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

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, 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. 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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. 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DAF-16 with of the mammalian nuclear as is the for the mammalian homologs of We the effect of AKT phosphorylation and 14-3-3 association on several of DAF-16 including its ability to to the DNA and activate transcription. We evidence for PI 3-kinase-dependent inhibition of DAF-16 DNA binding activity via 14-3-3-dependent and 14-3-3-independent Thus, our suggest a mode of DAF-16 previously the site of to site of to site mutants the the site of the GAL4 DNA binding to to in a M. of of and the has previously G. J. Nature. 1998; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). a J. DAF-16 in the Forkhead DNA binding of DAF-16 as the 14-3-3 binding to and the by 14-3-3 binding a M. to vitro, and in cells and on of as the kinase and of DAF-16 as the in a kinase and with at for 14-3-3 in cells to to and with of AKT- phosphorylated and for at the on and phosphorylated DAF-16 by and by of of nuclear as in the with for at in a and of in to the of the with of DAF-16 of fusion the for of DAF-16 in mammalian for at to the of demonstrate inhibition of DNA binding by DAF-16 phosphorylated with for at by of 14-3-3 at for at which on at for and the to the HepG2 cells the in with and of to for In the in and of GAL4 DNA binding fusion protein with the gene a gene by GAL4 DNA binding sites of the for with and the in the absence of the of the 3-kinase inhibition enhances DAF-16 DNA binding and activity via an AKT/14-3-3 of DAF-16 DNA binding activity in cell nuclear cells DAF-16 and and for binding to as in A. and to of the and is inhibition of PI 3-kinase activity enhances binding of DAF-16 to nuclear of cells DAF-16 in and in the of as in for binding to the as in A and of DAF-16 in the nuclear and of the shown by inhibition of PI 3-kinase with enhances binding of DAF-16 AKT site mutants to nuclear cells with DAF-16 DAF-16 and DAF-16 and in in the of and and to as in A. of DAF-16 in the nuclear and by growth factors DAF-16 transcription HepG2 hepatoma cells with a gene the of the and a with to for cells to B and and in the absence A and and of and for and to the of inhibition of PI 3-kinase activity enhances transcription activity of DAF-16 and AKT site mutants DAF-16 and DAF-16 on the IGFBP·IRE. HepG2 cells with an the A and and and the gene and the gene cells in with and cells with and effect of is shown as the of inhibition of PI 3-kinase activity not activity of DAF-16 mutants on the GAL4 site. HepG2 cells with and the cells in with cells with activity for gene and is as the of the for the DAF-16/14-3-3 association cells in with of and of the DAF-16 and DNA binding cells with of the with the genetic that DAF-16 downstream of the PI kinase AKT elegans, AKT phosphorylation sites in DAF-16 has for the mammalian DAF-16 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, 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 J. T. Arden S. A. 1999; 96: PubMed Scopus 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, Y. Y. N. T. M. Y. S. A. 1999; 96: PubMed Scopus Google Scholar), AKT DAF-16 on at least of its AKT and sites as the sites in DAF-16 phosphorylated by AKT but not DAF-16 to This phosphorylation of DAF-16 at of by AKT its binding to mammalian in This association is by a to a 14-3-3 binding site on but not by the of the with association with 14-3-3 is also by mutation of the sites on DAF-16 with and In the site at a site to the 14-3-3 binding a site phosphorylation is for 14-3-3 binding in with of DAF-16 and activate transcription the in the 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, 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). also that DAF-16 binds to the A DAF-16 with a to in the DNA binding not to the an of DAF-16 that the DNA binding to and A DAF-16 the We AKT phosphorylation association of DAF-16 with 14-3-3 the ability of DAF-16 to its target site. of DAF-16 by AKT not by binding and the of 14-3-3 to DAF-16 in an inhibition of DAF-16 DNA binding activity and of 14-3-3 effect on DAF-16 DNA binding AKT and and the kinase the 14-3-3 binding the ability of 14-3-3 to DAF-16 DNA binding the effect and the of the binding for the Thus, the ability of 14-3-3 to DAF-16 DNA binding the association of 14-3-3 with DNA binding activity of DAF-16 mutants in ability to DAF-16 and DAF-16 by AKT/14-3-3 and with and and the DNA binding activity of the DAF-16 that the ability to 14-3-3 and the DAF-16 the ability to 14-3-3 AKT phosphorylation not 14-3-3 not its ability to DNA and of the 14-3-3 to DAF-16 DNA binding with the ability of 14-3-3 to DAF-16 but not DAF-16 DNA suggests that 14-3-3 with DAF-16 at sites and This interaction in the DNA binding of We shown that AKT phosphorylation of DAF-16 association of 14-3-3 and that association inhibits binding of DAF-16 to In HepG2 cells, insulin inhibits transcription by DAF-16 and effect the AKT/14-3-3 sites in DAF-16 N. Ogg S. Cahill C., W., B. Nui S. Dore J. Calvo D. Shi Y. Ruvkun G. Alexander-Bridges S. A. PubMed Scopus Google Scholar). insulin inhibition of DAF-16 activity results an interaction of DAF-16 with 14-3-3 that inhibits DNA not to insulin inhibition of DAF-16 activity the protein to the by of a DNA binding the effect of insulin on the activity of a fusion protein the GAL4 DNA binding and DAF-16 the DNA site in GAL4 DNA In HepG2 cells, DAF-16 in a transcription of the by A and and effect is by insulin by of constitutively AKT AKT site DAF-16 is to the effect of insulin and AKT on gene transcription to and Thus, in HepG2 cells, the effect of insulin and AKT on DAF-16 is 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 S. A. PubMed Scopus Google Scholar). DAF-16 and DAF-16 as fusion with the GAL4 DNA binding and response to insulin the the GAL4 DNA site to transcription. fusion protein gene transcription to the DAF-16 in the not activity the the activity by insulin A and the activity of which to not by insulin the and fusion transcription on the GAL4 DNA binding site by insulin and and that to insulin its activity is an site, but not a site, indicates that the response of fusion protein is to that of the DAF-16 to DAF-16 activity a direct effect on the transcription activity of nuclear of to the negative effect of insulin on the GAL4 and the DNA binding as the effect of insulin on the that effect is mediated at the of DAF-16 DNA the that is to insulin signaling the protein is to the GAL4 DNA target site suggests that 14-3-3 inhibition of DAF-16 DNA binding a in the negative of DAF-16 activity in DAF-16 to in DNA binding results that the association of DAF-16 with 14-3-3 a role in the negative of DAF-16 DNA HepG2 cells not DAF-16 to by DNA binding to direct effects of insulin on DAF-16 DNA binding in studies of 14-3-3 and DAF-16 in cells demonstrate that 14-3-3 and DAF-16 in cells and in cells in and of cells with the PI inhibitor a in association with and that PI 3-kinase signaling to AKT and phosphorylation of DAF-16 its association with mammalian 14-3-3 as for 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). Accordingly, that the association on the of the site at on DAF-16 in as is the in the at which not association of DAF-16 with 14-3-3 in vitro, 14-3-3 association in of site and sites in DAF-16/14-3-3 association ability of DAF-16 to with mammalian 14-3-3 with the of In cells, DAF-16 in the and in the nucleus and of DAF-16 in 14-3-3 DAF-16 and to the nucleus with and and and and Thus, DAF-16 with mammalian of PI 3-kinase signaling with a of DAF-16 to the nucleus and an of DAF-16 the and with and and that the C. transcription factor DAF-16 to mammalian and the mammalian in an to its mammalian homologs and 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, J. T. Arden S. A. 1999; 96: PubMed Scopus Google Scholar). that inhibition of PI 3-kinase signaling with to of DAF-16/14-3-3 in cells, the effect of on DAF-16 DNA binding activity in cells with DAF-16 of DAF-16 in cells by the on DAF-16 We DAF-16 DNA binding activity in the nuclear of cells in inhibition of PI 3-kinase by DAF-16 DNA binding activity and This not a of an in DAF-16 protein in the nuclear to nuclear of DAF-16 and Thus, the in DAF-16 DNA binding activity shown an in its DNA binding activity that negative of DAF-16 DNA binding activity by a PI We also the effect of PI 3-kinase inhibition on of DAF-16 AKT site mutants that not 14-3-3 in DAF-16 and DAF-16 and the DNA binding activity of DAF-16 and In cells mutants of DAF-16 in 14-3-3 DAF-16 and to the nucleus and and and to DAF-16 and the DNA binding activity of DAF-16 and DAF-16 enhanced by PI 3-kinase inhibition and and In the HepG2 cell inhibits the effect of factors on gene transcription by to the activity in cells PI 3-kinase inhibitor enhances gene transcription that in cells DAF-16 the A to and inhibits the activity of DAF-16 by and DAF-16 activity and activity of and of DAF-16 by PI 3-kinase inhibition in HepG2 cells DAF-16 in the as GAL4 fusion activity is in response to and activity is the GAL4 DNA binding site to direct gene not activate the Thus, that the effect of is also mediated at the of DNA binding in effect of on is its effect on and on the B to and which suggests that DAF-16 is to 14-3-3-dependent and by in ability of to the activity of DAF-16 AKT/14-3-3 site mutants that to the nucleus DAF-16 and indicates that a PI DAF-16 DNA binding and transcription activity. results the of at least two that to DAF-16 DNA binding in response to factors that activate PI 3-kinase-dependent signaling show that in to its role in nuclear of 14-3-3 binding of AKT- phosphorylated DAF-16 to DNA and pathway a PI 3-kinase-dependent pathway that inhibits the DNA binding activity of DAF-16 an AKT/14-3-3 site that 14-3-3 and is not to PI 3-kinase-dependent nuclear and ability of PI 3-kinase signaling to DAF-16 DNA binding of 14-3-3 a interaction of DAF-16 with an This an to 14-3-3 and DAF-16 DNA binding activity in response to PI 3-kinase the other a that to DAF-16 DNA binding activity DAF-16 in response to PI 3-kinase In a a kinase downstream of PI 3-kinase DAF-16 DAF-16 and ability to of DAF-16 DNA binding via and in with of 14-3-3 to DNA in with inhibition on on of insulin on GAL4 with DAF-16 and its ability to to the DAF-16 with and is the nucleus to the inhibits transcription of DAF-16 activity is on but not GAL4 DNA pointing to the of binding as a mode of by not the activity of the site DAF-16 a mode of DAF-16 is by DAF-16 which AKT not is not the nucleus DAF-16 is to DNA binding by the kinase inhibitor enhances DNA binding and transcription activity of DAF-16 and and mediates its effect at least in via an AKT by of GAL4 DAF-16 and on an but not a GAL4 DNA site, indicates that effect is mediated at the of DNA in a with DAF-16 and its ability to to the DAF-16 with and is the nucleus to the inhibits transcription of DAF-16 activity is on but not GAL4 DNA pointing to the of binding as a mode of by not the activity of the site DAF-16 a mode of DAF-16 is by DAF-16 which AKT not is not the nucleus DAF-16 is to DNA binding by the kinase inhibitor enhances DNA binding and transcription activity of DAF-16 and and mediates its effect at least in via an AKT by of GAL4 DAF-16 and on an but not a GAL4 DNA site, indicates that effect is mediated at the of DNA In HepG2 cells, that insulin inhibition of DAF-16 via an AKT/14-3-3 pathway with the ability of 14-3-3 to AKT phosphorylated that insulin to the activity of to the GAL4 DNA site, as to the DNA site, that is not to inhibition of DNA binding nuclear is to GAL4 Thus, in HepG2 and cells, growth factors that PI 3-kinase activity to DAF-16 DNA binding via an interaction with 14-3-3 and that is for nuclear that insulin inhibition of DAF-16 is by mutation of its AKT sites in HepG2 cells that of Guo 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), results for In a role for a kinase other AKT in mediating the effect of PI 3-kinase signaling on DAF-16 DNA binding and function. suggest that the PI 3-kinase activity in HepG2 and cells via a pathway that which mediates the effect of insulin in HepG2 insulin signaling via PI 3-kinase inhibits DAF-16 via its AKT sites in HepG2 cells the effect of to PI 3-kinase activity and DAF-16 DNA binding and transcription is on DAF-16 and DAF-16 in our and DAF-16 activity the in and HepG2 cells the negative effect of insulin (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, 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). Thus, that the PI 3-kinase activity in and HepG2 cells to a kinase other PI 3-kinase signaling to AKT the phosphorylation of a that with that growth factor signaling downstream of PI 3-kinase to the activity of is by in the hepatoma cell insulin signaling via an AKT inhibits the transcription activity of effect activity is the GAL4 DNA binding site the site M. A. J. Accili D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This is with a direct effect of insulin on FKHR transcription activity and suggests that insulin signaling to in is that the of for DAF-16 on genetic in C. (3Paradis S. Ruvkun G. Genes Dev. 1998; 12: 2488-2498Crossref PubMed Scopus (556) Google Scholar). the DAF-16 FKHRL1 of the site and the negative effect of insulin in cells, mutation of the AKT sites in FKHRL1 inhibits the effect of insulin by Yamasaki T. T. R.M. Granner D.K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). insulin of AKT not to the effects of PI 3-kinase activity on gene negative of gene in hepatoma cells downstream of PI 3-kinase the protein kinase and K. M. T. Y. K. K. M. Mol. Cell. Biol. 1998; Google Scholar). insulin and AKT activity in and insulin insulin phosphorylation of FKHR at site in cells J. Barr Accili D. J. PubMed Scopus Google and not nuclear of FKHR in In HepG2 cells insulin and activity in the absence of This suggests that the for DAF-16 also for mammalian homologues as 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) in HepG2 is that of FKHR protein show in B that the effect of to DAF-16 DNA binding activity is not to an in DAF-16 protein nuclear Thus, to a direct effect on the DNA binding activity of of of DAF-16 at the of DNA binding as as of by 14-3-3 the of the PI 3-kinase signaling to results for phosphorylation sites to nuclear nuclear and transcription of the target gene for A. Science. 1999; PubMed Scopus Google Scholar). the of DAF-16 and its mammalian homologues the that the diverse effects of insulin on the metabolism, growth, and survival of its target We and for of the We Nui for of insulin-like growth factor protein kinase B phosphatidylinositol kinase insulin-like growth factor binding protein-1 insulin response element phosphoenolpyruvate carboxykinase