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 (Invitrogen) pcDNA3-Flag DAF-16a1. DAF-16a1 Bst YI from pGEM-FLAG-DAF-16a1 ligated the Bam HI site of pGEX-4T-1 (Amersham Pharmacia Biotech) gen- erate pGEX-DAF-16a1. Phosphorylation site mutants were prepared using the QuickChange site-directed mutagenesis kit (Stratagene). The DAF-16a1 Bst YI insert from pGEM-FLAG-DAF-16a1 was ligated into the Bam HI site of the GAL4 DNA binding domain plasmid to generate GAL4-DAF-16 derivatives. The rat IGF-BP-1 promoter (nucleotides 921 cloned in PGL3-LUC a gift from M. Preparation of pMT2-Myc-14-3-3, pGEX-GST-14-3-3, pGEX-GST-14-3-3 dimerization mutant pEBG-GST-AKT plasmid was a DAF-16 were using the Forkhead DNA binding domain of DAF-16 cloned into GST 14-3-3 phosphopeptide LPKINRSA(Sp)EPSLHR (PP, c-Raf-1 unphosphorylated syn- Anti-phosphopeptide 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.

In mammalian cells, insulin/IGF-1 signaling via PI 3-kinase and AKT mediates diverse effects on cell metabolism, growth, and survival (9 -11). 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 (12). In hepatoma cells, insulin-inhibition of IRE-directed gene transcription is mediated via a PI 3-kinase-dependent signaling pathway (13). 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 (14), AFX (15), and FKHR (16 -18) activate gene transcription via the IGFBP⅐IRE. Insulin/IGF-1 signaling (19 -21) or overexpression of AKT (17,19) stimulates phosphorylation of these factors and inhibits their activating effect (16,17).
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 (14). AKT preferentially phosphorylates substrates that carry the RXRXXS, which is contained within certain consensus 14-3-3 binding motifs RSXS p XP, or RXXXS p XP where S p represents phosphoserine (22). 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 (14). 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 (23)(24)(25). 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 (26,27), the DNA binding activity of p53 (28), and other targets (29 -31).
Kinase Assay-For experiments to phosphorylate DAF-16 in 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 g of GST-AKT as the kinase and 2 g 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 MgCl 2 , 2 mM dithiothreitol, and 100 M ATP (cold assay) supplemented with [␥-32 P]ATP (10 -20 Ci/reaction) (hot assay) at 30°C for 40 min.
Protein Interaction Assays-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 g 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.
Electrophoretic Mobility Shift Assay-Samples containing 2 g of GST-DAF-16 or 5-10 g of nuclear extracts, treated as indicated in the figure legends were incubated with 50,000 cpm of 32 P-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 mM MgCl 2 , 0.1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl, 10% glycerol, 0.1% bovine serum albumin, and 1 g of poly(dG/dC) in each sample. For competition assays, 10ϫ cold IRE or mutant IRE was added prior to the addition of 32 P-labeled IRE probe. For supershift assays, the reaction was pre-incubated with 1 g 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 32 P-labeled IRE probe. To demonstrate inhibition of DNA binding by 14-3-3, DAF-16 (2 g) was phosphorylated with GST-AKT (2 g) for 30 min at 30°C, followed by addition of 14-3-3 (2 g). The reaction was further incubated at 4°C for 15 min, at which time labeled 32 P-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.
Transfections-For transcriptional analysis, HepG2 cells were transfected using the CaPO 4 method in 30-mm six-well plates with IGFBP-LUC (15 g) reporter plasmid and pcDNA3-DAF-16 variants (2 g) or pcDNA3 control vector (2 g) per 1.5 ml of precipitate. The RSV-␤galactosidase vector (2 g) was used to control for transfection efficiency. In the experiments described in Figs

RESULTS
AKT Phosphorylates DAF-16 and Promotes Its Association with 14-3-3-Consistent with the genetic data that positions DAF-16 downstream of the PI 3-kinase-regulated serine/threonine kinase AKT in C. elegans, there are four consensus AKT phosphorylation sites in DAF-16 (Fig. 1A). As has been established for the mammalian DAF-16 orthologs FKHR (16,19,32), FKHRL1 (14), and AFX (15,34), 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. 1B, 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. 1C). 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 (compare lane 2 with lanes 3 and 4). The association with 14-3-3 is also inhibited by mutation of the AKT-phosphorylation sites on DAF-16 (compare lane 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).
14-3-3 Association with Wild-type DAF-16 Inhibits Its DNA Binding Activity-Homologues of DAF-16 bind and activate transcription through the IRE in the IGFBP gene (14,16). Accordingly we also find that DAF-16 binds specifically to the IRE ( Fig. 2A). A DAF-16 derivative L201P, with a leucine to proline substitution in the forkhead DNA binding domain, does not bind to the 32 P-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. 2A, 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. 2B, 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. 2B, compare lanes 3 and 4). The addition of 14-3-3 had no effect on DAF-16 DNA binding when AKT was omitted (Fig. 2B, compare lanes 2 and 4), or when ATP was omitted (Fig. 2C, 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 ( Insulin Inhibition of DAF-16 Activity Is Mediated at the Level of DNA Binding-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 (21). 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. 3A, 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 (16,21). DAF-16 WT and DAF-16 4A mutant were expressed as fusion proteins with the GAL4 DNA binding domain (Fig. 3, panel B) and their response to insulin was assessed using the IGFBP⅐IRE (bars A-D) or the GAL4 DNA site (bars E-H) to drive transcription. The GAL4-DAF-16 fusion protein stimulated basal IGFBP gene transcription 4-fold, identical to the DAF-16 derivatives expressed in the pcDNA expression system (data not shown). As expected, when activity was assessed using the IGFBP-1 promoter (containing the IRE), GAL4-DAF-16 activity was inhibited by insulin (Fig. 3, panel B,  compare bars A and B), while the activity of GAL4-DAF-16 - The observation that GAL4-DAF-16 responds to insulin when its activity is assessed using an IRE site, but not a GAL 4 site, indicates that the response of this fusion protein is analogous to that of the native DAF-16 protein. If insulin's action to inhibit DAF-16 activity resulted from a direct effect on the intrinsic transcription activity of GAL4-DAF-16 or from nuclear export of GAL4-DAF-16, we would expect to see the negative effect of insulin on both the GAL4 and the IRE DNA binding sites. Inasmuch as we observe the inhibitory effect of insulin on the IRE alone, we conclude that insulin's effect is mediated at the level of DAF-16 DNA binding. Furthermore, the observation that GAL4-DAF-16 is resistant to insulin signaling when the protein is tethered to the GAL4 DNA target site suggests that 14-3-3 inhibition of DAF-16 DNA binding may be a first step in the negative regulation of DAF-16 activity allowing subsequent changes in DAF-16 subcellular localization to occur.

PI 3-Kinase Signaling Regulates DAF-16/14-3-3 Interaction and Consequent Subcellular Distribution-Our in vitro DNA
binding results imply that the association of DAF-16 with 14-3-3 plays a crucial role in the negative regulation of DAF-16 DNA binding. As HepG2 cells do not express sufficient DAF-16 to enable detection by DNA binding assay, we were unable to study direct effects of insulin on DAF-16 DNA binding in these cells. However, coexpression studies of GST-tagged 14-3-3 and Flag-tagged DAF-16 proteins in 293 cells demonstrate that 14-3-3 and DAF-16 can associate both in serum-deprived cells and in cells growing exponentially in serum (Fig. 4A, compare  lanes 2 and 4). Treatment of serum-starved cells with the PI  5, 8, 11, and 14) or vehicle (all others) for 40 min at 30°C followed by 30-min incubation with prokaryotic recombinant GST-14-3-3 (lanes 2, 5, 8, 11, and 14) or vehicle (all others). The samples were assayed for binding to mutant (lanes 3, 6, 9, 12, and 15) or wild type (all others) 32 P-IRE probes as in panel A.
Inhibition of PI 3-kinase signaling with LY294002 caused a shift of DAF-16 to the nucleus and an almost complete disappearance of DAF-16 from the cytoplasm (Fig. 4B, compare lanes  15 and 16 with lanes 18 and 19 and lane 21 and 22). The finding that the C. elegans transcription factor DAF-16 can couple to mammalian AKT, 14-3-3, and the mammalian import/export machinery demonstrates it functions in an analogous manner to its mammalian homologs FKRH and FKHRL1 (14,32).

Inhibition of Endogenous PI 3-Kinase Signaling Enhances DAF-16 DNA Binding Activity Independent of DAF-16 AKT
Phosphorylation Sites-Having demonstrated that inhibition of PI 3-kinase signaling with LY294002 leads to dissociation of DAF-16/14-3-3 in 293 cells, we examined the effect of LY294002 on DAF-16 DNA binding activity in these cells. Nuclear extracts were isolated from 293 cells transiently transfected with expression plasmids encoding Flag-tagged DAF-16 (Fig. 5). The identity of DAF-16 overexpressed in HEK 293 cells was demonstrated by supershift experiments using antibodies against the Flag epitope tag on DAF-16 (Fig. 5A, compare lanes  2 and 5).
We detected low DAF-16 DNA binding activity in the nuclear extracts of 293 cells growing exponentially in serum (Fig. 5B,  lane 2); inhibition of PI 3-kinase by LY294002 markedly increased DAF-16 DNA binding activity (compare lanes 2 and 3). This increase was not simply a reflection of an increase in DAF-16 protein in the LY294002 treated nuclear extracts due to nuclear translocation, because extracts containing equal amounts of DAF-16 were employed (Fig. 5B, see Western blot (lower panel); compare lanes 4 and 6). Thus, the increase in DAF-16 DNA binding activity shown reflects an increase in its specific DNA binding activity indicating that LY294002 can prevent negative regulation of DAF-16 DNA binding activity by a PI 3-kinase-mediated mechanism.
In the insulin-responsive HepG2 cell line, serum inhibits the effect of endogenous factors on IGFBP gene transcription by 90% (Fig. 5D, bar B) relative to the activity seen in serumdeprived cells (bar A). The PI 3-kinase inhibitor enhances IG-

14-3-3-dependent and -independent Regulation of DAF-16
FBP-1 gene transcription 2-fold above that seen in serumstarved cells (compare bars A and C). DAF-16 activates the IGFBP promoter (compare bar A to bar D) and serum inhibits the activity of exogenous DAF-16 by 50% (compare bars D and E), while LY294002 increases DAF-16 activity 2.5-fold over control levels (compare bars D and F).
The transcriptional activity of both wild type and mutant derivatives of DAF-16 was similarly regulated by PI 3-kinase inhibition in HepG2 cells (Fig. 5E). Whether wild-type or mutant DAF-16 derivatives are expressed in the pcDNA ex-pression system (panel D) or as GAL4 fusion proteins compare (panel E), their activity is stimulated above basal in response to LY294002 (Fig. 5E, bars B, D, and F). However, when activity is assessed using the GAL4 DNA binding site to direct gene expression, LY294002 does not activate the GAL4-DAF-16 derivatives (Fig. 5F, bars B, D, and F). Thus, we conclude that the stimulatory effect of LY29004 is also mediated at the level of DNA binding in vivo.
on the IGFBP-IRE (Fig. 5E, compare bar B to bars D and F), which suggests that DAF-16 WT is subject to both 14-3-3-dependent and independent regulation by LY294002 in vivo. The ability of LY294002 to enhance the activity of DAF-16 AKT/14-3-3 site mutants that are confined strictly to the nucleus (Fig.  5C, lower panel, lanes 9 -14, DAF-16 2A, 3A, and 4A) indicates that a PI 3-kinase-responsive, 14-3-3/AKT site-independent mechanism can control DAF-16 DNA binding and transcription activity. 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 path-  Fig. 4B and assayed for binding to the IGFBP-IRE as described in Fig. 2A 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, and 9) or serum-deprived in the presence of LY294002 (10 M, lanes 2, 5, 8, and 10) or wortmannin (10 nM, lanes 3 and 6). Binding to IGFBP⅐IRE was assayed as in Fig. 2A. Lower panel,  ways. 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 (Table I 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-kinasedependent 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.
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 insulinmediated 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 FIG. 6. Proposed 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 binding in vivo may occur via a combination of pathways I and II.  14 -3-3-dependent (I) and -independent (II) pathways Pathway I, 14 -3-3 associates with AKT-phosphorylated DAF-16 WT in 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.

14-3-3-dependent and -independent Regulation of DAF-16
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. (16), 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 (14,16). 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 (35). 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 insulinregulated, AKT-independent mechanisms for DAF-16 regulation were proposed based on genetic data in C. elegans (3). 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% (36). 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 C and Rac (37). Third, although insulin and IGF-1 can stimulate AKT activity equivalently in wild-type and insulin receptor-deficient SV40transformed hepatocytes, respectively, only insulin stimulates phosphorylation of FKHR at site Thr-24 in these cells (33); 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 (16) in HepG2 cells. Although it is formally possible that LY294002 activation of endogenous FKHR could require new protein synthesis, we show in Fig. 5B 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 (38). 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.