Phosphatidylinositol 3-Kinase Signaling Inhibits DAF-16 DNA Binding and Function via 14-3-3-dependent and 14-3-3-independent Pathways
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m010042200
ISSN1083-351X
AutoresCatherine M. Cahill, Guri Tzivion, Nargis Nasrin, Scott Ogg, Justin Dore, Gary Ruvkun, Maria Alexander-Bridges,
Tópico(s)14-3-3 protein interactions
ResumoIn Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C.elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/1433 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, our results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function. In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. We show that in mammalian cells, C.elegans DAF-16 is a direct target of AKT and that AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/1433 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, our results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function. In Caenorhabditis elegans, genetic evidence indicates that an insulin-like signaling pathway, which includes an insulin/IGF-11-like receptor (DAF-2), phosphatidylinositol 3-kinase (PI 3-kinase; AGE-1), and protein kinase B (also known as AKT) controls life cycle, metabolism, and longevity (1Kimura K.D. Tissenbaum H.A. Liu Y. Ruvkun G. Science. 1997; 277: 942-946Crossref PubMed Scopus (1725) Google Scholar, 2Morris J.Z. Tissenbaum H.A. Ruvkun G. Nature. 1996; 382: 536-539Crossref PubMed Scopus (702) Google Scholar, 3Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (556) Google Scholar, 4Paradis S. Ailion M. Toker A. Thomas J.H. Ruvkun G. Genes Dev. 1999; 13: 1438-1452Crossref PubMed Scopus (322) Google Scholar, 5Ogg S. Ruvkun G. Mol. Cell. 1998; 2: 887-893Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar). This pathway negatively regulates the activity of DAF-16, a member of the Forkhead (FKH) family of transcription factors (3Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (556) Google Scholar, 6Lin K. Dorman J.B. Rodan A. Kenyon C. Science. 1997; 278: 1319-1322Crossref PubMed Scopus (1209) Google Scholar, 7Ogg S. Paradis S. Gottlieb S. Patterson G.I. Lee L. Tissenbaum H.A. Ruvkun G. Nature. 1997; 389: 994-999Crossref PubMed Scopus (1543) Google Scholar, 8Gottlieb S. Ruvkun G. Genetics. 1994; 137: 107-120Crossref PubMed Google Scholar). In mammalian cells, insulin/IGF-1 signaling via PI 3-kinase and AKT mediates diverse effects on cell metabolism, growth, and survival (9Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (675) Google Scholar, 10Cheatham B. Vlahos C. Cheatham L. Wang L. Blenis J. Khan C. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1001) Google Scholar, 11Avruch J. Mol. Cell. Biochem. 1998; 183: 31-48Crossref Scopus (323) Google Scholar). Biochemical studies to date suggest that PI 3-kinase is important to the metabolic actions of insulin including its effects on gene transcription. A common DNA sequence, referred to as the insulin response element (IRE), binds members of the Forkhead transcription factor family and mediates the negative effect of insulin on transcription of the insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK) genes (12O'Brien R.M. Noisin E.L. Suwanichkul A. Yamasaki T. Lucas P.C. Wang J. Powell D.R. Granner D.K. Mol. Cell. Biol. 1995; 15: 1747-1758Crossref PubMed Google Scholar). In hepatoma cells, insulin- inhibition of IRE-directed gene transcription is mediated via a PI 3-kinase-dependent signaling pathway (13Cichy S.B. Uddin S. Danilkovich A. Guo S. Klippel A. Unterman T.G. J. Biol. Chem. 1998; 273: 6482-6487Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Accordingly, work in several laboratories aimed at identifying the downstream targets of insulin signaling to the nucleus has focused on the role of mammalian homologues of DAF-16, FKHR, FKHRL1, and AFX in mediating the negative effect of insulin/IGF-1 signaling on gene transcription. In the absence of insulin/IGF-1, FKHRL1 (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar), AFX (15Kops G. de Ruiter N. De Vries-Smits A. Powell D. Bos J. Burgering B. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar), and FKHR (16Guo 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, 17Tang E. Nunez G. Barr F. Guan K.-L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 18Durham S. Suwanichkul A. Scheimann A. Yee D. Jackson J. Barr F. Powell D. Endocrinology. 1999; 140: 3140-3146Crossref PubMed Scopus (133) Google Scholar) activate gene transcription via the IGFBP·IRE. Insulin/IGF-1 signaling (19Rena G. Guo S. Cichy S. Unterman T. Cohen P. J. Biol. Chem. 1999; 274: 17179-17183Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 20Nakae J. Park B.-C. Accili D. J. Biol. Chem. 1999; 274: 15982-15985Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 21Nasrin N. Ogg S. Cahill C., W., B. Nui S. Dore J. Calvo D. Shi Y. Ruvkun G. Alexander-Bridges M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10412-10417Crossref PubMed Scopus (141) Google Scholar) or overexpression of AKT (17Tang E. Nunez G. Barr F. Guan K.-L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 19Rena G. Guo S. Cichy S. Unterman T. Cohen P. J. Biol. Chem. 1999; 274: 17179-17183Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar) stimulates phosphorylation of these factors and inhibits their activating effect (16Guo 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, 17Tang E. Nunez G. Barr F. Guan K.-L. J. Biol. Chem. 1999; 274: 16741-16746Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). The prevailing view of the mechanism underlying insulin/IGF-1 inhibition of FKHRL1 and other DAF-16 homologs is that phosphorylation of FKHRL1 by AKT at two sites, Thr-32 and Ser-253 promotes retention of these proteins in the cytoplasm (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar). AKT preferentially phosphorylates substrates that carry the RXRXXS, which is contained within certain consensus 14-3-3 binding motifs RSXSp XP, or RXXXSp XP where Sprepresents phosphoserine (22Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Smerdon S.J. Cantley L.C. Cell. 1997; 7: 961-971Abstract Full Text Full Text PDF Scopus (1348) Google Scholar). Hence, AKT phosphorylation of its target proteins may create 14-3-3 binding sites. For example, the AKT site at T32 in FKHRL1 is a 14-3-3 consensus binding sequence; AKT phosphorylation of FKHRL1 at sites Thr-32 and Ser-253 promotes interaction of FKHRL1 with 14-3-3 and cytoplasmic retention of FKHRL1 (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar). The 14-3-3 family of proteins has also been shown to play a role in nuclear export and/or cytoplasmic retention of the yeast protein Cdc25 (23Yaffe M.B. Rittinger K. Volinia S. Caron P.R. Aitken A. Leffers H. Smerdon S.J. Cantley L.C. Nature. 1999; 13: 172-175Google Scholar, 24Kumagai A. Dunphy W.G. Genes Dev. 1999; 13: 1067-1072Crossref PubMed Scopus (255) Google Scholar, 25Yang J. Winkler K. Yoshida M. Kornbluth S. EMBO J. 1999; 18: 2174-2183Crossref PubMed Scopus (205) Google Scholar). In addition to promoting changes in cellular localization, binding of 14-3-3 to certain of its target proteins directly affects their activity. For example, 14-3-3 can stimulate the catalytic activity of the serine/threonine kinase c-Raf-1 (26Tzivion G. Luo Z.J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (388) Google Scholar, 27Luo Z.-J. Zhang X.-F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), the DNA binding activity of p53 (28Waterman M. Stavridi E. Waterman J. Halazonetis T. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (404) Google Scholar), and other targets (29Matta-Yelin M. Aitken A. Ravid S. Mol. Biol. Cell. 1997; 8: 1889-1899Crossref PubMed Scopus (25) Google Scholar, 30Datta S.R. Dudek H. Tao X. Masters S. Fu H. Gotoh Y. Greenberg M.E. Cell. 1997; 91: 231-241Abstract Full Text Full Text PDF PubMed Scopus (4946) Google Scholar, 31Zha J. Harda H. Yang E. Jockel J. Korsmeyer S.J. Cell. 1996; 87: 619-628Abstract Full Text Full Text PDF PubMed Scopus (2255) Google Scholar). The Thr-32 and Ser-253 sites are conserved within DAF-16 (Thr-54 and Ser-240/Thr-242), FKHRL1 (Thr-32, Ser-253) (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar), FKHR (Thr-24, Ser-253) (32Biggs 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 (942) Google Scholar), and AFX (Thr-28, Ser-258) (15Kops G. de Ruiter N. De Vries-Smits A. Powell D. Bos J. Burgering B. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar). Accordingly, regulation of nuclear export by growth factor signaling to PI 3-kinase and AKT has been demonstrated for FKHR1 (32Biggs 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 (942) Google Scholar), FKHR (33Nakae J. Barr V. Accili D. EMBO J. 2000; 19: 989-996Crossref PubMed Scopus (255) Google Scholar), and AFX (34Takaishi 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). We questioned whether the Thr-54 site in DAF-16 would function as a 14-3-3 binding site and, if so, whether PI 3-kinase signaling would regulate the interaction of C. elegans DAF-16 with elements of the mammalian nuclear import/export machinery as is the case for the mammalian homologs of DAF-16. We therefore examined the effect of AKT phosphorylation and 14-3-3 association on several aspects of DAF-16 function, including its ability to localize to the nucleus, bind DNA and activate transcription. We find evidence for PI 3-kinase-dependent inhibition of DAF-16 DNA binding activity via 14-3-3-dependent and 14-3-3-independent mechanisms. Thus, our observations suggest a more complex mode of DAF-16 regulation than previously anticipated. The DAF-16a1HindIII/NheI insert from pGEM-FLAG-DAF-16a1 was ligated into the HindIII/XbaI site of pcDNA3 (+) (Invitrogen) to generate pcDNA3-Flag DAF-16a1. The DAF-16a1BstYI insert from pGEM-FLAG-DAF-16a1 was ligated into theBamHI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to generate pGEX-DAF-16a1. Phosphorylation site mutants were prepared using the QuickChange site-directed mutagenesis kit (Stratagene). The DAF-16a1 BstYI insert from pGEM-FLAG-DAF-16a1 was ligated into the BamHI site of the GAL4 DNA binding domain plasmid to generate GAL4-DAF-16 derivatives. The rat IGF-BP-1 promoter (nucleotides −921 to +79) cloned in PGL3-LUC was a gift from M. Rechler (National Institutes of Health, Bethesda, MD). Preparation of pMT2-Myc-14-3-3, pGEX-GST-14-3-3, and the pGEX-GST-14-3-3 dimerization mutant has been described previously (26Tzivion G. Luo Z.J. Avruch J. Nature. 1998; 394: 88-92Crossref PubMed Scopus (388) Google Scholar, 27Luo Z.-J. Zhang X.-F. Rapp U. Avruch J. J. Biol. Chem. 1995; 270: 23681-23687Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The pEBG-GST-AKT plasmid was a gift from J. R. Woodgett (Toronto, Canada). Specific DAF-16 antibodies were produced in rabbits using the Forkhead DNA binding domain of DAF-16 cloned into GST as the antigen. The competitor 14-3-3 binding phosphopeptide LPKINRSA(Sp)EPSLHR (PP, corresponding to c-Raf-1 amino acids 613–627) and the unphosphorylated version (P) were synthesized by QCB (Boston, MA). Anti-phosphopeptide specific antibodies against 14-3-3 binding consensus were a gift from M. Comb (New England Biolabs, Beverly, MA). For experiments to phosphorylate DAF-16in vitro, GST-DAF-16 proteins were purified from bacteria and GST-AKT was expressed in 293 cells and subsequently affinity-purified on GSH beads (Amersham Pharmacia Biotech). Kinase assays were performed using 2 μg of GST-AKT as the kinase and 2 μg of GST-DAF-16 or DAF-16 mutant as the substrate in a kinase buffer containing 40 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 5 mm MgCl2, 2 mm dithiothreitol, and 100 μm ATP (cold assay) supplemented with [γ-32P]ATP (10–20 μCi/reaction) (hot assay) at 30 °C for 40 min. Myc epitope-tagged 14-3-3 expressed in 293 cells was absorbed to anti-Myc epitope antibodies (clone 9E10) pre-coupled to protein-A beads and incubated with 2 μg of AKT- phosphorylated wild-type and mutant GST-DAF-16 for 90 min at 4 °C. Following extensive washes, the associated proteins were separated on SDS-PAGE and phosphorylated DAF-16 was detected by autoradiography. Both wild-type and mutant GST-DAF-16 variants were detected by anti-GST immunoblotting. Samples containing 2 μg of GST-DAF-16 or 5–10 μg of nuclear extracts, treated as indicated in the figure legends were incubated with 50,000 cpm of32P-labeled IGFBP-IRE probe (caaaacaaacttattttgaa) or G-C/A-C mutant probe (caaaagaaacttcttttgaa) for 15 min at 4 °C in a buffer containing 40 mm Tris-HCl, pH 7.5, 5 mmMgCl2, 0.1 mm EDTA, 1 mmdithiothreitol, 50 mm KCl, 10% glycerol, 0.1% bovine serum albumin, and 1 μg of poly(dG/dC) in each sample. For competition assays, 10× cold IRE or mutant IRE was added prior to the addition of 32P-labeled IRE probe. For supershift assays, the reaction was pre-incubated with 1 μg of either specific DAF-16 antibody (for detection of GST fusion proteins) or M2 antibody (against the Flag tag for detection of DAF-16 expressed in mammalian cells) for 15 min at 4 °C prior to the addition of 32P-labeled IRE probe. To demonstrate inhibition of DNA binding by 14-3-3, DAF-16 (2 μg) was phosphorylated with GST-AKT (2 μg) for 30 min at 30 °C, followed by addition of 14-3-3 (2 μg). The reaction was further incubated at 4 °C for 15 min, at which time labeled32P-IRE probe was added. Samples were resolved on 4% Tris-glycine PAGE at 100 V for 3 h. Nuclear and cytoplasmic extracts were prepared using the NE-PER kit (Pierce) according to the manufacturer's instructions. For transcriptional analysis, HepG2 cells were transfected using the CaPO4 method in 30-mm six-well plates with IGFBP-LUC (15 μg) reporter plasmid and pcDNA3-DAF-16 variants (2 μg) or pcDNA3 control vector (2 μg) per 1.5 ml of precipitate. The RSV-β-galactosidase vector (2 μg) was used to control for transfection efficiency. In the experiments described in Figs. 3 and 5, 2 μg of GAL4 DNA binding domain control vector or GAL4-DAF-16 fusion protein vector variants cotransfected with either the IGFBP-luciferase reporter gene or a luciferase reporter gene driven by five GAL4 DNA binding sites cloned upstream of the TK109 promoter. Cells were shocked for 1 min with 10% Me2SO and the incubation continued in the absence of serum. Insulin was added during the last 16 h of the incubation.Figure 5PI 3-kinase inhibition enhances DAF-16 DNA binding and transcriptional activity via an AKT/14-3-3 site-independent pathway. A, identification of DAF-16 DNA binding activity in 293 cell nuclear extract. Nuclear extract was isolated from 293 cells expressing Flag epitope-tagged DAF-16 (M2-DAF-16) (lanes 3–5) or pcDNA alone (lanes 1 and 2) and assayed for binding to the32P-labeled IGFBP-IRE as in Fig. 2 A. Preimmune serum (PI, lanes 1 and 4) or anti-Flag antibody (M2, lanes 2 and5) was used to supershift DAF-16/DNA complexes. The location of the DAF-16/DNA complex and M2/DAF-16/DNA complex (supershift) is indicated. B, inhibition of endogenous PI 3-kinase activity enhances binding of DAF-16 to IRE DNA. Upper panel, nuclear extracts of 293 cells expressing Flag-epitope-tagged DAF-16 (M2-DAF-16) (lanes 2and 3) or vehicle (lane 1) grown in serum (lanes 1 and 2) or serum-deprived in the presence of LY294002 (10 μm,lane 3) were prepared as in Fig. 4 Band assayed for binding to the IGFBP-IRE as described in Fig.2 A and "Experimental Procedures." Lower panel, expression of DAF-16 in the nuclear (N) and cytoplasmic (C) fractions of the extracts shown was determined by anti-Flag immunoblotting. C, inhibition of endogenous PI 3-kinase with LY294002 enhances binding of DAF-16 AKT site mutants to IRE DNA. Upper panel, nuclear extract was isolated from 293 cells transfected with pcDNA alone (lanes 1–3), Flag-epitope-tagged DAF-16 (lanes 4–6), DAF-16 2A (lanes 7 and 8), or DAF-16 4A (lanes 9 and 10). Cells were grown in serum (lanes 1, 4, 7, and9) or serum-deprived in the presence of LY294002 (10 μm, lanes 2, 5,8, and 10) or wortmannin (10 nm,lanes 3 and 6). Binding to IGFBP·IRE was assayed as in Fig. 2 A. Lower panel, expression of DAF-16 in the nuclear (N) and cytoplasmic (C) fractions was determined by anti-Flag immunoblotting. D, serum growth factors regulate DAF-16 transcription activation. Insulin-responsive HepG2 hepatoma cells were cotransfected with a luciferase reporter gene under the control of the native IGFBP promoter (15 μg) and pcDNA3-DAF-16 (2 μg/ml) (bars D–F) or a control pcDNA3 vector (2 μg/ml) (bars A-C) together with RSV- β-galactosidase to correct for transfection efficiency. 4 h after transfection, cells were changed to serum-containing media (bars B and E) or serum deprivation media (starved) (bars A, C,D, and F) in the absence (bars A and D) or presence (bars C and F) of LY294002 (10 μm). Cells were harvested and assayed for luciferase (Promega kit) and β-galactosidase (Tropix kit) expression according to the manufacturers instructions. The mean ratios ± S.E. of luciferase/β-galactosidase triplicates are presented. E, inhibition of endogenous PI 3-kinase activity enhances transcription activity of DAF-16 WT and AKT site mutants DAF-16 3A and DAF-16 4A on the IGFBP·IRE. HepG2 cells were transiently cotransfected with an expression vector encoding the wild-type GAL4·DAF-16 (bars A and B), or mutant GAL4·DAF-16 derivatives 3A (bars C and D) or 4A (bars E and F) (2 μg), the IGFBP-luciferase reporter gene (15 μg), and the RSV-β galactosidase reporter gene (2 μg). Control cells growing exponentially in serum were stimulated with vehicle (bars A, C, and E) or serum-starved cells were stimulated with LY294002 (bars B, D, and F). The effect of LY294002 is shown as the percentage of control value. F, inhibition of endogenous PI 3-kinase activity does not affect transcriptional activity of DAF-16 WT or mutants on the GAL4 site. HepG2 cells were transiently cotransfected with GAL4·DAF-16 derivatives (2 μg/ml) and the GAL4-LUC (15 μg) reporter gene. Control cells growing exponentially in serum (bars A, C, andE) were compared with serum-starved cells stimulated with LY294002 (bars B, D, andF). Luciferase activity was normalized for β-galactosidase gene expression and is presented as the percentage of the serum value for each plasmid.View Large Image Figure ViewerDownload (PPT) For the DAF-16/14-3-3 association experiments, 293 cells were transfected using LipofectAMINE (Life Technologies, Inc.) in 10-cm plates with 2 μg each of GST-14-3-3 or GST-AKT and 4 μg of pcDNA3-DAF-16 variants. For the DAF-16 localization and DNA binding experiments, 293 cells were transfected with 5 μg of the pcDNA3-DAF-16 variants or pcDNA3 alone. Consistent with the genetic data that positions DAF-16 downstream of the PI 3-kinase-regulated serine/threonine kinase AKT inC. elegans, there are four consensus AKT phosphorylation sites in DAF-16 (Fig. 1 A). As has been established for the mammalian DAF-16 orthologs FKHR (16Guo 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, 19Rena G. Guo S. Cichy S. Unterman T. Cohen P. J. Biol. Chem. 1999; 274: 17179-17183Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar,32Biggs 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 (942) Google Scholar), FKHRL1 (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar), and AFX (15Kops G. de Ruiter N. De Vries-Smits A. Powell D. Bos J. Burgering B. Nature. 1999; 398: 630-634Crossref PubMed Scopus (952) Google Scholar, 34Takaishi 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), AKT can phosphorylate DAF-16 on at least three of its four potential AKT sites, and these sites serve as the only AKT-phosphorylation sites in vitro (Fig.1 B, top). Phosphospecific antibodies generated against 14-3-3-binding consensus sequences can specifically recognize DAF-16 phosphorylated by AKT but not unphosphorylated DAF-16 (Fig.1 B, compare lane 2 to lane 1). This antibody recognizes phosphorylation of DAF-16 at threonine 54 (Fig. 1 B, middle, lane 2 versus lane 3). Phosphorylation of recombinant prokaryotic GST-DAF-16 by AKT induces its binding to recombinant mammalian 14-3-3ζ in vitro(Fig. 1 C). This association is inhibited by a competitor phosphopeptide corresponding to a 14-3-3 binding site on c-Raf-1 but not by the unphosphorylated form of the peptide (comparelane 2 with lanes 3 and4). The association with 14-3-3 is also inhibited by mutation of the AKT-phosphorylation sites on DAF-16 (comparelane 2 with lanes 5,7, and 8). In particular, the AKT-phosphorylation site at threonine 54, a site matching closest to the 14-3-3 binding consensus, represents a site whose phosphorylation is indispensable for 14-3-3 binding in vitro (compare lane 2 with lane 5). Homologues of DAF-16 bind and activate transcription through the IRE in the IGFBP gene (14Brunet A. Bonni A. Zigmond M. Lin M. Juo P. Hu L. Anderson M. Arden K. Blenis J. Greenberg M. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 16Guo 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). Accordingly we also find that DAF-16 binds specifically to the IRE (Fig.2 A). A DAF-16 derivative L201P, with a leucine to proline substitution in the forkhead DNA binding domain, does not bind to the 32P-labeled IRE, nor does an amino-terminal fragment (1–69) of DAF-16 that lacks the forkhead DNA binding domain (Fig. 2, compare lane 4 to lanes 6 and 7). A specific antibody raised against DAF-16 supershifts the DAF-16/DNA complex (Fig. 2 A, lane 5). We examined whether AKT phosphorylation and/or subsequent association of DAF-16 with 14-3-3 could alter the ability of DAF-16 to bind its target IRE site. Phosphorylation of DAF-16 by AKT did not by itself affect DAF-16-DNA binding (Fig. 2 B, compare lanes 1 and 3); however, the addition of 14-3-3 to AKT-phosphorylated DAF-16 resulted in an almost complete inhibition of DAF-16 DNA binding activity (Fig. 2 B, comparelanes 3 and 4). The addition of 14-3-3 had no effect on DAF-16 DNA binding when AKT was omitted (Fig.2 B, compare lanes 2 and 4), or when ATP was omitted (Fig. 2 C, compare lanes 7 and 3) from the kinase reaction. Moreover, the competitor 14-3-3 binding phosphopeptide selectively blocked the ability of 14-3-3 to inhibit DAF-16 DNA binding while the unphosphorylated version had no effect (Fig. 2 B, comparelanes 5 and 6) demonstrating the requirement of the 14-3-3-phosphopeptide binding domain for the inhibition. Thus, the ability of 14-3-3 to inhibit DAF-16 DNA binding required the association of 14-3-3 with phospho-DAF-16. The DNA binding activity of DAF-16 mutants impaired in their ability to bind 14-3-3, DAF-16 54A (T54A), and DAF-16 4A (T54A, S240A, T242A, S314A) was unaffected by AKT/14-3-3 (Fig. 2 D, compare lanes 1 and 2 with lanes 4 and5 and lanes 7 and 8). Conversely, the DNA binding activity of the DAF-16 2A (240/242A) mutant that retains the ability to bind 14-3-3 was inhibited (Fig.2 D, lanes 13 and 14). Although the DAF-16 (S314A) mutant retains the ability to bind 14-3-3 following AKT phosphorylation (data not shown), 14-3-3 does not inhibit its ability to bind DNA (Fig. 2 D, lanes 10 and 11). The inability of the dimerization-deficient 14-3-3 mutant to inhibit DAF-16 DNA binding (Fig. 2 C, compare lane 3 withlane 6), together with the ability of wild- type 14-3-3 to inhibit mutant DAF-16 2A (S240A/T242A), but not mutant DAF-16 (T54A) or (S314A) DNA binding, suggests that dimeric 14-3-3 interacts with DAF-16 at sites Thr-54 and Ser-314. This interaction may, in turn, mask the forkhead DNA binding domain of DAF-16. We have shown that AKT phosphorylation of DAF-16 WT allows association of 14-3-3 and that this association inhibits binding of DAF-16 to DNA. In HepG2 cells, insulin inhibits transcription activation by DAF-16 and this effect requires the AKT/14-3-3 sites in DAF-16 (21Nasrin N. Ogg S. Cahill C., W., B. Nui S. Dore J. Calvo D. Shi Y. Ruvkun G. Alexander-Bridges M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10412-10417Crossref PubMed Scopus (141) Google Scholar). If insulin inhibition of DAF-16 activity results from an interaction of DAF-16 with 14-3-3 that inhibits DNA binding, we would not expect to see insulin inhibition of DAF-16 activity if the protein were tethered to the promoter by way of a heterologous DNA binding domain. Therefore, we compared the effect of insulin on the activity of a fusion protein encoding the GAL4 DNA binding domain and DAF-16 using the IRE DNA site in IGFBP-1 or GAL4 DNA (Fig.3). In HepG2 cells, DAF-16 expressed in a pcDNA vector activates transcription of the IGFBP promoter by 4-fold (Fig. 3 A, compare bars A and D) and this effect is inhibited by insulin (bar E) or by overexpression of constitutively active AKT (bar F). The AKT site mutant DAF-16 4A is resistant to the effect of insulin and AKT on IGFBP gene transcription (compare bar G to bars H and I, respectively). Thus, in HepG2 cells, the inhibitory effect of insulin and AKT on DAF-16 is dependent on its AKT/14-3-3 sites (16Guo 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, 21Nasrin N. Ogg S. Cahill C., W., B. Nui S. Dore J. Calvo D. Shi Y. Ruvkun G. Alexander-Bridges M.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10
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