a of that the protein is in tuberin, antisera against different regions of tuberin were for immunoprecipitation and for of a in the of the 180-kDa was of the with an from E. coli GST-Tub-N, not by with of E. coli or GST-DH Immunoprecipitation of from and cells with these antisera revealed a 180-kDa which specifically by with from E. coli the fusion protein, not by with control not immunoprecipitation also revealed a 180-kDa protein in the cell the when was was than in the which to the in most of the To the subcellular of tuberin, cells were lysed by in the of The was centrifuged at × g to membrane/particulate and lysates from an of cells were with and of the was by immunoprecipitation or by A of the tuberin was in the the possibility that tuberin may with the cell plasma or with or The wide variety of organ affected in TSC patients suggests that the 180-kDa tuberin protein is expressed in many different cell expression of the TSC2 in diverse cell has been demonstrated by We a of different human cell lines for tuberin expression These lines a variety of cell (see to were and portions of an of protein were subjected to the of and cells, tuberin was expressed in of the cell lines of the cell lines a different of the abnormalities in this have not been linked to TSC M.R. Tuberous Sclerosis. Raven Press, New York1988Google Scholar, M. F. 1991; PubMed Google Scholar). in the of tuberin were in of the cell lines and These may of tuberin, or the of of the TSC2 have been demonstrated for the TSC2 in S. The region of tuberin bearing homology to Rap1GAP amino acids) is than the Rap1GAP catalytic domain and is also than the homology between different GAP proteins M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar, B. I. L. R. McCormick F. P. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar). tuberin function as a GAP for of tuberin contain this type of activity. native tuberin was by immunoprecipitation from cell lysates, and the were incubated with Rap1a to potential GAP activity We of GAP activity Rap1a in the tuberin not in in which a control protein was a positive we the GAP activity Rap1a in lysates of cells. of GAP activity Rap1a than tuberin from of These may to of tuberin from the lysates, the reaction of Sepharose tuberin are different from of proteins in the the cells may express other of GAP as one of the characterized Rap1GAP K. Papageorge P. Vass W.C. P. McCormick F. Lowy D.R. 1991; PubMed Scopus Google Scholar, B. T. McCormick F. Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). The results strongly suggest that tuberin GAP activity for we not the possibility that this activity is to a Rap1GAP. To between these we the of the fusion protein, which contains the amino acids the putative catalytic of tuberin, to stimulate the GTPase activity of Rap1a. The protein, expressed in both E. coli and Sf9 cells, was for GAP activity Rap1a. lysates of bacteria were a GAP activity was E. coli lysates a control protein GAP activity lysates of Sf9 cells GAP activity when with lysates of Sf9 cells a control protein cells express which for the activity in the control cell Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). To the of tuberin GAP we incubated of native tuberin with Rap2, Ras, or proteins. The GAP activity of tuberin four of the GTPase proteins was in the with the results of with antisera of GAP activity to control the intrinsic GTPase activity of Ras, Rap2, and was not by with and the GTPase activity of was not by tuberin not we have the identification of the TSC2 gene product, tuberin, and the of GAP activity Rap1a suggests that this activity a function of tuberin. native tuberin, by GAP activity than a C-terminal fragment of tuberin, suggesting that this activity may regulated by of tuberin in cells. GAP activity may a region of the C terminus than that which was (amino acids is that tuberin with and the of this the tuberin C that the C-terminal region of tuberin is critical for biological function from the of tumor in the Eker rat strain, which is caused by an in of tuberin from the catalytic 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 Rap1a the or the for the GAP activity of tuberin, as than 50 small molecular weight GTP-binding proteins are known to M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1762) Google Scholar). which the other of is to to tuberin GAP as Rap1b is to Rap1a and between the have been V. Lerosey I. Chardin P. Tavitian A. Nucleic Acids Res. 1988; 16: 7719Crossref PubMed Scopus (100) Google Scholar, P. Chardin P. Lerosey I. B. N. Tavitian A. 1988; Google Scholar). The that Ras, Rac, and homology with are to the GAP activity of tuberin strongly suggests that Rap1 is the for this activity. The of tuberin was found in the of cells, consistent with the possibility that tuberin with which is associated with V. M. M. J. Cell Sci. 1994; Google Scholar). Rap1 as the for the GAP activity of tuberin, the role of this protein in the development of tumors in TSC patients may one of different that Rap1 a positive signal has from studies in which was shown to induce DNA synthesis and morphological when 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). a in the of was shown to in a of this expression of the of were found to suggesting a positive role for the product in cell proliferation or cell Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). of these findings suggesting a positive signaling role for the of tuberin in tumors of patients with TSC and in renal cell tumors of the Eker rat may of in constitutive tuberin is not the of Rap1GAP activity in cells, activity critical in cell in cells that are to tumor in TSC this tuberin to the NF1 product neurofibromin, a critical for of Ras in tumor cells of NF1 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). studies have demonstrated that neurofibromin is critical as a regulator of in cells, is not as a regulator in other cell M.R. J.E. S. Vass W.C. G. R. Lowy D.R. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar, H. K. M. Ratner N. J. 1995; Scholar). of this of NF1 the of M.R. J.E. S. Vass W.C. G. R. Lowy D.R. Mol. Cell. Biol. 1994; PubMed Scopus Google Scholar). One for these findings is that neurofibromin to Ras, Ras from with mitogenic a also to tuberin and loss of tuberin expression in cells to a positive signal for the function of tuberin and Rap1 is by studies which have raised the possibility that Rap1 has a negative on cell These studies with the that was identified as a cDNA when of the in 3T3 bearing an H. Sugimoto Y. Matsuzaki T. Ikawa Y. Noda M. Cell. 1989; 56: 77-84Abstract Full Text PDF PubMed Scopus (763) Google Scholar). expression of active was shown to the activation of with and with than a of B. I. McCormick F. EMBO J. 1993; 12: PubMed Scopus Google Scholar). studies have demonstrated that Rap1 is of binding to of the putative mitogenic for and M. J. V. Chardin P. Tavitian A. R. McCormick F. A. 1990; PubMed Scopus Google Scholar, J. E. M.S. J. Nature. 1993; PubMed Scopus Google These results have been as a Rap1 Ras binding of Rap1 to Ras molecules. not possibility is that Rap1 proteins in the transduction of the mitogenic from B. S. R. L. K. McCormick F. P. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). with this tuberin function as an protein well as a for and the loss of tuberin expression of the from Rap1. Rap1 is to a for the GAP activity of tuberin, we the possible of other small molecular weight GTP-binding proteins. a protein with homology to Rap1GAP and tuberin, was shown to have GAP activity both Rap1 and the Ras-related GTPase M. N. B. K. H. H. N. Mol. Cell. Biol. 1995; 15: PubMed Scopus Google Scholar). the that Rap1 to with is possible that tuberin cell by binding to Ras, stimulating we the possibility that the tumor suppressor function of tuberin is to to or other of the Ras in to Rap1. Cell lines derived from tumors of the Eker rat these of the tumor suppressor function of tuberin. We and for proteins expression We also and Papageorge for and Lowy for and