Interaction of FoxO1 and TSC2 Induces Insulin Resistance through Activation of the Mammalian Target of Rapamycin/p70 S6K Pathway
2006; Elsevier BV; Volume: 281; Issue: 52 Linguagem: Inglês
10.1074/jbc.m608116200
ISSN1083-351X
AutoresYongheng Cao, Yuji Kamioka, Norihide Yokoi, Toshiyuki Kobayashi, Okio Hino, Masafumi Onodera, Naoki Mochizuki, Jun Nakae,
Tópico(s)Hippo pathway signaling and YAP/TAZ
ResumoBoth TSC2 (tuberin) and forkhead transcription factor FoxO1 are phosphorylated and inhibited by Akt and play important roles in insulin signaling. However, little is known about the relationship between TSC2 and FoxO1. Here we identified TSC2 as a FoxO1-binding protein by using a yeast two-hybrid screening with a murine islet cDNA library. Among FoxOs, only FoxO1 can be associated with TSC2. The physical association between the C terminus of TSC2 (amino acids 1280-1499) and FoxO1 degrades the TSC1-TSC2 complex and inhibits GTPase-activating protein activity of TSC2 toward Rheb. Overexpression of wild type FoxO1 enhances p70 S6K phosphorylation, whereas overexpression of TSC2 can reverse these effects. Knockdown of endogenous FOXO1 in human vascular endothelial cells decreased phosphorylation of p70 S6K. Prolonged overexpression of wild type FoxO1 enhanced phosphorylation of serine 307 of IRS1 and decreased phosphorylation of Akt and FoxO1 itself even in the presence of serum. These data suggest a novel mechanism by which FoxO1 regulates the insulin signaling pathway through negative regulation of TSC2 function. Both TSC2 (tuberin) and forkhead transcription factor FoxO1 are phosphorylated and inhibited by Akt and play important roles in insulin signaling. However, little is known about the relationship between TSC2 and FoxO1. Here we identified TSC2 as a FoxO1-binding protein by using a yeast two-hybrid screening with a murine islet cDNA library. Among FoxOs, only FoxO1 can be associated with TSC2. The physical association between the C terminus of TSC2 (amino acids 1280-1499) and FoxO1 degrades the TSC1-TSC2 complex and inhibits GTPase-activating protein activity of TSC2 toward Rheb. Overexpression of wild type FoxO1 enhances p70 S6K phosphorylation, whereas overexpression of TSC2 can reverse these effects. Knockdown of endogenous FOXO1 in human vascular endothelial cells decreased phosphorylation of p70 S6K. Prolonged overexpression of wild type FoxO1 enhanced phosphorylation of serine 307 of IRS1 and decreased phosphorylation of Akt and FoxO1 itself even in the presence of serum. These data suggest a novel mechanism by which FoxO1 regulates the insulin signaling pathway through negative regulation of TSC2 function. Forkhead transcription factors of the FoxO (Forkhead box-containing protein, O subfamily) family are conserved across many species. In Caenorhabditis elegans, DAF-16, the FoxO orthologue, is downstream of the DAF-2/AGE-1/AKT signaling pathway (1Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1532) Google Scholar, 2Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1202) Google Scholar). Loss-of-function of daf-2, age-1, or akt causes life span extension in a daf-16-dependent manner (3Lin K. Hsin H. Libina N. Kenyon C. Nat. Genet. 2001; 28: 139-145Crossref PubMed Scopus (750) Google Scholar). In Drosophila, the life span is extended over 50% by ablation of insulin-producing cells or mutations of genes encoding the insulin-like receptor (dInR) or its receptor substrate (chico) (4Stocker H. Hafen E. Curr. Opin. Genet. Dev. 2000; 10: 529-535Crossref PubMed Scopus (207) Google Scholar). Similarly, dInR phosphorylates and inactivates dFOXO, the Drosophila homologue of DAF-16/FOXO (5Puig O. Marr M.T. Ruhf M.L. Tjian R. Genes Dev. 2003; 17: 2006-2020Crossref PubMed Scopus (471) Google Scholar). In mammals, InsR/IGF1R-PI3K-Akt signaling inhibits transcription by FoxO1, FoxO3a, and FoxO4 (6Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1092) Google Scholar). These proteins possess a fork-head DNA binding domain consisting of around 110 amino acids and a transactivation domain in the C terminus. FoxOs bind to consensus FoxO-binding sites (GTAAA(C/T)A, T(G/A)TTTAC) in the promoter region of their target genes and activate gene expression (7Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (545) Google Scholar). It has been reported that FoxOs cause cell cycle arrest through induction of p27, p21, cyclin B, polo-like kinase, the retinoblastoma family-related protein p130 and cyclin G2, apoptosis through induction of Fas ligand and Bim, DNA repair through GADD45, stress resistance through MnSOD, and regulation of glucose and lipid metabolism through G6pase, apoC-III, and Igfbp-1 (8Greer E.L. Brunet A. Oncogene. 2005; 24: 7410-7425Crossref PubMed Scopus (1052) Google Scholar). Several FoxO-binding proteins, which include co-activators, transcription factors, signaling molecules, and Sirt1, a NAD-dependent deacetylase, have also been identified (8Greer E.L. Brunet A. Oncogene. 2005; 24: 7410-7425Crossref PubMed Scopus (1052) Google Scholar). These FoxO-binding molecules regulate FoxO-dependent transcription and vice versa. However, there have been few reports that described identification of FoxO-binding proteins by comprehensive strategies. The PI3K 2The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; DMEM, Dulbecco's modified Eagle's medium; HUVEC, human vascular endothelial cell; siRNA, small interfering RNA; RNAi, RNA interference; ANOVA, analysis of variance; WT, wild type; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; GAP, GTPase-activating protein; HA, hemagglutinin. 2The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; mTOR, mammalian target of rapamycin; TSC, tuberous sclerosis complex; DMEM, Dulbecco's modified Eagle's medium; HUVEC, human vascular endothelial cell; siRNA, small interfering RNA; RNAi, RNA interference; ANOVA, analysis of variance; WT, wild type; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; GAP, GTPase-activating protein; HA, hemagglutinin.-Akt pathway is also important for growth factor stimulation of mammalian target of rapamycin (mTOR) signaling (9Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1048) Google Scholar). The primary mechanism by which Akt activates mTOR signaling appears to be through direct phosphorylation and inhibition of TSC2 (also known as tuberin). TSC1 (also known as hamartin) and TSC2 were first identified as genes mutated in patients with tuberous sclerosis complex (TSC), an autosomal dominant disease. Affected patients suffer from hamartomas in a wide spectrum of organs. TSC1 and TSC2 physically associate in vivo and form a heterodimeric complex (10The European Chromosome 16 Tuberous Sclerosis Consortium.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1500) Google Scholar, 11Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3425) Google Scholar, 12Sparagana S.P. Roach E.S. Curr. Opin. Neurol. 2000; 13: 115-119Crossref PubMed Scopus (113) Google Scholar). TSC2 has been directly linked to cell size regulation by the discovery that mutation in dTsc2 leads to the gigas (large cell) phenotype (13Ito N. Rubin G.M. Cell. 1999; 96: 529-539Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Recent studies revealed that the TSC1-TSC2 complex functions downstream of Akt and upstream of target of rapamycin to restrict cell growth and cell proliferation (14Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell Biol. 2002; 4: 699-704Crossref PubMed Scopus (567) Google Scholar, 15Goncharova E.A. Goncharov D.A. Eszterhas A. Hunter D.S. Glassberg M.K. Yeung R.S. Walker C.L. Noonan D. Kwiatkowski D.J. Chou M.M. Panettieri Jr., R.A. Krymskaya V.P. J. Biol. Chem. 2002; 277: 30958-30967Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 16Kwiatkowski D.J. Zhang H. Bandura J.L. Heiberger K.M. Glogauer M. el-Hashemite N. Onda H. Hum. Mol. Genet. 2002; 11: 525-534Crossref PubMed Scopus (533) Google Scholar, 17Tee A.R. Fingar D.C. Manning B.D. Kwiatkowski D.J. Cantley L.C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13571-13576Crossref PubMed Scopus (690) Google Scholar). Akt-phosphorylation of TSC2 leads to the functional inactivation of the TSC1-TSC2 complex and results in mTOR activation leading to phosphorylation of two main mTOR substrates, ribosomal p70 S6 kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein (4E-BP1), and elevated mRNA translation (18Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2378) Google Scholar, 19Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar, 20Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (774) Google Scholar). The TSC2 C-terminal region has homology with the catalytic domain of GTPase-activating proteins (GAPs). An inhibitory target of TSC1-TSC2 has been identified as Ras homologue enriched in brain (Rheb), a small GTPase. GTP-bound Rheb is bound to and activates mTOR (21Manning B.D. Cantley L.C. Trends Biochem. Sci. 2003; 28: 573-576Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 22Li Y. Corradetti M.N. Inoki K. Guan K.L. Trends Biochem. Sci. 2004; 29: 32-38Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). There have been several reports about molecules, which regulate TSC2 function. Energy depletion inhibits mTOR signaling through AMP-activated kinase phosphorylation of TSC2, although it is not know how AMP-activated kinase phosphorylation of TSC2 enhances the ability of TSC1-TSC2 to inhibit downstream signaling to Rheb (23Corradetti M.N. Inoki K. Bardeesy N. DePinho R.A. Guan K.L. Genes Dev. 2004; 18: 1533-1538Crossref PubMed Scopus (465) Google Scholar, 24Inoki K. Zhu T. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (2971) Google Scholar, 25Shaw R.J. Bardeesy N. Manning B.D. Lopez L. Kosmatka M. DePinho R.A. Cantley L.C. Cancer Cell. 2004; 6: 91-99Abstract Full Text Full Text PDF PubMed Scopus (875) Google Scholar). The hypoxia-inducible gene, regulated in development and damage responses (REDD1), is also induced by energy depletion, and this leads to inhibition of mTOR complex 1 signaling to p70 S6K in a TSC2-dependent manner (26Sofer A. Lei K. Johannessen C.M. Ellisen L.W. Mol. Cell. Biol. 2005; 25: 5834-5845Crossref PubMed Scopus (346) Google Scholar). It is important to identify molecules that regulate TSC2 function because these molecules may affect the activity of mTOR/p70 S6K signaling and finally determine the activity of PI3K/Akt pathway through a negative feedback loop (27Um S.H. D'Alessio D. Thomas G. Cell Metab. 2006; 3: 393-402Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). In this study, we identified TSC2 as a novel FoxO1-binding protein by a yeast two-hybrid screening using a murine islet cDNA library. Binding of FoxO1 to TSC2 in cytoplasm inhibits TSC2 function and results in activation mTOR/p70 S6K and inhibition of Akt activity through negative feedback on IRS protein, leading eventually to feedback activation of FoxO1. Here we demonstrate a novel mechanism by which FoxO1 regulates activity of mTOR/p70 S6K signaling pathway and of FoxO1 itself through association with TSC2. Antibodies and Plasmids—We purchased anti-FLAG (M2) and anti-tubulin from Sigma; anti-c-Myc (9E10), anti-TSC2 (C-20 and N-19), anti-FOXO1 (H128), anti-FOXO1 (N18), anti-IRS2 from Santa Cruz Biotechnology; anti-HA (12CA5) from Roche Applied Science; anti-p70 S6K, anti-phospho-p70 S6K (Thr(P)-389), anti-phospho-FOXO1 (Ser(P)-256), anti-Akt, anti-phospho-Akt (Thr(P)-308 and Ser(P)-473), and anti-phospho-IRS-1 (Ser(P)-307) from Cell Signaling Technology; anti-TSC1 from Zymed Laboratories Inc.; anti-IRS1 from Upstate Biotechnology, Inc.; and anti-GFP from Dr. Michiyuki Matsuda, Osaka University. We used pCAG/FLAG-rTSC2DEE, pMT2/FLAG-p70 S6K (28Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.P. Kasuga M. Nishimoto I. Avruch J. J. Biol. Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar), pCMV5/cMyc/FoxO1 (29Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), pCMV5/cMyc/FoxO3a (30Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (501) Google Scholar), pTB701-FLAG-FOXO4 (31Takaishi H. Konishi H. Matsuzaki H. Ono Y. Shirai Y. Saito N. Kitamura T. Ogawa W. Kasuga M. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11836-11841Crossref PubMed Scopus (217) Google Scholar), pCA-EGFP-Rheb (32Kamioka Y. Fukuhara S. Sawa H. Nagashima K. Masuda M. Matsuda M. Mochizuki N. J. Biol. Chem. 2004; 279: 40091-40099Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and adenoviral vectors encoding wild type or mutant FoxO1 (30Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (501) Google Scholar). The pCAG/FLAG-rTSC2DEE was constructed by ligation of N-terminal FLAG-tagged full-length cDNA of rTSC2 with pCAG-GS vector (33Tsuchiya H. Orimoto K. Kobayashi K. Hino O. Cancer Res. 1996; 56: 429-433PubMed Google Scholar, 34Niwa H. Yamamura K. Miyazaki J. Gene (Amst.). 1991; 108: 193-199Crossref PubMed Scopus (4562) Google Scholar). Construction of Expression Vectors—For mutagenesis of FoxO1, we performed overlap extension PCR using pCMV5/cMyc/ADA FoxO1 as a template as described previously (35Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (250) Google Scholar). For construction of pCMV5/cMyc/T24A/S253A/S316A (3A), the following mutagenic primers were used: primer 1, 5′-AGA GCT GCG GCC ATG GAC AAC-3′, corresponding to nucleotide 844-864, and primer 2, 5′-GTT GTC CAT GGC CGC AGC TCT-3′, corresponding to nucleotide 864-844. Yeast Two-hybrid Screen—Amino acids 424-550 of the murine FoxO1 were cloned in-frame into the GAL4 DNA-binding domain plasmid pGBKT7 (Clontech). The GAL4 activation domain cDNA library of murine islets was constructed as described previously (36Nishimura M. Yokoi N. Miki T. Horikawa Y. Yoshioka H. Takeda J. Ohara O. Seino S. DNA Res. 2004; 11: 315-323Crossref PubMed Scopus (7) Google Scholar). AH109 yeast strain was used for the library search. The transformation was performed as described in the Clontech Matchmaker two-hybrid system 3 protocol. The transformants were plated on SD/-Ade/-His/-Leu/-Tyr plates in the presence of galactose and then were incubated at 30 °C for 3-4 days. Positive interaction was identified by strong β-galactosidase activity. Individual positive clones were isolated by YEASTMAKER™ yeast plasmid isolation kit (BD Biosciences) and were sequenced by ABI310 automated DNA sequencer and analyzed for homology with sequences in the GenBank™ data base using the BLAST algorithm. Cell Culture, Transfection, and Viral Transduction— HEK293 cells were cultured in DMEM containing 10% fetal calf serum. SV40-transformed hepatocytes used in these studies have been described in previous publication (29Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Human vascular endothelial cells (HUVEC) were cultured in HuMedia-EB2 (KURABO) supplemented with 2% fetal calf serum, 10 ng/ml human recombinant epidermal growth factor, 1 μg/ml hydrocortisone, 5 ng/ml human recombinant fibroblast growth factor, and 10 μg/ml heparin. Transient transfection was performed using Lipofectamine (Invitrogen) according to manufacturer's protocol. Adenoviral infection was described in a previous publication (30Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (501) Google Scholar). We transduced SV40-transformed hepatocytes by incubating them with adenoviral preparations at 10-50 multiplicities of infection for 2 h. Cell Isolation and Culture—Brown adipocytes and their precursor cells were isolated from newborn wild type mice by collagenase digestion as described previously (37Klein J. Fasshauer M. Ito M. Lowell B.B. Benito M. Kahn C.R. J. Biol. Chem. 1999; 274: 34795-34802Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Preadipocytes were immortalized by infection with the retroviral vector pGCDNsamIRES-Puro, encoding SV40T antigen (38Nabekura T. Otsu M. Nagasawa T. Nakauchi H. Onodera M. Mol. Ther. 2006; 13: 301-309Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and selected with puromycin (1 μg/ml). Preadipocytes were grown to confluence in culture medium supplemented with 50 nm insulin and 50 nm triiodothyronine (differentiation medium) (day 0). Adipocyte differentiation was induced by treating confluent cells for 24 h in differentiation medium further supplemented with 0.5 mm isobutylmethylxanthine, 0.5 μm dexamethasone, and 0.125 mm indomethacin. After induction, cells were changed back to differentiation medium, which was then changed every day. At day 5, cells were harvested and used for experiments. Western Blot Analysis—We lysed cells in buffer containing 50 mm Tris-HCl (pH 7.6), 250 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and protease inhibitors (Roche Applied Science). After centrifugation to remove insoluble material, each 30 μg of sample was electrophoresed in SDS-PAGE, and Western blotting was performed. For immunoprecipitation, cell lysates were diluted with buffer containing 50 mm Tris-HCl (pH 7.6), 150 mm NaCl, 0.1% Nonidet P-40, 10% glycerol, 5 mm MgCl2, and protease inhibitors. In Vitro Translation and Glutathione S-Transferase Fusion Protein Pulldown Assay—The TSC2 deletions were generated by PCR using specific primers, and they were cloned in-frame into the EcoRI and SalI sites of pGEX-4T-1. These fusion proteins were expressed in 20 μl of 50% slurry beads containing ∼2 μg of protein (either GST or alone, or fused to deleted TSC2 mutants), resuspended in 350 μl of binding buffer (50 mm Tris-HCl (pH 8.0), 120 mm NaCl, 1 mm EDTA, and 0.5% Nonidet P-40). This was mixed with 10 μlof in vitro translated wild type FoxO1 (Promega TnT reticulocyte lysate system kit). Binding was performed for 6 h at 4 °C. The beads were then washed four times with the binding buffer and resuspended in 2× SDS-PAGE sample buffer. Samples were then subjected to SDS-PAGE and transferred to a nitrocellulose membrane; the blot was incubated with anti-FOXO1 antibody and developed with an ECL detection system (Amersham Biosciences). Immunofluorescence—Immunofluorescence using SV40-transformed hepatocytes was performed as described previously (35Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (250) Google Scholar). After transient transfection with pCAG/FLAG-TSC2 using Lipofectamine (Invitrogen), SV40-transformed hepatocytes were transduced with adenovirus encoding HA-tagged wild type or ADA-FoxO1. HA-tagged FoxO1 was visualized with anti-HA monoclonal antibody and fluorescein isothiocyanate-conjugated anti-mouse IgG, and TSC2 was visualized with anti-TSC2 polyclonal antibody and rhodamine-conjugated anti-rabbit IgG. Measurement of GTP- and GDP-bound Rheb—SV40-transformed hepatocytes were cultured in 6-well plates and co-transfected with pCAG/FLAG-rTSC2DEE and pCA-EGFP-Rheb using Lipofectamine 2000 reagent (Invitrogen) and subsequently transduced with an adenovirus encoding WT FoxO1. At 48 h after transfection, the cells were washed once with phosphate-free DMEM (DMEM without sodium phosphate and sodium pyruvate; Invitrogen) and incubated with 1 ml of phosphate-free DMEM for 90 min. Cells were then incubated with 25 μCi of [32P]phosphate/ml (GE healthcare) for 4 h. After the labeling, cells were lysed with prechilled lysis buffer (0.5% Triton X-100, 20 mm Tris (pH 7.5), 150 mm NaCl, 20 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 10 μg of leupeptin/ml, 10 μg of aprotinin/ml; 600 μl per well of a 6-well plate). To avoid lysing the nuclei, the cells were incubated with lysis buffer for just 30 s with gentle shaking. The lysates were then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant (500 μl) was transferred to a fresh tube. Sixteen microliters of NaCl (500 mm) was added to 160 μl of supernatant to inhibit GAP activity in the lysates. To immunoprecipitate pCA-EGFP-Rheb, anti-green fluorescent protein and protein A-agarose (GE Healthcare) were added to the supernatant and incubated for 3 h at 4°C. The beads were washed with lysis buffer two times and with wash buffer (20 mm Tris (pH 7.5), 150 mm NaCl, 20 mm MgCl2) one time at 4 °C. The Rheb-bound nucleotides were eluted with 15 μl of elution buffer (20 mm Tris (pH 7.5), 20 mm EDTA, 2% SDS) at 68 °C for 10 min. Ten microliters of eluted nucleotides was then applied onto polyethyleneimine-cellulose plates. Before applying sample, the plate was soaked in methanol and dried with a hair dryer. The bottom portion of the plate was immersed in methanol again, and the plate was placed in a sealed chromatography chamber that was filled with 0.75 m KH2PO4 (pH 3.4) to a depth of 1 cm. The chamber was closed, and the solvent was allowed to ascend to the top of the plate. The plate was then removed and air-dried. GTP and GDP resolved by thin layer chromatography were visualized and quantified by a BAS-5000 (Fuji Film). Design and Transfection of siRNAs—We used BLOCK iT RNAi Designer (Invitrogen) to identify target siRNAs and used Stealth RNAi (Invitrogen). The FOXO1-specific sequence was 5′-AACUGCAGAUGUCUGCUGAGCAUGU-3′. HUVECs were transfected with Stealth RNAi using Lipofectamine 2000 according to the manufacturer's instruction (Invitrogen). At 48 h after transfection, cells were harvested and used for Western blotting. Statistics—We calculated descriptive statistics and ANOVA followed by Fisher's test using the Statview software (SAS Institute Inc.). Identification of TSC2 as a FoxO1-binding Protein—To identify proteins that bind to FoxO1, we performed a yeast two-hybrid screen using GAL4-FoxO1 fragment (amino acids 424-550) as bait and a murine islet cDNA library as prey. About 2 × 106 transformants were tested. The strongest colony was isolated and was found to encode the C-terminal fragment of murine TSC2 (amino acids 1280-1815). To confirm the interaction between TSC2 and FoxO1, we co-transfected HEK293 cells with pCAG/FLAG-rTSC2DEE and pCMV5/cMyc-WT FoxO1, and we immunoprecipitated cell lysates using normal mouse IgG or anti-c-Myc mouse antibody and blotted with anti-FLAG antibody. Reciprocal immunoprecipitation/immunoblotting using anti-FLAG and anti-c-Myc antibodies showed that the exogenously expressed FoxO1 could interact with FLAG-tagged full-length-TSC2 (Fig. 1a). Furthermore, to investigate whether endogenous FoxO1 is physically associated with TSC2 or not, cell lysates from brown adipocytes immortalized by SV40-T antigen were immunoprecipitated with anti-FOXO1 (Fig. 1b, lane 2) or anti-TSC2 (Fig. 1b, lane 5) and followed by an immunoblotting with using antibodies against TSC2 (Fig. 1b, lanes 1-3, top panel) or FOXO1 (Fig. 1b, lanes 4-6, top panel). In this cell line, both FoxO1 and TSC2 are expressed abundantly. Our results showed that endogenous FoxO1 was associated with endogenous TSC2 (Fig. 1b). These results suggest that FoxO1 interacts physically with TSC2 in vivo. TSC2 Binds to Only FoxO1 among FoxO Family Members— FoxOs consist of FoxO1, FoxO3a, and FoxO4. These molecules have highly conserved motifs, which include forkhead DNA binding domain, three Akt phosphorylation sites, and several acetylation sites. Therefore, it is interesting to investigate whether TSC2 is associated with other FoxOs or not. To examine whether all three FoxOs can interact with TSC2 equally, we transfected SV40-transformed hepatocytes with cMyc-FoxO1, -FoxO3a, or FLAG-FOXO4 and immunoprecipitated lysates with antibody against TSC2 or normal rabbit serum and immunoblotted with antibody against c-Myc or FLAG. This co-immunoprecipitation study demonstrated that only FoxO1 could interact with endogenous TSC2 (Fig. 2, lane 3, top panel). These data suggest that FoxO1 interacts with TSC2 among FoxOs specifically. Identification of FoxO1-binding Site in TSC2 Protein—To examine whether FoxO1 interacts with TSC2 directly, we constructed several glutathione S-transferase (GST) fusion TSC2 fragments. Because the yeast two-hybrid screening identified the C-terminal fragment of TSC2 (amino acids 1280-1815), we constructed several deleted Tsc2 mutants in this region (Fig. 3a). Using in vitro translated wild type FoxO1, pulldown assays with GST-deleted TSC2 fusion proteins were performed and showed that FoxO1 interacted with the C-terminal TSC2 fragment (amino acids 1280-1686) directly. The FoxO1-binding site on TSC2 protein encompasses amino acids 1280-1499 (Fig. 3b, lane 3), located near the GAP domain of TSC2. These data suggest that FoxO1 binds to TSC2 directly. FoxO1 Co-localizes with TSC2 in Cytoplasm—To examine subcellular localization of the FoxO1/TSC2 interaction, we transfected SV40-transformed hepatocytes with pCAG/FLAG-rTSC2DEE, transduced them with adenovirus encoding with HA-WT or ADA FoxO1 (30Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Investig. 2001; 108: 1359-1367Crossref PubMed Scopus (501) Google Scholar), and performed immunofluorescence using anti-TSC2 polyclonal and anti-HA monoclonal antibodies. In this cell line, even in the absence of serum, 70-80% of wild type FoxO1 was located in cytoplasm until 48 h after transduction (data not shown), where it co-localized with TSC2. In contrast, FLAG-TSC2 failed to co-localize with constitutively nuclear HA-tagged ADA FoxO1 (Fig. 4a). These data demonstrate that FoxO1 co-localizes with TSC2 in cytoplasm. Furthermore, to examine whether FoxO1 interacts with TSC2 in cytoplasm, we used a constitutively active mutant FoxO1 (3A FoxO1) in which all three Akt phosphorylation sites were mutated to alanine (T24A/S253A/S316A; 3A FoxO1) and performed co-immunoprecipitation experiments in the same cell line. Although exogenous WT FoxO1 interacted with TSC2 as well as in HEK293 cells (Fig. 1a), the 3A FoxO1 interacted with TSC2 weakly compared with wild type FoxO1 (Fig. 4b, lanes 2 and 3). These data suggest the possibility that FoxO1 may associate with TSC2 in cytoplasm. FoxO1 Inhibits TSC2 and Enhances p70 S6K Phosphorylation— TSC2 regulates cellular function mainly by their inhibitory effects on mTOR and its targets p70 S6K and 4E-BP1. It is important to elucidate whether binding of FoxO1 to TSC2 affects the activity of the mTOR/p70 S6K pathway. To investigate the effects of the FoxO1/TSC2 interaction on the mTOR pathway, we transfected SV40-transformed hepatocytes with FLAG-p70 S6K followed by transduction with adenovirus encoding HA-WT FoxO1 or -ADA FoxO1, which is localized in the nucleus and active constitutively and is immunoprecipitated with anti-FLAG monoclonal antibody and blotted with anti-phospho-p70 S6K (Thr(P)-389) antibody. After serum deprivation for 24 h, p70 S6K is dephosphorylated, and insulin increases phosphorylation of p70 S6K (Fig. 5a, lanes 1 and 2 and lanes 6 and 7). However, even in the absence of insulin, p70 S6K was phosphorylated in a dose-dependent manner of transduced WT FoxO1 (Fig. 5, a, lanes 3-5, and b, left panel). As described above, in this cell line, even after serum deprivation for 24 h, around 70-80% of transduced wild type FoxO1 is located in the cytoplasm (data not shown) and is phosphorylated (Fig. 5a, lanes 3-5). These data indicate that FoxO1 is constantly phosphorylated in this cell line even in the absence of serum and insulin. In contrast, p70 S6K was dephosphorylated in cells transduced with the ADA-FoxO1 (Fig. 5, a, lanes 8-10, and b, right panel). These data suggest that FoxO1 in cytosol enhances p70 S6K phosphorylation. To confirm whether enhanced phosphorylation of p70 S6K by FoxO1 is mediated through binding to TSC2, we overexpressed FLAG-TSC2 in SV40-transformed hepatocytes transduced with adenovirus encoding HA-WT FoxO1, and we investigated the effects on phosphorylation of p70 S6K. Overexpression of TSC2 decreased phosphorylation of p70 S6K (Fig. 5c, lanes 1 and 2). Overexpression of WT FoxO1 enhanced phosphorylation of p70 S6K (Fig. 5c, lanes 1 and 3). Even in the presence of WT FoxO1, overexpression of TSC2 decreased phosphorylation of p70 S6K (Fig. 5c, lanes 3 and 4). These data suggest that cytoplasmic FoxO1 enhances phosphorylation of p70 S6K through association with endogenous TSC2. These data also suggest the possibility that cytoplasmic FoxO1 may affect an inhibitory action of TSC2 onto mTOR and activate mTOR. FoxO1 Functions Upstream of mTOR for Activation of p70 S6K—To investigate whether enhanced phosphorylation of p70 S6K by overexpression of WT FoxO1 is mediated through mTOR activation, we treated cells with rapamycin (25 nm) and examined effects on p70 S6K phosphorylation. Overexpression of WT FoxO1 enhanced phosphorylation of p70 S6K (Fig. 6, lanes 2 and 3). In contrast, treatment with rapamycin abolished p70 S6K phosphorylation induced by overexpression of FoxO1 (Fig. 6, lane 4). These data suggest that FoxO1 enhances phosphorylation of p70 S6K through activation of mTOR and mTOR functions downstream of FoxO1 for p70 S6K phosphorylation. FoxO1 Reduces the Association between TSC1 and TSC2— Dimerization of TSC2 with TSC1 is important for functional inhibition on the mTOR/p70 S6K pathway. To elucidate the mechanism of how FoxO1 inhibits TSC2, we transduced HUVEC with adenovirus encoding WT FoxO1 and immunoprecipitated cell lysates with anti-TSC2 (Fig. 7a, lanes 3-5), anti-TSC1 (Fig. 7a, lanes 8-10), or anti-HA antibody (Fig. 7a, lanes 13-15) and immunoblotted with anti-TSC1, anti-TSC2, or anti-HA antibody. Overexpression of WT Fox
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