Characterization of Insulin Inhibition of Transactivation by a C-terminal Fragment of the Forkhead Transcription Factor Foxo1 in Rat Hepatoma Cells
2003; Elsevier BV; Volume: 278; Issue: 28 Linguagem: Inglês
10.1074/jbc.m212750200
ISSN1083-351X
AutoresValérie Perrot, Matthew M. Rechler,
Tópico(s)FOXO transcription factor regulation
ResumoThe transcription factor Foxo1 controls the expression of genes involved in fundamental cellular processes. In keeping with its important physiological roles, Foxo1 activity is negatively regulated in response to growth factors and cytokines that activate a phosphatidylinositol 3-kinase (PI 3-kinase) protein kinase B (PKB)/Akt pathway. PKB/Akt-mediated phosphorylation of Foxo1 has been shown to result in the inhibition of target gene transcription and to trigger the export of Foxo1 from the nucleus, which is generally believed to explain the subsequent decrease of transcription. In the present study, using a chimeric protein in which a C-terminal fragment of Foxo1 (amino acids 208–652) containing the transactivation domain is fused to the yeast Gal4 DNA binding domain, we present evidence showing that insulin can directly regulate transactivation by Foxo1 in H4IIE rat hepatoma cells. Insulin inhibition of Foxo1-(208–652)-stimulated transactivation is mediated by PI 3-kinase but in contrast to full-length Foxo1, does not require either of the two PKB/Akt phosphorylation sites (Ser253 and Ser316) present in the protein fragment. Using mutational and deletion studies, we identify two potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364 that are critical for insulin inhibition of transactivation by Foxo1-(208–652). We conclude that the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding and nuclear exclusion. These different regulatory mechanisms allow the precise control of transcription of Foxo1 target genes by insulin. The transcription factor Foxo1 controls the expression of genes involved in fundamental cellular processes. In keeping with its important physiological roles, Foxo1 activity is negatively regulated in response to growth factors and cytokines that activate a phosphatidylinositol 3-kinase (PI 3-kinase) protein kinase B (PKB)/Akt pathway. PKB/Akt-mediated phosphorylation of Foxo1 has been shown to result in the inhibition of target gene transcription and to trigger the export of Foxo1 from the nucleus, which is generally believed to explain the subsequent decrease of transcription. In the present study, using a chimeric protein in which a C-terminal fragment of Foxo1 (amino acids 208–652) containing the transactivation domain is fused to the yeast Gal4 DNA binding domain, we present evidence showing that insulin can directly regulate transactivation by Foxo1 in H4IIE rat hepatoma cells. Insulin inhibition of Foxo1-(208–652)-stimulated transactivation is mediated by PI 3-kinase but in contrast to full-length Foxo1, does not require either of the two PKB/Akt phosphorylation sites (Ser253 and Ser316) present in the protein fragment. Using mutational and deletion studies, we identify two potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364 that are critical for insulin inhibition of transactivation by Foxo1-(208–652). We conclude that the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding and nuclear exclusion. These different regulatory mechanisms allow the precise control of transcription of Foxo1 target genes by insulin. The forkhead transcription factor Foxo1 is a member of the FOXO subfamily of hepatocyte nuclear factor-3/forkhead ("winged helix") transcription factors (1Lai E. Clark K.L. Burley S.K. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10421-10423Crossref PubMed Scopus (301) Google Scholar, 2Kaufmann E. Knochel W. Mech. Dev. 1996; 57: 3-20Crossref PubMed Scopus (572) Google Scholar, 3Kaestner K.H. Knochel W. Martinez D.E. Genes Dev. 2000; 14: 142-146PubMed Google Scholar, 4Biggs III, W.H. Cavenee W.K. Mamm. Genome. 2001; 12: 416-425Crossref PubMed Scopus (173) Google Scholar). 1The human (FOXO) and mouse (Foxo) members of the FOXO subfamily of forkhead transcription factors were previously known as FKHR (FOXO1, Foxo1), FKHRL1 (FOXO3a, Foxo3), and AFX (FOXO4, Foxo4).1The human (FOXO) and mouse (Foxo) members of the FOXO subfamily of forkhead transcription factors were previously known as FKHR (FOXO1, Foxo1), FKHRL1 (FOXO3a, Foxo3), and AFX (FOXO4, Foxo4). FOXO1 was initially identified in an alveolar rhabdomyosarcoma following a chromosomal translocation that fused its C-terminal transactivation domain (amino acids 211–655) to the DNA binding domain of Pax3 (5Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher Jr., F. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (771) Google Scholar). Other members of the FOXO subfamily were subsequently identified and include FOXO3a (6Anderson M.J. Viars C.S. Czekay S. Cavenee W.K. Arden K.C. Genomics. 1998; 47: 187-199Crossref PubMed Scopus (283) Google Scholar) and FOXO4 (7Parry P. Wei Y. Evans G. Genes Chromosomes Cancer. 1994; 11: 79-84Crossref PubMed Scopus (139) Google Scholar, 8Borkhardt A. Repp R. Haas O.A. Leis T. Harbott J. Kreuder J. Hammermann J. Henn T. Lampert F. Oncogene. 1997; 14: 195-202Crossref PubMed Scopus (212) Google Scholar), as well as the Caenorhabditis elegans ortholog of FOXO1, Daf-16 (9Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1529) Google Scholar, 10Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1200) Google Scholar). The FOXO1 transcription factors regulate the expression of a wide variety of genes involved in the control of key cellular processes, such as cell survival, apoptosis (11Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 12Takaishi 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, 13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 14Dijkers P.F. Medema R.H. Lammers J.W. Koenderman L. Coffer P.J. Curr. Biol. 2000; 10: 1201-1204Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 15Suhara T. Kim H.S. Kirshenbaum L.A. Walsh K. Mol. Cell. Biol. 2002; 22: 680-691Crossref PubMed Scopus (134) Google Scholar, 16Tang T.T. Dowbenko D. Jackson A. Toney L. Lewin D.A. Dent A.L. Lasky L.A. J. Biol. Chem. 2002; 277: 14255-14265Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), cell cycle progression (17Medema R.H. Kops G.J. Bos J.L. Burgering B.M. Nature. 2000; 404: 782-787Crossref PubMed Scopus (1216) Google Scholar, 18Nakamura N. Ramaswamy S. Vazquez F. Signoretti S. Loda M. Sellers W.R. Mol. Cell. Biol. 2000; 20: 8969-8982Crossref PubMed Scopus (493) Google Scholar, 19Kops G.J. Medema R.H. Glassford J. Essers M.A. Dijkers P.F. Coffer P.J. Lam E.W. Burgering B.M. Mol. Cell. Biol. 2002; 22: 2025-2036Crossref PubMed Scopus (371) Google Scholar, 20Alvarez B. Martinez A.C. Burgering B.M. Carrera A.C. Nature. 2001; 413: 744-747Crossref PubMed Scopus (237) Google Scholar, 21Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Crossref PubMed Scopus (702) Google Scholar, 22Furukawa-Hibi Y. Yoshida-Araki K. Ohta T. Ikeda K. Motoyama N. J. Biol. Chem. 2002; 277: 26729-26732Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar), DNA repair (21Tran H. Brunet A. Grenier J.M. Datta S.R. Fornace Jr., A.J. DiStefano P.S. Chiang L.W. Greenberg M.E. Science. 2002; 296: 530-534Crossref PubMed Scopus (702) Google Scholar), protection against oxidative stress (22Furukawa-Hibi Y. Yoshida-Araki K. Ohta T. Ikeda K. Motoyama N. J. Biol. Chem. 2002; 277: 26729-26732Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 23Nemoto S. Finkel T. Science. 2002; 295: 2450-2452Crossref PubMed Scopus (732) Google Scholar), and differentiation (24Leenders H. Whiffield S. Benoist C. Mathis D. Eur. J. Immunol. 2000; 30: 2980-2990Crossref PubMed Scopus (54) Google Scholar, 25Nakae J. Kitamura T. Kitamura Y. Biggs W.H. Arden K.C. Accili D. Dev. Cell. 2003; 4: 119-129Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). Forkhead proteins also influence insulin sensitivity (26Nakae J. Biggs W.H.R. Kitamura T. Cavanee W.K. Wright C.V. Arden K.C. Accili D. Nat. Genet. 2002; 32: 245-253Crossref PubMed Scopus (531) Google Scholar) and participate in the transcriptional control of genes regulated by insulin, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, and insulin-like growth factor-binding protein-1 (IGFBP-1) 2The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; GFP, green fluorescent protein; MAP, mitogen-activated protein.2The abbreviations used are: IGFBP, insulin-like growth factor-binding protein; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; GFP, green fluorescent protein; MAP, mitogen-activated protein. (26Nakae J. Biggs W.H.R. Kitamura T. Cavanee W.K. Wright C.V. Arden K.C. Accili D. Nat. Genet. 2002; 32: 245-253Crossref PubMed Scopus (531) Google Scholar, 27Durham S.K. Suwanichkul A. Scheimann A.O. Yee D. Jackson J.G. Barr F.G. Powell D.R. Endocrinology. 1999; 140: 3140-3146Crossref PubMed Scopus (133) Google Scholar, 28O'Brien R.M. Streeper R.S. Ayala J.E. Stadelmaier B.T. Hornbuckle L.A. Biochem. Soc. Trans. 2001; 29: 552-558Crossref PubMed Scopus (0) Google Scholar, 29Hall R.K. Yamasaki T. Kucera T. Waltner-Law M. O'Brien R. Granner D.K. J. Biol. Chem. 2000; 275: 30169-30175Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 30Barthel A. Schmoll D. Kruger K.D. Bahrenberg G. Walther R. Roth R.A. Joost H.G. Biochem. Biophys. Res. Commun. 2001; 285: 897-902Crossref PubMed Scopus (95) Google Scholar, 31Nakae J. Kitamura T. Silver D.L. Accili D. J. Clin. Invest. 2001; 108: 1359-1367Crossref PubMed Scopus (501) Google Scholar). The FOXO proteins recognize and bind to an insulin response element (IRE) located in the promoter of these genes and activate transcription (27Durham S.K. Suwanichkul A. Scheimann A.O. Yee D. Jackson J.G. Barr F.G. Powell D.R. Endocrinology. 1999; 140: 3140-3146Crossref PubMed Scopus (133) Google Scholar, 29Hall R.K. Yamasaki T. Kucera T. Waltner-Law M. O'Brien R. Granner D.K. J. Biol. Chem. 2000; 275: 30169-30175Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 32Streeper R.S. Svitek C.A. Chapman S. Greenbaum L.E. Taub R. O'Brien R.M. J. Biol. Chem. 1997; 272: 11698-11701Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 33Furuyama T. Nakazawa T. Nakano I. Mori N. Biochem. J. 2000; 349: 629-634Crossref PubMed Scopus (544) Google Scholar). Members of the FOXO subfamily share several structural features. In addition to the highly conserved central DNA binding forkhead domain (Fox Box), they contain a C-terminal transactivation domain and three consensus phosphorylation sites for the serine/threonine protein kinase B/Akt (34Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (552) Google Scholar). For example, in Foxo1 used in the present study, these sites are located at Thr24, Ser253, and Ser316 (35Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). In vivo, the activity of these transcription factors is tightly regulated by phosphorylation of their PKB/Akt sites in response to insulin, insulin-like growth factor-I (IGF-I), and other growth factors that activate a PI 3-kinase/phosphoinositide-dependent kinase-1 (PDK-1) pathway leading to PKB/Akt activation. PKB/Akt-mediated phosphorylation of the forkhead proteins was shown to result in inhibition of target gene transcription. Phosphorylation of the PKB/Akt sites also caused the redistribution of the FOXO proteins from the nucleus to the cytoplasm (12Takaishi 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, 13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 36Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (250) Google Scholar, 37Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (939) Google Scholar). The resulting decrease in nuclear FOXO proteins has been proposed as a possible mechanism for the inhibition of FOXO-stimulated transcription (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar). The present study was initiated to understand the mechanism of insulin inhibition of transactivation of the IGFBP-1 promoter by Foxo1 and, in particular, to identify the sites in the forkhead protein that are mediating the insulin effect. We have used a fusion protein in which the yeast Gal4 DNA binding domain (1–147) was coupled to a C-terminal fragment of Foxo1 (Foxo1-(208–652)) that includes the transactivation domain and corresponds to the FOXO1 fragment (amino acids 211–655) originally identified in an alveolar rhabdomyosarcoma (5Galili N. Davis R.J. Fredericks W.J. Mukhopadhyay S. Rauscher Jr., F. Emanuel B.S. Rovera G. Barr F.G. Nat. Genet. 1993; 5: 230-235Crossref PubMed Scopus (771) Google Scholar). The Foxo1-(208–652) fusion protein was shown to stimulate transcription in an insulin-inhibitable manner (38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here, we demonstrate that in rat hepatoma cells, insulin inhibition of Foxo1-(208–652)-stimulated promoter activity occurs at the transactivation level rather than by affecting subcellular localization. We show that insulin inhibition is mediated through a PI 3-kinase pathway, but that the two consensus PKB/Akt phosphorylation sites present in the protein are not required. Using mutational and deletion studies, we identify two additional potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364 that are critical for insulin inhibition of transactivation by Foxo1-(208–652). Thus, the inhibition of Foxo1 by insulin is complex and involves multiple sites. The direct regulation of the transactivation activity of Foxo1-(208–652) by insulin provides an additional mechanism by which insulin promotes Foxo1 inhibition, thereby regulating the expression of genes controlled by this transcription factor. Materials—Restriction enzymes were purchased from Invitrogen. Taq polymerase was obtained from PerkinElmer Life Sciences-Applied Biosystems (Foster City, CA). Human recombinant insulin (Humulin U-100 regular) was obtained from Eli Lilly & Co. (Indianapolis, IN). LY294002, PD98059, and SB203580 were purchased from Calbiochem (San Diego, CA). Cell Lines—H4IIE rat hepatoma cells (39Ooi G.T. Brown D.R. Suh D.S. Tseng L.Y. Rechler M.M. J. Biol. Chem. 1993; 268: 16664-16672Abstract Full Text PDF PubMed Google Scholar) were grown as monolayer cultures in low glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and antibiotics (50 units/ml of penicillin and 50 μg/ml of streptomycin). Plasmid Constructs—The rat IGFBP-1 promoter-luciferase reporter plasmid (p925GL3) and the expression vector pCMV5/c-Myc-Foxo1, which contains the entire 652 amino acid coding region of Foxo1 were previously described (35Nakae J. Park B.C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). pG5E1b, a plasmid encoding a luciferase gene driven by an E1b promoter and five copies of the Gal4 binding element, kindly provided by Dr. A. Roberts (NCI, National Institutes of Health), was used as a reporter in Gal4 promoter assays (40Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (471) Google Scholar). C-terminal fragments of Foxo1 to be tested were expressed as Gal4 fusion proteins by subcloning the corresponding sequences in a pM2 expression vector (generously provided by Dr. I. Sadowski (University of British Columbia)) encoding the DNA binding domain of the yeast transcription factor Gal4 (40Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Crossref PubMed Scopus (471) Google Scholar, 41Sadowski I. Bell B. Broad P. Hollis M. Gene (Amst.). 1992; 118: 137-141Crossref PubMed Scopus (201) Google Scholar). C-terminal fragments of Foxo1, corresponding to amino acids 208–652, 256–652, 317–652, 332–652, 350–652, 365–652, 412–652, 461–652, 476–652, and 501–652 were obtained by PCR using the sense primers, 5′-ATACGTAGATATCAATTCAATTCGCCACAATCTG-3′; 5′-ATACGTAGATATCAACAACAGTAAATTTGCTAAGAGC-3′; 5′-ATACGTAGATATCAATGCTAGTACCATCAGTGGG-3′; 5′-ATACGTAGATATCCAGGATGACCTGGGAGAT-3′; 5′-ATACGTAGATATCGCCAAGATGGCGTCTACG-3′; 5′-ATACGTAGATATCCCAGAAAACATGGAGAAC-3′; 5′-ATACGTAGATATCCCAAACTACTCAAAGTAC-3′; 5′-ATACGTAGATATCGAGTTGTTGACTTCTGAC-3′; 5′-ATACGTAGATATCCCGGTTGATCCCGGAGTG-3′; 5′-ATACGTAGATATCATGCCAGCGTATGGCAGC-3′, respectively, and the antisense primer, 5′-GCTCTAGATTAGCCTGACACCCAGCTGTG-3′. The PCR fragments were digested with BamHI and XbaI and sequentially ligated into the BamHI and XbaI restriction sites of pM2 to generate pM2-Foxo1-(208–652), pM2-Foxo1-(256–652), pM2-Foxo1-(317–652), pM2-Foxo1-(332–652), pM2-Foxo1-(350–652), pM2-Foxo1-(365–652), pM2-Foxo1-(412–652), pM2-Foxo1-(461–652), pM2-Foxo1-(476–652), and pM2-Foxo1-(501–652). Overlap extension PCR was used to generate pM2-Foxo1-(208–652Δ379–460). The fragments corresponding to amino acids 208–378 and 461–652 contained a BamHI restriction site at the 5′-end and an XbaI site at the 3′-end, respectively. Following amplification of each individual fragment, a second PCR was carried out to generate a single fragment containing the deletion. This fragment was then subcloned into the BamHI-XbaI site of pM2. pM2-Foxo1-(208–652Δ350–364) was generated according to the same procedure. In addition, Foxo1-(208–652) and Gal4DBD-Foxo1-(208–652) were ligated into the EcoRI and XbaI sites of a pCEF expression vector containing a GFP epitope tag. Expression vectors encoding wild-type p85α (SRα-p85) and a deletion mutant of p85α (SRα-Δp85) that lacks a binding site for the p110 catalytic subunit of PI 3-kinase, were described previously (42Hara K. Yonezawa H. Sakaue A. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (416) Google Scholar, 43Quon M.J. Chen H. Ing B.L. Liu M.L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) and kindly provided by Dr. K. Yonezawa (Kobe University School of Medicine, Japan). The expression vector encoding FLAG-p300 was a generous gift from Dr. M. Kawabata (Cancer Institute of Japanese Foundation for Cancer and Research, Tokyo, Japan). The sequence of all constructs was confirmed by automated DNA sequencing using a rhodamine fluorescent terminator sequencing kit (PerkinElmer Life Sciences). Site-directed Mutagenesis—Mutations were introduced using a PCR-based method (QuikChange™ site-directed mutagenesis kit; Stratagene, La Jolla, CA) following the manufacturer's instructions. The two consensus PKB/Akt phosphorylation sites (Ser253 and Ser316) in pM2-Foxo1-(208–652) and pM2-Foxo1-(208–652Δ379–460) were mutated to alanine. Ser319/Thr320, Ser322/Ser326/Thr330, Ser354/Thr355/Ser358/Ser360/Ser363, Thr464/Ser465/Ser467/Ser475, and Ser499 were substituted with alanine in pM2-Foxo1-(208–652Δ379–460) and pM2-Foxo1-(208–652/Ser253Ala/Ser316AlaΔ379–460). Ser319 and Ser499, alone or together, were also mutated to alanine in pM2-Foxo1-(208–652) and pCEF-GFP-Foxo1-(208–652). Mutated oligonucleotides used for DNA amplification are available on request. The introduction of mutations was confirmed by DNA sequencing. Transient Transfection—H4IIE cells were transfected as described in previous publications (38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 44Suh D.S. Zhou Y. Ooi G.T. Rechler M.M. Mol. Endocrinol. 1996; 10: 1227-1237PubMed Google Scholar). Briefly, cells were seeded in 60-mm tissue culture dishes at a density of 3 × 106 cells/dish 24 h prior to transfection and were 70–80% confluent at the time of transfection. Transient transfection was carried out with a total of 3–10 μg of DNA using diethylaminoethyl (DEAE)-dextran (Amersham Biosciences). Plasmid DNA (100 μl) was mixed with 100 μl of DEAE dextran stock solution (2 mg/ml in 0.15 m NaCl) previously diluted with an equal volume of Tris-buffered saline (25 mm Tris, pH 7.5, 137 mm NaCl, 5 mm KCl, 0.7 mm CaCl2, 0.5 mm MgCl2, 0.6 mm Na2HPO4) and incubated at room temperature. After 15 min, the mixture was added to each dish, and, 15 min later, 3 ml of Dulbecco's modified Eagle's medium supplemented with 10% serum was added, and the incubation continued overnight. Forty-eight hours after transfection, the medium was replaced with serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin with or without recombinant human insulin (0.25 μg/ml), and the incubation was continued for 20 h before harvest. In some experiments, transfected cells were preincubated for 30 min with LY294002 (50 μm), a selective inhibitor of PI 3-kinase (45Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1000) Google Scholar), PD98059 (50 μm), a selective inhibitor of mitogen-activated protein kinase kinase (46Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3249) Google Scholar) or SB203580 (20 μm), a selective inhibitor of p38 MAP kinase (47Macfarlane W.M. Smith S.B. James R.F. Clifton A.D. Doza Y.N. Cohen P. Docherty K. J. Biol. Chem. 1997; 272: 20936-20944Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), before insulin was added. Transfection efficiency was monitored by cotransfecting pRSV-β-galactosidase (40 ng) kindly provided by Dr. P. Yen (NIDDK, National Institutes of Health). In each experiment, the total amount of DNA was adjusted by adding empty expression vectors. Luciferase and β-Galactosidase Assays—Cell lysates for luciferase and β-galactosidase assays were prepared according to the manufacturer's instructions (Tropix, PE Applied Biosystems, Bedford, MA) using a Lumat LB 9507 luminometer (EG&G Berthold, Germany). Luciferase and β-galactosidase activities were measured in the same tube using the Dual Light chemiluminescent reporter gene assay kit, allowing 1 h at room temperature for luciferin fluorescence to decay before measuring β-galactosidase activity. Assays were performed in duplicate. Immunoprecipitation and Western Blot Analysis—H4IIE cells were transfected with GFP-tagged Foxo1 208–652 and Foxo1 208–652/Ser253Ala/Ser316Ala using the LipofectAMINE™ Plus reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, cells were switched to serum-free medium overnight before incubation with recombinant human insulin (0.25 μg/ml) for the indicated times. Lysis was performed using 0.5 ml of lysis buffer containing 150 mm NaCl, 0.8 mm MgCl2, 5 mm EGTA, 50 mm Hepes, pH 7.5, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. Lysates clarified by centrifugation at 13,000 rpm for 10 min at 4 °C, were either directly separated by SDS-PAGE and transferred onto nitrocellulose membrane, and/or first immunoprecipitated with anti-GFP antibody (Covance). Western blots were detected using anti-FKHR phospho-Ser253 antibody (Cell Signaling Technology, Beverly, MA) and visualized by chemiluminescence (Pierce). The expression level of Foxo1 constructs was examined by immunoblotting analysis using anti-FKHR antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclei from HEK-293T human embryonic kidney cells transfected with GFP-tagged Foxo1-(208–652) or Foxo1-(208–652/Ser319Ala/Ser499Ala) and FLAG-tagged p300 were prepared using the nuclei EZ prep isolation buffer (Sigma). After immunoprecipitation with anti-GFP antibody, proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-FLAG antibody to detect p300 (Covance). Immunofluorescence—H4IIE cells were transiently transfected with the LipofectAMINE™ Plus reagent (Invitrogen) using c-Myc-tagged full-length Foxo1, GFP-tagged Foxo1-(208–652), Gal4DBD/Foxo1-(208–652) or the corresponding empty vector. After 48 h, cells were serum-starved overnight, before incubation with recombinant human insulin (1 μg/ml) for 1 h at 37 °C. Then, cells were washed twice with phosphate-buffered saline, fixed with 2% paraformaldehyde and mounted with mounting medium from Molecular Probes. Cells transfected with c-Myc-tagged full-length Foxo1, were serum-starved overnight, treated with or without insulin, and fixed as described above, before permeabilization with Triton X-100 and detection with anti-c-Myc antibody (clone 9E10) (Covance) followed by incubation with goat anti-mouse FITC-labeled secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA). Cells were visualized using a fluorescence microscope. Insulin Inhibition of Foxo1-(208–652)-stimulated Transactivation Does Not Result from Nuclear Exclusion—The nuclear export of the FOXO proteins was proposed as a mechanism to explain the decrease of transcription of their selected target genes in response to growth factors (13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar). We previously reported that insulin potently inhibits transactivation by a C-terminal fragment of Foxo1, residues 208–652 (38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Here, we examined whether insulin regulates the subcellular localization of Foxo1-(208–652) in H4IIE cells in the same way that it affects the distribution of full-length Foxo1 (residues 1–652). As shown in Fig. 1, in contrast to full-length Foxo1, which was mainly localized to the nucleus in the absence of insulin, GFP-Foxo1-(208–652) was predominantly cytoplasmic in all cells. When insulin was added, we observed, as reported by others (36Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (250) Google Scholar, 37Biggs III, W.H. Meisenhelder J. Hunter T. Cavenee W.K. Arden K.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7421-7426Crossref PubMed Scopus (939) Google Scholar), a dramatic redistribution of full-length Foxo1 to the cytoplasm with almost no nuclear staining remaining, whereas the subcellular localization of GFP-Foxo1-(208–652) showed little if any change. Similar results were obtained with GFP-Gal4DBD/Foxo1-(208–652). Thus, unlike full-length Foxo1, changes in the subcellular localization of Foxo1-(208–652) in response to insulin cannot account for the inhibition of transcription observed (38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Our results suggest that inhibition of the C-terminal Foxo1 fragment by insulin occurs at the transactivation level. Insulin Inhibition of Foxo1-(208–652)-stimulated Transactivation Does Not Require the Two Consensus PKB/Akt Phosphorylation Sites—Several studies reported that insulin inhibition of transcription stimulated by full-length Foxo1 requires the phosphorylation of three consensus sites (Thr24, Ser253, and Ser316) by PKB/Akt (11Tang E.D. Nunez G. Barr F.G. Guan K.L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (659) Google Scholar, 13Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5380) Google Scholar, 48Guo S. Rena G. Cichy S. He X. Cohen P. Unterman T. J. Biol. Chem. 1999; 274: 17184-17192Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). In contrast to what has been shown for full-length Foxo1, we suggested in a previous publication (38Tomizawa M. Kumar A. Perrot V. Nakae J. Accili D. Rechler M.M. J. Biol. Chem. 2000; 275: 7289-7295Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) that PKB/Akt was not involved in insulin inhibition of Foxo1-(208–652)-stimulated transactivation. To explore further the mechanism by which insulin inhibits Foxo1 activi
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