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Phosphatidylinositol 3-Kinase Signaling Inhibits DAF-16 DNA Binding and Function via 14-3-3-dependent and 14-3-3-independent Pathways*

  • Catherine M. Cahill
    Footnotes
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‡Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the
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  • Guri Tzivion
    Footnotes
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ¶Diabetes Research Laboratory, Department of Molecular Biology, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, and the
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  • Nargis Nasrin
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‡Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the
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  • Scott Ogg
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‖Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114
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  • Justin Dore
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‡Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the
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  • Gary Ruvkun
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‖Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02114
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  • Maria Alexander-Bridges
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the

    ‡Diabetes Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, the
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  • Author Footnotes
    * This work was supported by National Institutes of Health NCI Grant CA73818-1, by National Institutes of Health Grant DK57200A01, by institutional support from Massachusetts General Hospital, and by National Institutes of Health Training Grant T32 DK07028–24 (to N. N.) and Grants AG05790 (to N. N.) and AG14161 and GM58012 (to S. O. and G. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § These authors contributed equally to this work.
Open AccessPublished:April 20, 2001DOI:https://doi.org/10.1074/jbc.M010042200
      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 (
      • Kimura K.D.
      • Tissenbaum H.A.
      • Liu Y.
      • Ruvkun G.
      ,
      • Morris J.Z.
      • Tissenbaum H.A.
      • Ruvkun G.
      ,
      • Paradis S.
      • Ruvkun G.
      ,
      • Paradis S.
      • Ailion M.
      • Toker A.
      • Thomas J.H.
      • Ruvkun G.
      ,
      • Ogg S.
      • Ruvkun G.
      ). This pathway negatively regulates the activity of DAF-16, a member of the Forkhead (FKH) family of transcription factors (
      • Paradis S.
      • Ruvkun G.
      ,
      • Lin K.
      • Dorman J.B.
      • Rodan A.
      • Kenyon C.
      ,
      • Ogg S.
      • Paradis S.
      • Gottlieb S.
      • Patterson G.I.
      • Lee L.
      • Tissenbaum H.A.
      • Ruvkun G.
      ,
      • Gottlieb S.
      • Ruvkun G.
      ).
      In mammalian cells, insulin/IGF-1 signaling via PI 3-kinase and AKT mediates diverse effects on cell metabolism, growth, and survival (
      • Alessi D.R.
      • Cohen P.
      ,
      • Cheatham B.
      • Vlahos C.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Khan C.
      ,
      • Avruch J.
      ). 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 (
      • O'Brien R.M.
      • Noisin E.L.
      • Suwanichkul A.
      • Yamasaki T.
      • Lucas P.C.
      • Wang J.
      • Powell D.R.
      • Granner D.K.
      ). In hepatoma cells, insulin- inhibition of IRE-directed gene transcription is mediated via a PI 3-kinase-dependent signaling pathway (
      • Cichy S.B.
      • Uddin S.
      • Danilkovich A.
      • Guo S.
      • Klippel A.
      • Unterman T.G.
      ). 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 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ), AFX (
      • Kops G.
      • de Ruiter N.
      • De Vries-Smits A.
      • Powell D.
      • Bos J.
      • Burgering B.
      ), and FKHR (
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ,
      • Tang E.
      • Nunez G.
      • Barr F.
      • Guan K.-L.
      ,
      • Durham S.
      • Suwanichkul A.
      • Scheimann A.
      • Yee D.
      • Jackson J.
      • Barr F.
      • Powell D.
      ) activate gene transcription via the IGFBP·IRE. Insulin/IGF-1 signaling (
      • Rena G.
      • Guo S.
      • Cichy S.
      • Unterman T.
      • Cohen P.
      ,
      • Nakae J.
      • Park B.-C.
      • Accili D.
      ,
      • Nasrin N.
      • Ogg S.
      • Cahill C., W., B.
      • Nui S.
      • Dore J.
      • Calvo D.
      • Shi Y.
      • Ruvkun G.
      • Alexander-Bridges M.C.
      ) or overexpression of AKT (
      • Tang E.
      • Nunez G.
      • Barr F.
      • Guan K.-L.
      ,
      • Rena G.
      • Guo S.
      • Cichy S.
      • Unterman T.
      • Cohen P.
      ) stimulates phosphorylation of these factors and inhibits their activating effect (
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ,
      • Tang E.
      • Nunez G.
      • Barr F.
      • Guan K.-L.
      ).
      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 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ). 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 (
      • Yaffe M.B.
      • Rittinger K.
      • Volinia S.
      • Caron P.R.
      • Aitken A.
      • Leffers H.
      • Smerdon S.J.
      • Cantley L.C.
      ). 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 (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ). 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 (
      • Yaffe M.B.
      • Rittinger K.
      • Volinia S.
      • Caron P.R.
      • Aitken A.
      • Leffers H.
      • Smerdon S.J.
      • Cantley L.C.
      ,
      • Kumagai A.
      • Dunphy W.G.
      ,
      • Yang J.
      • Winkler K.
      • Yoshida M.
      • Kornbluth S.
      ). 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 (
      • Tzivion G.
      • Luo Z.J.
      • Avruch J.
      ,
      • Luo Z.-J.
      • Zhang X.-F.
      • Rapp U.
      • Avruch J.
      ), the DNA binding activity of p53 (
      • Waterman M.
      • Stavridi E.
      • Waterman J.
      • Halazonetis T.
      ), and other targets (
      • Matta-Yelin M.
      • Aitken A.
      • Ravid S.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ,
      • Zha J.
      • Harda H.
      • Yang E.
      • Jockel J.
      • Korsmeyer S.J.
      ).
      The Thr-32 and Ser-253 sites are conserved within DAF-16 (Thr-54 and Ser-240/Thr-242), FKHRL1 (Thr-32, Ser-253) (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ), FKHR (Thr-24, Ser-253) (
      • Biggs W.H.
      • Meisenhelder J.
      • Hunter T.
      • Cavenee W.K.
      • Arden K.C.
      ), and AFX (Thr-28, Ser-258) (
      • Kops G.
      • de Ruiter N.
      • De Vries-Smits A.
      • Powell D.
      • Bos J.
      • Burgering B.
      ). Accordingly, regulation of nuclear export by growth factor signaling to PI 3-kinase and AKT has been demonstrated for FKHR1 (
      • Biggs W.H.
      • Meisenhelder J.
      • Hunter T.
      • Cavenee W.K.
      • Arden K.C.
      ), FKHR (
      • Nakae J.
      • Barr V.
      • Accili D.
      ), and AFX (
      • Takaishi H.
      • Konishi H.
      • Matsuzaki H.
      • Ono Y.
      • Shirai Y.
      • Saito N.
      • Kitamura T.
      • Ogawa W.
      • Kasuga M.
      • Kikkawa U.
      • Nishizuka Y.
      ). 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.

      DISCUSSION

      Our results reveal the existence of at least two mechanisms that cooperate to inhibit DAF-16 DNA binding in response to factors that activate PI 3-kinase-dependent signaling pathways. First, we show that in addition to its proposed role in promoting nuclear export/cytoplasmic retention of forkhead proteins, 14-3-3 can directly inhibit binding of AKT- phosphorylated DAF-16 to DNA (TableI and Fig.6, pathway I). Second we describe a novel PI 3-kinase-dependent pathway that inhibits the DNA binding activity of DAF-16 4A, an AKT/14-3-3 site mutant that cannot bind 14-3-3 and is not subject to PI 3-kinase-dependent nuclear export (Table I and Fig. 6,pathway II). The ability of endogenous PI 3-kinase signaling to prevent DAF-16 DNA binding independent of 14-3-3 may involve a phosphorylation-dependent interaction of DAF-16 with an interacting protein. This cofactor could have an analogous function to 14-3-3 and inhibit DAF-16 DNA binding activity in response to PI 3-kinase signaling. On the other hand, a cofactor that acts to stabilize DAF-16 DNA binding activity might dissociate from DAF-16 in response to PI 3-kinase signaling. In a third scenario, a non-AKT kinase (or phosphatase) downstream of endogenous PI 3-kinase could directly phosphorylate DAF-16 or DAF-16 4A and inhibit their ability to bind DNA.
      Table IInhibition of DAF-16 DNA binding via 14–3-3-dependent (I) and -independent (II) pathways
      PathwayDAF-16Association in vitro with 14-3-3Ability of 14-3-3 to inhibit DNA bindingin vitroAssociation in vivo with 14-3-3TranslocationInsulin inhibition on IRE siteLY294002 activation on IRE siteEffect of insulin or LY294002 on GAL4 site
      IWT++++++
      II4A+
      Pathway I, 14–3-3 associates with AKT-phosphorylated DAF-16 WTin vitro and blocks its ability to bind to the IRE DNA.In vivo DAF-16 WT associates with 14–3-3 and is translocated from the nucleus to the cytoplasm. Insulin inhibits transcription activation of DAF-16 WT when activity is assessed on IRE DNA, but not GAL4 DNA pointing to the importance of DAF-16/IRE binding as a mode of regulation by insulin. Insulin does not regulate the activity of the AKT/14–3-3 site mutant DAF-16 4A. Pathway II, a 14–3-3-independent mode of DAF-16 regulation is manifested by DAF-16 4A, which lacks all four AKT sites, does not bind 14–3-3, is not exported from the nucleus but, like DAF-16 WT, is subject to DNA binding regulation by the PI3 kinase inhibitor LY294002. LY294002 enhances DNA binding and transcription activity of both DAF-16 WT and 4A and therefore mediates its effect at least in part via an AKT site/14–3-3-independent pathway. Again regulation by LY294002 of GAL4 DAF-16 WT and 4A on an IRE but not a GAL4 DNA site, indicates that this effect is mediated primarily at the level of DNA binding.
      Figure thumbnail gr6
      Figure 6Proposed model of DAF-16 regulation by growth factor signaling to PI 3-kinase. Under conditions in which PI 3-kinase is inactive, DAF-16 is found in the nucleus and is bound to DNA. Pathway I, following growth factor stimulation and activation of PI 3-kinase, AKT phosphorylates DAF-16 on Thr-54, Ser-240/242, and Ser-314, 14-3-3 binds the Thr-54 and Ser-314 sites and prevents the interaction of DAF-16 with DNA. DAF-16 is then translocated to the cytoplasm. Pathway II, endogenous PI 3-kinase signaling to DAF-16 WT and DAF-16 4A, which lacks AKT/14-3-3 binding sites, inhibits their ability to binding DNA. This effect occurs in the absence of 14-3-3 association or DAF-16 translocation. We propose that endogenous PI 3-kinase activates a kinase (or phosphatase) other than AKT that phosphorylates DAF-16 4A and inhibits DAF-16 4A DNA binding activity directly or by recruiting a cofactor that interacts with DAF-16 in a manner analogous to 14-3-3. Alternatively AKT or another kinase could phosphorylate the cofactor that interacts with DAF-164A. Regulation of DAF-16 WT DNA bindingin vivo may occur via a combination of pathways I and II.
      In HepG2 cells, we find that insulin inhibition of DAF-16 function occurs via an AKT/14-3-3 site-dependent pathway (Fig. 6,pathway I), consistent with the observed ability of dimeric 14-3-3 to bind AKT phosphorylated DAF-16. Our observation that insulin fails to inhibit the activity of GAL4-DAF16 bound to the GAL4 DNA site, as opposed to the IRE DNA site, implies that GAL4-DAF-16 is not subject to insulin-mediated inhibition of DNA binding or nuclear export when it is tethered to GAL4 DNA. Thus, we propose that, in HepG2 and 293 cells, growth factors that regulate PI 3-kinase activity may act primarily to inhibit DAF-16 DNA binding via an interaction with 14-3-3 and that this step is permissive for nuclear export.
      Our finding that insulin inhibition of DAF-16 is prevented by mutation of its AKT sites in HepG2 cells confirms that of Guo et al.(
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ), who reported similar results for FKHR. In Fig. 6(pathway II), we propose a role for a kinase (or phosphatase) other than AKT in mediating the effect of PI 3-kinase signaling on DAF-16 DNA binding and function. Two observations suggest that the endogenous PI 3-kinase activity observed in serum-starved HepG2 and 293 cells may act via a distinct pathway from that which mediates the effect of insulin in HepG2 cells. First, whereas insulin signaling via PI 3-kinase inhibits DAF-16 function via its AKT sites in HepG2 cells (Fig. 3), the effect of LY294002 to inhibit endogenous PI 3-kinase activity and enhance DAF-16 DNA binding and transcription function is seen on both wild-type DAF-16 and DAF-16 4A (Fig. 5). Second, in our hands LY294002 stimulated wild-type and mutant DAF-16 4A activity over the control levels observed in serum-starved 293 and HepG2 cells (Fig. 5) rather than simply reversing the negative effect of serum or insulin (
      • Brunet A.
      • Bonni A.
      • Zigmond M.
      • Lin M.
      • Juo P.
      • Hu L.
      • Anderson M.
      • Arden K.
      • Blenis J.
      • Greenberg M.
      ,
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ). Thus, we conclude that the endogenous PI 3-kinase activity expressed in serum-starved 293 and HepG2 cells signals to a kinase other than AKT. Alternatively, endogenous PI 3-kinase signaling to AKT could modify the phosphorylation of a cofactor that interacts with DAF-16/Daf-16 4A.
      The observation that growth factor signaling activates distinct effectors downstream of PI 3-kinase to regulate the activity of DAF-16-like proteins is supported by three published reports. First, in the insulin-responsive H4 hepatoma cell line, insulin signaling via an AKT site-independent mechanism inhibits the transcription activity of GAL4-FKHR; this effect occurs whether activity is assessed using the GAL4 DNA binding site or the IGFBP-IRE site (
      • Tomizawa M.
      • Kumar A.
      • Perrot V.
      • Nakae J.
      • Accili D.
      • Rechler M.M.
      ). This observation is consistent with a direct effect of insulin on FKHR transcription activity or localization and suggests that distinct insulin signaling pathways to DAF-16-like FKH proteins may be operative in specific cells. It is notable that the existence of insulin-regulated, AKT-independent mechanisms for DAF-16 regulation were proposed based on genetic data in C. elegans (
      • Paradis S.
      • Ruvkun G.
      ). Second, although the DAF-16 homolog FKHRL1 can bind multimers of the PEPCK-IRE site and mediate the negative effect of insulin in H4IIE cells, mutation of the AKT sites in FKHRL1 inhibits the effect of insulin by 50% (
      • Hall R.K.
      • Yamasaki T.
      • Kucera T.
      • O'Brien R.M.
      • Granner D.K.
      ). Furthermore, insulin activation of AKT does not appear to explain all the effects of insulin-stimulated PI 3-kinase activity on PEPCK gene transcription; negative regulation of this gene in H4 hepatoma cells requires downstream effectors of PI 3-kinase distinct from AKT, the atypical protein kinase Cλ and Rac (
      • Kotani K.
      • Ogawa W.
      • Matsumoto M.
      • Kitamura T.
      • Sakaue H.
      • Hino Y.
      • Miyake K.
      • Sano W.
      • Akimoto K.
      • Kasuga M.
      ). Third, although insulin and IGF-1 can stimulate AKT activity equivalently in wild-type and insulin receptor-deficient SV40-transformed hepatocytes, respectively, only insulin stimulates phosphorylation of FKHR at site Thr-24 in these cells (
      • Nakae J.
      • Barr V.
      • Accili D.
      ); thus, only insulin, and not IGF-1, stimulates nuclear export of FKHR in these cells.
      In HepG2 cells both insulin and LY294002 regulate IGFBP promoter activity in the absence of exogenously expressed DAF-16. This observation suggests that the pathways we describe for DAF-16 are also relevant for endogenously expressed mammalian homologues such as FKHR (
      • Guo S.
      • Rena G.
      • Cichy S.
      • He X.
      • Cohen P.
      • Unterman T.
      ) in HepG2 cells. Although it is formally possible that LY294002 activation of endogenous FKHR could require new protein synthesis, we show in Fig. 5 B that the effect of LY294002 to enhance DAF-16 DNA binding activity is not due to an increase in DAF-16 protein expression or nuclear content. Thus, LY294002 appears to have a direct effect on the specific DNA binding activity of DAF-16.
      The proposed model of multistep regulation of DAF-16 at the level of DNA binding as well as regulation of subcellular localization by 14-3-3 underscores the complexity of the PI 3-kinase signaling pathways to forkhead proteins. Analogous results have been described for PHO4, where four distinct phosphorylation sites cooperate to regulate nuclear import, nuclear export, and transcription activation of the target gene for PHO5 (
      • Komeili A.
      • O'Shea E.K.
      ). Understanding the complex regulation of DAF-16 and its mammalian homologues will provide valuable insights into the mechanism that underlie the diverse effects of insulin on the metabolism, growth, and survival of its target tissues.

      Acknowledgments

      We thank Joseph Avruch, Jack Rogers, and Phil Daniel for critical reading of the manuscript. We thank Simin Nui for construction of GAL4·DAF-16 plasmids.

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