Title: Tuberin Regulates p70 S6 Kinase Activation and Ribosomal Protein S6 Phosphorylation
Abstract: Although the cellular functions ofTSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2−/− smooth muscle cells (ELT3) and TSC2−/− epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM. Although the cellular functions ofTSC2 and its protein product, tuberin, are not known, somatic mutations in the TSC2 tumor suppressor gene are associated with tumor development in lymphangioleiomyomatosis (LAM). We found that ribosomal protein S6 (S6), which exerts translational control of protein synthesis and is required for cell growth, is hyperphosphorylated in the smooth muscle-like cell lesions of LAM patients compared with smooth muscle cells from normal human blood vessels and trachea. Smooth muscle (SM) cells derived from these lesions (LAMD-SM) also exhibited S6 hyperphosphorylation, constitutive activation of p70 S6 kinase (p70S6K), and increased basal DNA synthesis. In parallel, TSC2−/− smooth muscle cells (ELT3) and TSC2−/− epithelial cells (ERC15) also exhibited hyperphosphorylation of S6, constitutive activation of p70S6K, and increased basal DNA synthesis. Re-introduction of wild type tuberin into LAMD-SM, ELT3, and ERC15 cells abolished phosphorylation of S6 and significantly inhibited p70S6K activity and DNA synthesis. Rapamycin, an immunosuppressant, inhibited hyperphosphorylation of S6, p70S6K activation, and DNA synthesis in LAMD-SM cells. Interestingly, the basal levels of phosphatidylinositol 3-kinase, Akt/protein kinase B, and p42/p44 MAPK activation were unchanged in LAMD-SM and ELT3 cells relative to levels in normal human tracheal and vascular SM. These data demonstrate that tuberin negatively regulates the activity of S6 and p70S6K specifically, and suggest a potential mechanism for abnormal cell growth in LAM. lymphangioleiomyomatosis phosphatidylinositol 3-kinase GTPase-activating protein mitogen-activated protein kinase p70 S6 kinase mammalian target of rapamycin smooth muscle fetal bovine serum airway smooth muscle cells vascular smooth muscle cells human lung fibroblasts green fluorescent protein Tris-buffered saline 5-bromo-2′-deoxyuridine, MOPS, 4-morpholinepropanesulfonic acid analysis of variance Lymphangioleiomyomatosis (LAM)1 is a disorder characterized by benign lesions of smooth muscle-like cells in the lung (1Taylor J.R. Ryu J. Colby T.V. Raffin T.A. N. Engl. J. 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Care Med. 2001; 164: 1072-1076Crossref PubMed Scopus (107) Google Scholar). The molecular mechanisms promoting LAM cell proliferation remain largely unknown (7Cheadle J.P. Reeve M.P. Sampson J.R. Kwiatkowski D.J. Hum. Genet. 2000; 107: 97-114Crossref PubMed Scopus (290) Google Scholar). Genetic studies demonstrate that somatic mutations in the tumor suppressor TSC2 gene are associated with pulmonary LAM (8Franz D.N. Brody A. Meyer C. Leonard J. Chuck G. Dabora S. Sethuraman G. Colby T.V. Kwiatkowski D.J. McCormack F.X. Am. J. Respir. Crit. Care Med. 2001; 164: 661-668Crossref PubMed Scopus (235) Google Scholar, 9Carsillo T. Astrinidis A. Henske E.P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6085-6090Crossref PubMed Scopus (563) Google Scholar, 10Yu J. Astrinidis A. Henske E.P. Am. J. Respir. Crit. Care Med. 2001; 164: 1537-1540Crossref PubMed Scopus (140) Google Scholar). Evidence suggests that TSC2 gene activation alters the proliferation of mammalian cells. Soucek et al. 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Mice lacking tuberin die as embryos and show hypoplasia of the liver and developmental abnormalities of other abdominal organs (15Onda H. Lueck A. Marks P.W. Warren H.B. Kwiatkowski D.J. J. Clin. Invest. 1999; 104: 687-695Crossref PubMed Scopus (327) Google Scholar, 16Kobayashi E. Minowa O. Kuno J. Mitani H. Hino O. Noda T. Cancer Res. 1999; 59: 1206-1211PubMed Google Scholar). Heterozygous mice develop cysts and slow growing tumors in a variety of tissues. The Eker rat, a model for hereditary renal cancer, has an insertion in the TSC2 locus resulting in a tuberin mutation and tumor development in multiple organs (17Yeung R. Xiao G. Jin F. Lee W. Testa J. Knudson A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11413-11416Crossref PubMed Scopus (278) Google Scholar, 18Kobayashi T. Nishizawa M. Hirayama Y. Kobayashi E. Hino O. Nucleic Acids Res. 1995; 23: 2608-2613Crossref PubMed Scopus (46) Google Scholar). Thus, as in the human disease, loss or mutation ofTSC2 modulates cell proliferation. TSC2 encodes a 1784-amino acid 200-kDa protein, tuberin, that contains a region with sequence similarity to the GTPase-activating protein (GAP) for Rap1 GTPase (19European Chromosome 16 Tuberous Sclerosis ConsortiumCell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1542) Google Scholar). The putative GAP domain has been reported to increase the intrinsic GTPase activity of both Rab5 and Rap1A GTPases (20Wienecke R. Konig A. DeClue J.E. J. Biol. Chem. 1995; 270: 16409-16414Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 21Xiao G.H. Shoarinejad F. Jin F. Golemis E.A. Yeung R.S. J. Biol. Chem. 1997; 272: 6097-6100Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). Further downstream, tuberin modulates the transcriptional activity of AP1 and the steroid hormone receptor family (22Henry K.W. Yuan X. Koszewski N.J. Onda H. Kwiatkowski D.J. Noonan D.J. J. Biol. Chem. 1998; 273: 20535-20539Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 23Tsuchiya H. Orimoto K. Kobayashi K. Hino O. Cancer Res. 1996; 56: 429-433PubMed Google Scholar, 24Noonan D.J. Lou D. Griffith N. Vanaman T.C. Arch. Biochem. Biophys. 2002; 398: 132-140Crossref PubMed Scopus (48) Google Scholar). However, little else is known about how tuberin is regulated or about its other downstream signaling targets. Given the phenotypic similarity between loss of TSC2 and overexpression of PI 3-kinase in Drosophila (25Leevers S.J. Weinkove D. MacDougall L.K. Hafen E. Waterfield M.D. EMBO J. 1996; 15: 6584-6594Crossref PubMed Scopus (421) Google Scholar), we hypothesized that deregulation of PI 3-kinase effectors might be important for the abnormal cell proliferation associated withTSC2 deficiency. One critical downstream target of PI 3-kinase, p70 S6 kinase (p70S6K), affects cell and organ size inDrosophila (26Montagne J. Stewart M.J. Stocker H. Hafen E. Kozma S.C. Thomas G. Science. 1999; 285: 2126-2129Crossref PubMed Scopus (629) Google Scholar). Activation of p70S6K and its subsequent phosphorylation of ribosomal protein S6 are required for biosynthesis of the cellular translational apparatus, a critical component for cell growth and proliferation. Conditional deletion of the S6 gene in the liver of adult mice abrogates cell proliferation and cyclin E expression despite the formation of cyclin D-CDK4 complexes (27Volarevic S. Steward M.J. Lefderman B. Zilberman F. Terracciano L. Montini E. Grompe M. Kozma S.C. Thomas G. Science. 2000; 288: 2045-2047Crossref PubMed Scopus (331) Google Scholar). The central role of p70S6K and ribosomal protein S6 in cellular proliferation has been further demonstrated by the use of rapamycin, a macrolide that specifically and directly inhibits the mammalian target of rapamycin (mTOR), an obligated upstream activator of p70S6K (28Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1724) Google Scholar, 29Sabatini D.M. Erjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1273) Google Scholar, 30Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1755) Google Scholar). In the current study, we demonstrate that tuberin specifically regulates the activity of p70S6K and ribosomal protein S6. S6 hyperphosphorylation and p70S6K activation and increased DNA synthesis were observed in LAM tissue samples, primary LAM-derived smooth muscle (LAMD-SM) cultures, and established cell lines lacking the functional tuberin protein. These effects were reversed by the re-introduction of tuberin. Surprisingly, other signaling molecules such as PI 3-kinase, Akt, and MAPK were unaffected by cellular levels of tuberin. These results suggest that tuberin may modulate its effects on cellular proliferation through specific modulation of the p70S6K pathway. LAMD-SM cells were dissociated from LAM nodules obtained from the lungs of LAM patients who have undergone lung transplant. LAM tissue was obtained in compliance with the University of Pennsylvania Institutional Review Board approved protocol and the protocol approved by NHLBI, National Institutes of Health LAM Registry Tissue Committee. Briefly, nodules was subjected to an enzymatic digestion in 10 ml of M199 medium containing 0.2 mm CaCl2, 2 mg/ml collagenase D (Roche Molecular Biochemicals), 1 mg/ml trypsin inhibitor (Sigma), and 3 mg/ml elastase (Worthington, Lakewood, NJ) for 60 min in a shaking water bath at 37 °C. The cell suspension was filtered and then washed with equal volumes of cold DF8 medium consisting of equal amounts of Ham's F-12 and Dulbecco's modified Eagle's medium with 1.6 × 10−6mferrous sulfate, 1.2 × 10−5 units/ml vasopressin, 1.0 × 10−9m triiodothyronine, 0.025 mg/ml insulin, 1.0 × 10−8m cholesterol, 2.0 × 10−7m hydrocortisone, and 10 pg/ml transferrin supplemented with 10% FBS. Aliquots of the cell suspension were plated at a density of 1.0 × 104cells/cm2 on tissue culture plates covered with Vitrogen (Cohesion Technologies Inc., Palo Alto, CA). The cells were cultured in DF8 medium and were passaged twice a week. LAMD-SM cells in subculture during the third through twelfth cell passages were used. All experiments were performed using in parallel three primary cell lines derived from two LAM patients. LAMD-SM-1 cell line represents the total population of cells derived from one nodule of one patient. LAMD-SM-2 and LAMD-SM-7 are clonal lines, two of eight clones that were derived from a LAM nodule of another patient. Each cell line was characterized on the basis of TSC2 mutational analysis, tuberin expression, smooth muscle α-actin expression, the level of DNA synthesis, and HMB45 immunoreactivity. TSC2 gene mutational analysis of these cell lines was performed on DNA and RNA samples by two methods. First, reverse transcriptase-PCR analysis of the entire coding region of the TSC2 mRNA was performed using a set of eight primer pairs to generate eight overlapping fragments (31Dabora S.L. Jozwiak S. Franz D.N. Roberts P.S. Nieto A. Chung J. Choy Y.S. Reeve M.P. Thiele E. Egelhoff J.C. Karprzyk-Obara J. Domanska-Pakiela D. Kwiatkowski D.J. Am. J. Hum. Genet. 2001; 68: 64-80Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). These fragments were subjected to bi-directional sequencing and compared with the TSC2 consensus sequence using the program Sequencher version 2.0. Analysis of selected individual exons of TSC2 by denaturing high-performance liquid chromatography and sequencing was also performed as described (31Dabora S.L. Jozwiak S. Franz D.N. Roberts P.S. Nieto A. Chung J. Choy Y.S. Reeve M.P. Thiele E. Egelhoff J.C. Karprzyk-Obara J. Domanska-Pakiela D. Kwiatkowski D.J. Am. J. Hum. Genet. 2001; 68: 64-80Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). Comparative analysis of morphological, biochemical, and functional characteristics demonstrated similarities between LAMD-SM-1, LAMD-SM-2, and LAMD-SM-7 cell lines. Morphologically LAMD-SM cells were spindle-like with high levels of basal DNA synthesis (see “Results”), and they showed positive immunoreactivity to anti-smooth muscle α-actin antibody (data not shown) and tuberin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) (See “Results”). However, LAMD-SM cells showed no immunoreactivity for HMB-45 (DAKO Co., Carpinteria, CA) (data not shown), which showed positive immunostaining of the epithelioid cells in LAM nodules (data not shown) (3Matsui K. Takeda K., Yu, Z.X. Travis W.D. Moss J. Ferrans V.J. Arch. Pathol. Lab. Med. 2000; 124: 267-275PubMed Google Scholar, 4Matsui K. Takeda K., Yu, Z.X. Valencia J. Travis W.D. Moss J. Ferrans V.J. Am. J. Respir. Crit. Care Med. 2000; 161: 1002-1009Crossref PubMed Scopus (125) Google Scholar, 32Matsumoto Y. Horiba K. Usuki J. Chu S.C. Ferrans V.J. Moss J. Am. J. Respir. Cell Mol. Biol. 1999; 21: 327-336Crossref PubMed Scopus (119) Google Scholar) and in human melanoma, used as a positive control. All assays were performed on cells maintained for 48 h in serum-free DF basal media containing 1% bovine serum albumin before starting the experiment. A TSC2−/− ELT3 smooth muscle cell line was derived from the Eker rat uterine leiomyoma (33Howe S.R. Gottardis M.M. Everitt J.I. Goldsworthy T.L. Wolf D.C. Walker C.L. Am. J. Pathol. 1995; 146: 1568-1579PubMed Google Scholar). The TSC2−/− ERC15 cell line was derived from a Eker rat renal carcinoma (34Hino O. Klein-Szanto A.J.P. Freed J.J. Testa J.R. Brown D.J. Vilensky M. Yeung R.S. Tartof K.D. Knudson A.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 327-331Crossref PubMed Scopus (167) Google Scholar). The TSC2+/+ TRKE2 cell line was derived from rat primary kidney epithelial cells (35Walker C.L. Ginsler J. Carcinogenesis. 1992; 13: 25-32Crossref PubMed Scopus (22) Google Scholar). ELT3, ERC15, and TRKE2 cells were maintained in DF8 medium (33Howe S.R. Gottardis M.M. Everitt J.I. Goldsworthy T.L. Wolf D.C. Walker C.L. Am. J. Pathol. 1995; 146: 1568-1579PubMed Google Scholar). Human airway smooth muscle (ASM) cells, human pulmonary arterial vascular smooth muscle (VSM) cells, and human lung fibroblasts (HLF) were used as TSC2+/+ cell lines. Expression of tuberin in these cells was confirmed by immunoblot analysis with anti-tuberin antibody (data not shown). ASM and VSM cells and HLF were dissociated from human trachea, human pulmonary artery, and human lung, respectively, which were obtained from human lung transplant donors and were previously described (36Panettieri R.A. DePalo L.R. Murray R.K. Yadvich P.A. Kotlikoff M.I. Am. J. Physiol. 1989; 256: C329-C335Crossref PubMed Google Scholar, 37Goncharova E. Ammit A.J. Irani C. Carroll R.G. Eszterhas A.J. Panettieri R.A. Krymskaya V.P. Am. J. Physiol. Lung Cell. Mol. Physiol. 2001; 283: L354-L363Crossref Scopus (168) Google Scholar). ASM cells were maintained in Ham's F-12 with 10% FBS; VSM were maintained in Ham's F-12 with 10% FBS supplemented with 15 μg/ml endothelial cell growth supplement (BD Biosciences), HLF were maintained in RPMI supplemented with 10% FBS. All assays were performed on cells maintained for 48 h in serum-free medium before starting the experiment. ELT3 and ERC15 were maintained in serum-free DF8 basal media containing 1% bovine serum albumin; ASM and VSM cells were in serum-free Ham's F-12 supplemented with 0.1% bovine serum albumin. Microinjection was performed using Eppendorf Microinjection System (Hamburg, Germany). Briefly, cells plated on 2-well glass chamber slides (Nalgene Nunc International, Naperville, IL) and maintained in serum-free medium were microinjected with pEGFP or pEGFP-TSC2 plasmids expressing green fluorescent protein (GFP) or GFP-tagged tuberin, respectively, or with inhibitory anti-VPS34 antibody, which specifically recognize and inhibit class III PI 3-kinase (38Siddhanta U. McIlroy J. Shah A. Zhang Y. Backer J.M. J. Cell Biol. 1998; 143: 1647-1659Crossref PubMed Scopus (139) Google Scholar) simultaneously with dextran Rhodamine Green (Molecular Probes, Eugene, OR) to identify microinjected cells. Eighteen hours after injection of plasmids or 30 min after the injection of antibody, the cells were washed 3 times in phosphate-buffered saline, fixed with 3.7% paraformaldehyde (Polysciences, Inc., Warrington, PA) for 15 min, and treated with 0.1% Triton X-100 (Sigma) for 30 min at room temperature. The cells were blocked with 0.5% TSA Fluoresce™ System blocking reagent (PerkinElmer Life Sciences) in 20 mm Tris (pH 7.5) and 150 mm NaCl (TBS) for 1 h at 37 °C. After incubation with primary antibodies (anti-phospho-ribosomal protein S6 (S235) antibody (Upstate Biotechnology, Lake Placid, NY; 1:50) or anti-GFP, rabbit serum (Molecular Probes; 1:200 dilution) and then secondary antibodies (Alexa Fluor 594 donkey anti-sheep IgG conjugate, 1:400 dilution, or Alexa Fluor 488 goat anti-rabbit IgG conjugate (Molecular Probes) 1:400) for 1 h at 37 °C, the cells were mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The cells were visualized on the Bio-Rad 1024-MP confocal microscopic system or Nikon Eclipse E400 microscope under appropriate filters. LAM tissue sections were stained with hematoxylin and eosin or immunohistochemically with anti-smooth muscle α-actin clone 1A4 fluorescein isothiocyanate conjugate (Sigma), primary anti-phospho-ribosomal protein S6 antibody (Upstate Biotechnology), and secondary Alexa Fluor 594 donkey anti-sheep IgG conjugate (Molecular Probes) antibodies. Negative controls included omission of the primary antibody or replacement of the primary antibody with isotype matched IgG. Plasmids were prepared using EndoFree Plasmid Maxi Kit (Qiagen Inc., Valencia, CA). Transient transfection was performed using the Effectene transfection reagent (Qiagen) according to the manufacturer's protocol. Briefly, cells were incubated with pcDNA3 or pcDNA3-TSC2 (21Xiao G.H. Shoarinejad F. Jin F. Golemis E.A. Yeung R.S. J. Biol. Chem. 1997; 272: 6097-6100Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 39Kleymenova E. Ibragimov-Beskrovnaya O. Kugoh H. Everitt J., Xu, H. Kiguchi K. Landes G. Harris P. Walker C. Mol. Cell. 2001; 7: 823-832Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) for 6 h and then washed with phosphate-buffered saline and maintained for the next 24 h in medium supplemented with 10% FBS. Cells were maintained for 48 h in serum-free media before the immunoblot, p70S6K activity, or DNA synthesis assays. Transient transfection of pcDNA-TSC2 plasmid was verified by immunoblot assay using anti-tuberin (C20) (Santa Cruz Biotechnology) antibody. Near confluent cells, grown on 2-well glass chamber slides, were maintained for 48 h in serum-free medium, then after 16 h, 10 μm 5-bromo-2′-deoxyuridine (BrdUrd), a thymidine analogue, was added to all wells. Twenty-four hours after the addition of BrdUrd, the cell monolayers were fixed with 3.7% paraformaldehyde and then permeabilized with 0.1% Triton X-100. After denaturation of DNA with 4 n HCl for 3 min at room temperature, the monolayers were incubated for 1 h at 37 °C with 2 μg/ml mouse anti-BrdUrd antibody (BD Biosciences) and then with 10 μg/ml Texas Red-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at 37 °C to detect BrdUrd-positive cells. The cells were then incubated with 4,6-diamidino-2-phenylindole (1 μg/ml) in 0.9% NaCl to detect the total number of nuclei. The cells were examined using a fluorescent microscope (Nikon Eclipse E400) under 200× magnification with the appropriate fluorescent filters. Mitotic index was defined as the percentage of BrdUrd-positive nuclei/field/total number of cells/field. A total of 200 cells were counted/each condition in each experiment. Cells were washed twice with ice-cold TBS and lysed in p70S6K lysis buffer (20 mm Tris (pH 7.5), 150 mm NaCl, 20 mm NaF, 2.5 mmNa4P2O7·10 H2O, 1 mm β-glycerophosphate, 1% Nonidet P-40, 1 mmbenzamidine, 10 mmp-nitrophenyl phosphate, 0.1 mm phenylmethylsulfonyl fluoride (Sigma)) or radioimmune precipitation buffer-TBS lysis buffer (25 mm Tris-base HCl (pH 7.4), 150 mm NaCl, 0.5% sodium deoxycholate, 1.0% Nonidet P-40 (Calbiochem), 0.1% SDS, 1 mm EGTA, 5 mm EDTA, 200 μmNa3VO4, 10 μg/ml aprotinin, 10 μg/ml leupeptin (Sigma), 1 mm phenylmethylsulfonyl fluoride) for 30 min at 4 °C. Frozen tissue samples were homogenized with radioimmune precipitation buffer-TBS lysis buffer and lysed for 30 min at 4 °C. The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. Protein contents were measured using a Bio-Rad protein assay reagent kit (Bio-Rad). Equal amounts of lysate, adjusted to protein content, were subjected to SDS-PAGE and immunoblot analysis. The blots were exposed to either anti-p70 S6 kinase, anti-tuberin (C20) sc-893 (Santa Cruz Biotechnology), anti-phospho-p70 S6 kinase (Thr-389), anti-phospho-p70S6 kinase (Thr-421/Thr-424), anti-S6 ribosomal protein, anti-phospho-Akt (Ser-473), anti-Akt (Cell Signaling Technology, Inc., Beverly, MA), anti-phospho p42/p44 MAPK (Thr-202/Thr-204), or anti-p42/p44 MAPK (New England Biolabs, Inc., Beverly, MA) antibodies. All antibodies were in TBS plus 0.5% Tween 20 (TBST), and all incubations were overnight at 4 °C. After three washes in TBST, the nitrocellulose filters were exposed to either anti-rabbit, anti-mouse (Boehringer-Mannheim) or anti-sheep (Upstate Biotechnology) horseradish peroxide-conjugated secondary antibodies. Filters were washed five times in TBST and visualized using Enhanced Chemiluminescence (ECL) (Amersham Biosciences). Image analysis was performed using the Gel-Pro analyzer program (Media Cybernetics, Silver Spring, MD). In vitro p70S6K activity assay was performed as described previously (40Masuda-Robens J.M. Krymskaya V.P., Qi, H. Chou M.M. Methods Enzymol. 2001; 333: 45-55Crossref PubMed Scopus (4) Google Scholar). Briefly, serum-free medium-maintained cells were washed twice in ice-cold phosphate-buffered saline and then lysed in lysis buffer (20 mm Tris (pH 8.0), 150 mm NaCl, 5 mmEGTA, 1 mm EDTA, 10 mmNa4P2O7 × 10 H2O, 1 mm benzamidine, 1% Nonidet P-40, 10 mmp-nitrophenyl phosphate, 0.1 mmphenylmethylsulfonyl fluoride). After incubation for 30 min at 4 °C, lysates were centrifuged at 14,000 rpm for 10 min. Supernatants were incubated with 2 μg of anti-p70S6K antibody with gentle rocking overnight at 4 °C. The immunocomplexes were collected by 50 μl of protein A-Sepharose (Amersham Biosciences) for 2 h at 4 °C. The immunoprecipitates were washed twice with ice-cold cell lysis buffer, twice with lysis buffer without Nonidet P-40, and twice with an assay dilution buffer (20 mm MOPS (pH 7.2), 25 mmβ-glycerol phosphate, 5 mm EGTA, and 1 mmdithiothreitol (Sigma)). The immunoprecipitates/protein A-Sepharose beads were then resuspended in assay dilution buffer containing 50 μm substrate peptide, 4 μm protein kinase C inhibitor peptide, 0.4 μm protein kinase A inhibitor peptide, and [γ-32P]ATP (PerkinElmer Life Sciences). The samples were incubated for 10 min at 30 °C, and then 20 μl of the reaction mixture was spotted onto p81 phosphocellulose filters, which were washed 3 times with 0.75% phosphoric acid and 1 time with acetone. The radioactivity of samples was measured using a Beckman LS 6500 scintillation counter. Phosphatidylinositol 3-kinase activity assays were performed as previously described (41Krymskaya V.P. Penn R.B. Orsini M.J. Scott P.H. Plevin R.J. Walker T.R. Eszterhas A.J. Amrani Y. Chilvers E.R. Panettieri R.A. Am. J. Physiol. 1999; 277: L65-L78PubMed Google Scholar). The cells were washed twice with ice-cold wash buffer (137 mm NaCl, 20 mm Tris-HCl, 1 mmMgCl2, 1 mm CaCl2, 0.2 mm Na3VO4 (pH 7.5)) and lysed in lysis buffer (wash buffer plus 10% (v/v) glycerol, 1% (v/v) Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin) (42Krymskaya V.P. Hoffman R. Eszterhas A. Ciocca V. Panettieri R.A. Am. J. Physiol. 1997; 273: L1220-L1227Crossref PubMed Google Scholar). The lysates were centrifuged at 14,000 rpm for 10 min. Supernatants were incubated with anti-phosphotyrosine (Upstate Biotechnology) (2 μg/ml), anti-p85 (2 μg/ml) (Upstate Biotechnology), anti-p110α (2 μg/ml) (Santa Cruz Biotechnology), or anti-p110β (2 μg/ml) (Santa Cruz Biotechnology) antibodies. Protein A-Sepharose, 50 μl, was then added to the lysates for 2 h at 4 °C. The immunoprecipitates were washed 3 times in phosphate-buffered saline containing 1% Nonidet P-40, 2 times in 0.1m Tris-HCl (pH 7.5), 0.5 m LiCl, and 2 times in TNE (10 mm Tris-HCl, 100 mm NaCl, 1 mm EDTA (pH 7.5)). All solutions contained 0.2 mm vanadate. Sonicated phosphatidylinositol (Sigma) in Tris-HCl/EGTA (0.2 mg/ml, final concentration) was added to immunoprecipitates, and the phosphorylation reactions were started by the addition of MgCl2, ATP, and [γ-32P]ATP (30 μCi/sample) for 10 min at 30 °C. Reactions were stopped by the addition of 100 μl of 1 n HCl and extracted with 160 μl of chloroform-methanol (1:1). Lipids were separated on oxalate-coated TLC plates (Merck) using a solvent system of chloroform-methanol-water-ammonium hydroxide (60:40:11.3:2) and then detected by autoradiography. The position of [32P]phosphatidylinositol 3-monophosphate was determined by the position of a phosphatidylinositol phosphate standard that is separated on a TLC in parallel and developed in iodine vapor. Data points from individual assays represent the mean values ± S.E. Statistically significant differences among groups were assessed with the analysis of variance (ANOVA) (Bonferroni Dunn test), with values of p < 0.05 sufficient to reject the null hypothesis for all analyses. All experiments were designed with matched control conditions within each experiment to enable statistical comparison as paired samples. Because somatic mutation of the tumor suppressor TSC2 gene is associated with LAM disease, mutational analysis of the gene in LAMD-SM cells derived from LAM patients was performed and compared with the location of the mutation in the Eker rat and the mutations inDrosophila affecting cell growth. The TSC2 gene, comprising 41 exons, encodes for tuberin, a 200-kDa protein with predicted leucine zipper motive at amino acids 81–98 (exon 3), two small coiled-coil domains at amino acids 346–371 (exon 10) and 1008–1021 (exon 26), and a small region of homology to the Rap1 GAP at amino acids 1593–1631 (exons 34–38) (19European Chromosome 16 Tuberous Sclerosis ConsortiumCell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1542) Google Scholar, 43Halley D.J.J. Acta Genet. Med. Gemellol. 1996; 45: 63-75Crossref PubMed Google Scholar, 44Maheshwar M.M. Sandford R. Nellist M. Cheadle J.P. Sgotto B. Vauldin M. Sampson J.R. Hum. Mol. Genet. 1996; 5: 131-137Crossref PubMed Scopus (43) Google Scholar) (Fig.1). Mutational analysis of theTSC2 gene in LAMD-SM cells was performed on DNA and RNA derived from these cell cultures. Analysis of LAMD-SM-1 cells by reverse transcriptase-PCR indicated the presence of the rare variant C2580T in exon 22 of TSC2. This polymorphism has been seen previously at a frequency of 0.5% in TSC2(zk.bwh.harvard.edu/projects/tsc/polymorphisms.html), suggesting that it was present in hemizygous state in LAMD-SM-1 cells and that there was loss of one TSC2 allele due to a large genomic deletion. Further evidence for th