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
Department of Molecular Biology,
Massachusetts General Hospital and Department of Genetics, Harvard
Medical School, Boston, Massachusetts 02114
Received for publication, November 3, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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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 (1-5). This pathway negatively regulates the activity of
DAF-16, a member of the Forkhead (FKH) family of transcription factors
(3, 6-8).
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 IGFBP·IRE.
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
RSXSpXP, or
RXXXSpXP where Sp
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 (23-25). 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).
The Thr-32 and Ser-253 sites are conserved within DAF-16 (Thr-54 and
Ser-240/Thr-242), FKHRL1 (Thr-32, Ser-253) (14), FKHR (Thr-24, Ser-253)
(32), and AFX (Thr-28, Ser-258) (15). Accordingly, regulation of
nuclear export by growth factor signaling to PI 3-kinase and AKT has
been demonstrated for FKHR1 (32), FKHR (33), and AFX (34). 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.
Plasmids and Reagents--
The DAF-16a1
HindIII/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-16a1
BstYI insert from pGEM-FLAG-DAF-16a1 was ligated into the
BamHI 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 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 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
[ 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 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.
Electrophoretic Mobility Shift Assay--
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 of
32P-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
MgCl2, 0.1 mM EDTA, 1 mM
dithiothreitol, 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 labeled
32P-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 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-
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.
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.
1B, compare lane 2 to lane
1). This antibody recognizes phosphorylation of DAF-16 at
threonine 54 (Fig. 1B, middle, lane
2 versus lane 3).
Phosphorylation of recombinant prokaryotic GST-DAF-16 by AKT induces
its binding to recombinant mammalian 14-3-3 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 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. 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 (Fig. 2B, compare
lanes 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. 2D, compare lanes
1 and 2 with lanes 4 and
5 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.
2D, 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. 2D, lanes
10 and 11). The inability of the
dimerization-deficient 14-3-3 mutant to inhibit DAF-16 DNA binding
(Fig. 2C, compare lane 3 with
lane 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.
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 IGFBP·IRE
(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-16-4A,
which fails to bind 14-3-3, was not affected by insulin (Fig. 3,
panel B, compare bars C and
D). By contrast, although the GAL4-DAF-16 and GAL4-DAF-16 4A
fusion proteins activated transcription similarly when assessed on the
GAL4 DNA binding site (bars E-H), neither
wild-type GAL4-DAF-16, nor GAL4-DAF-16-4A were inhibited by insulin
(panel B, bars E and
F and bars G and H).
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 3-kinase-specific inhibitor LY294002 caused a marked decrease in
14-3-3/DAF-16 association (Fig. 4A, compare lane
2 with 3 and lane 11 with
12). These findings suggested that PI 3-kinase signaling to
AKT and phosphorylation of DAF-16 could regulate its association with
mammalian 14-3-3 as it does for FKHRL1 (14).
Accordingly, we found that the 14-3-3/DAF-16 association depended on
the presence of the AKT-phosphorylation site at residue Thr-54 on
DAF-16 (Fig. 4A, lanes 13-15)
in vivo, as is the case in vitro. However, the
alanine mutations at residues 240/242, which did not prevent
association of DAF-16 2A with 14-3-3 in vitro, greatly
reduced 14-3-3 association in vivo (lanes
16-18). Mutation of site Thr-54 and sites Ser-240/Thr-242
in DAF 3A completely prevented DAF-16/14-3-3 association
(lanes 19-21).
The ability of DAF-16 derivatives to associate with mammalian 14-3-3 correlated with the subcellular localization of DAF-16. In
exponentially growing cells, recombinant DAF-16 was present both in the
cytoplasm and in the nucleus (Fig. 4B, lanes
2-4 and 15-17). Mutants of DAF-16 impaired in
14-3-3 binding, DAF-16 T54A, 2A, and 4A were confined strictly to the
nucleus (Fig. 4B, compare lanes 2 and
3 with lanes 5 and 6,
lanes 8 and 9, and lanes
11 and 12). Thus, DAF-16 can interact with
mammalian import/export proteins.
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.
We also examined the effect of PI 3-kinase inhibition on regulation of
DAF-16 AKT site mutants that do not bind 14-3-3 in vivo,
DAF-16 2A and DAF-16 4A (Fig. 5C, upper
panel). Both LY294002 and wortmannin increased the DNA
binding activity of wild-type DAF-16 (Fig. 5C,
upper panel, compare lane 4 to lanes 5 and 6). In exponentially
growing cells mutants of DAF-16 impaired in 14-3-3 binding, DAF-16 2A,
3A, and 4A were confined strictly to the nucleus (Fig. 5C,
lower panel, compare lanes
9 and 10, lanes 11 and
12, and lanes 13 and 14).
Nevertheless, similar to DAF-16 WT (Fig. 5C,
lanes 4 and 5), the DNA binding
activity of DAF-16 2A and DAF-16 4A was enhanced by PI 3-kinase
inhibition (Fig. 5C, upper panel,
compare lanes 7 and 8 and
lanes 9 and 10).
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 serum-deprived cells (bar A). The PI
3-kinase inhibitor enhances IGFBP-1 gene transcription 2-fold above
that seen in serum-starved 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 expression 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.
The enhancing effect of LY294002 on GAL4-DAF-16 WT is greater than its
enhancing effect on GAL4-DAF-16 3A and 4A 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.
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 (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-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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 (26, 27). 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).
-32P]ATP (10-20 µCi/reaction) (hot assay) at
30 °C for 40 min.
-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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in vitro
(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).
View larger version (30K):
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Fig. 1.
AKT phosphorylates DAF-16 on four distinct
sites and mediates 14-3-3 binding. A, linear map of
DAF-16 protein showing the amino acid sequence of the putative AKT
consensus (RXRXXS) phosphorylation sites at
Thr-54, Ser-240, Thr-242, and Ser-314. The indicated mutants were
constructed for expression in both mammalian and bacterial cells: 1A
(T54A); 2A (S240A and T242A); 3A (T54A, S240A, and T242A); 4A (all four
sites mutated to alanine). B, phosphorylation of GST-DAF-16
in vitro by AKT. Recombinant prokaryotic GST-fused DAF-16
(lanes 1 and 2) and the indicated
mutants (lanes 3-6), 2 µg each, were incubated
in a kinase buffer containing 2 µg of active recombinant mammalian
GST-AKT (lanes 2-6) or vehicle (lane
1) for 40 min at 30 °C. Following SDS-PAGE, the samples
were blotted with phosphopeptide antibodies against degenerated 14-3-3 binding consensus
(XXXXXRSXS(p)XPXXXXX) (a
gift from M. Comb). Autoradiogram showing GST-DAF-16 phosphorylation
(top) and immunoblot showing reactivity with the
phosphospecific antibodies (middle) and an anti-GST
immunoblot showing equal protein loading (bottom) are
presented. C, in vitro binding of AKT
phosphorylated GST-DAF-16 to 14-3-3. Unphosphorylated GST-DAF-16
(lane 1), AKT-phosphorylated GST-DAF-16
(lanes 2-4 and 9), or the indicated
GST-DAF-16 mutants (lanes 5-8) were incubated
with immobilized Myc-tagged 14-3-3 (prepared using anti-Myc antibodies
and protein A beads) from 293 cells (lanes 1-8)
or control beads (lane 9) in the presence of
competitor 14-3-3 binding phosphopeptide (PP, 1 mM, lane 3) or unphosphorylated
control peptide (P, 1 mM, lane
4) for 2 h at 4 °C. Following washes to remove
nonspecific binding, bound proteins retained on the immobilized
Myc-tagged 14-3-3 beads were subjected to SDS-PAGE, transferred to a
polyvinylidene difluoride membrane, and tested for phospho-GST-DAF-16
by autoradiography (top). Anti-GST immunoblot
(middle) was used for the detection of both DAF-16 WT and
AKT site mutant DAF-16 derivatives bound to the Myc-14-3-3 column. The
DAF-16 mutants 3A and 4A are only partially phosphorylated or not at
all and can not be detected by autoradiography. A Coomassie stain of
the blot (bottom) is shown to demonstrate equal 14-3-3 input.
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Fig. 2.
AKT phosphorylation and subsequent 14-3-3 binding inhibit DAF-16 DNA binding. A, DAF-16 binds to
IRE DNA. Prokaryotic recombinant GST-DAF-16 (lanes
1-5), GST-DAF-16 (L201P, mutation in the DNA binding
domain) (lane 6), and GST-DAF-16 (amino acids
1-69, lane 7), 2 µg each, were incubated with
32P-labeled IGFBP IRE (50,000 cpm) in electrophoretic
mobility shift assay binding buffer alone (lanes
1, 4, 6, and 7) or in the
presence of 10× cold competitor wild-type IRE (lane
2) or mutant IRE (lane 3) or in the
presence of anti-DAF-16 antibody (lane 5) and
resolved on a 4% nondenaturating gel as described under
"Experimental Procedures." An autoradiogram of the gel is
presented. The positions of DAF-16/DNA complexes and complexes
supershifted with antibody are indicated. B, AKT
phosphorylation of DAF-16 and 14-3-3 association prevents DAF-16
binding to IRE DNA. GST-DAF-16 (2 µg) was incubated in a kinase
buffer containing 2 µg of active GST-AKT (lanes
3-6) or vehicle (lane 1 and
2) for 40 min at 30 °C, followed by a 30-min incubation
with prokaryotic recombinant GST-14-3-3 (lanes 2 and 4-6), or vehicle (lanes 1 and
3). The presence of competitor phosphopeptide
(pp, 1 mM, lane 5) or
unphosphorylated peptide (p, 1 mM,
lane 6) is indicated. GST-DAF-16 was assayed for
DNA binding as in panel A. C,
Inhibition of DAF-16 binding to the IRE requires active AKT and an
intact 14-3-3 dimer. GST-DAF-16 (2 µg) was incubated in a kinase
buffer containing 2 µg of active GST-AKT in the presence
(lanes 1-6) or absence (lanes
7-10) of ATP, followed by a 30-min incubation with
GST-14-3-3 (lanes 2-5 and 7-9),
vehicle (lane 1), or a dimerization-deficient
GST-14-3-3 (dm, lanes 6 and
10) in the presence of competitor phosphopeptide
(pp, 1 mM, lanes 4 and
8) or unphosphorylated peptide (p, 1 mM, lanes 5 and 9). The
samples were assayed for DNA binding as in panel
A. D, inhibition of DAF-16 binding to the IRE by
AKT/14-3-3 requires intact AKT sites Thr-54 and Ser-314 on DAF-16.
GST-DAF-16 (lanes 1-3), GST-DAF-16(T54A)
(lanes 4-6), GST-DAF-16(4A) (lanes
7-9), GST-DAF-16(314A) (lanes
10-12), and GST-DAF-16(2A) (lanes
13-15), 2 µg each, were incubated in a kinase buffer
containing 2 µg of active GST-AKT (lanes 2,
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) 32P-IRE probes as in panel
A.
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Fig. 3.
Insulin inhibition of DAF-16 activity in
HepG2 cells is dependent on the AKT sites and is mediated at the level
of DNA binding. A, insulin and AKT/PKB inhibits the
transcription activity of DAF-16 WT but not AKT site mutant DAF-16 4A.
HepG2 cells were transiently cotransfected with the IGFBP-luciferase
reporter gene (15 µg) and the expression vector pcDNA3 (1 µg/ml) (bars A-C), wild-type
pcDNA·DAF-16 (1 µg/ml) (bars D-F), or a mutant of
DAF-16 in which an alanine residue was inserted in place of serine or
threonine at the four putative AKT sites, DAF-16 4A (1 µg/ml)
(bars G-I), and constitutively active PKB (2 µg/ml) (bars C, F, and
I). Serum-starved cells were incubated with insulin (1 milliunit/ml) (bars B, E, and
H) or vehicle (bars A, C,
D, F, G, and I) during the
last 16 h of the incubation. Luciferase activity is shown
corrected for -galactosidase gene expression. B, insulin
mediates its inhibitory effects on DAF-16 at the level of DNA binding.
HepG2 cells were transiently cotransfected with an expression vector
encoding wild-type GAL4·DAF-16 (bars A,
B, E, and F), or mutant GAL4·DAF-16
derivative 4A (bars C, D,
G, and H) and either the IGFBP-luciferase
reporter containing the IRE (bars A-D) or the
GAL4-LUC reporter gene (bars E-H) (15 µg)
together with the RSV-
-galactosidase reporter gene. Cells were
stimulated with vehicle (bars A, C,
E, and F) or with insulin (bars
B, D, F, and H) as
described in panel A. Luciferase activity was
corrected for
-galactosidase and plotted as percentage of the
unstimulated control.
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Fig. 4.
Serum growth factors signaling to PI 3-kinase
regulate DAF-16 nuclear/cytoplasmic distribution by regulating
DAF-16/14-3-3 association. A, in vivo
pull-down of Flag epitope-tagged DAF-16 with GST-tagged 14-3-3 or
GST-AKT. Flag epitope-tagged DAF-16 (lanes 2-4,
6-8, and 10-12), DAF-16 mutants; T54A
(lanes 13-15), 2A (lanes
16-18), 3A (lanes 19-21), or control vector
(lanes 1, 5, and 9) were
coexpressed in 293 cells with GST-14-3-3 (lanes
1-4 and 9-21) or GST-AKT (lanes
5-8). After transfection (24 h), cells were either grown in
serum (lanes 4, 8, 10,
13, 16, and 19) or were serum-deprived
in the presence of the PI 3-kinase inhibitor LY294002 (lanes
3, 7, 12, 15,
18, and 21) or vehicle (lanes
1, 2, 5, 6, 9,
11, 14, 17, and 20) for
24 h. Following affinity purification of GST fusions on GSH beads,
associated DAF-16 was detected by anti-Flag immunoblot (using
monoclonal antibody M2 (Sigma), top panel). The
blots were Coomassie-stained for GST fusion recovery evaluation
(middle panel). Representative samples of total
lysates were analyzed for DAF-16 expression by anti-Flag immunoblot
(bottom panel). B, subcellular
localization of DAF-16 WT and AKT site mutant derivatives in HEK 293 cells. Flag-epitope-tagged DAF-16 (lanes 2-4 and
15-23), DAF-16 mutants T54A (lanes
5-7), 2A (lanes 8-10), 4A
(lanes 11-13), or control vector
(lane 1) were expressed in 293 cells. 24 h
after transfection, cells were maintained in serum (lanes
1-17) or deprived of serum in the presence of PI 3-kinase
inhibitors: LY294002 (10 µM, lanes
18-20) or wortmannin (10 nM, lanes
21-23). Cells were harvested and fractionated into nuclear
and cytoplasmic fractions using the NE-PER kit (Pierce). 30 µg of
nuclear (N, lanes 3, 6,
9, 12, 16, 19, and
22), 100 µg of cytoplasmic (C, lanes
2, 5, 8, 11, 15,
18, and 21), and a combination of 15 µg of
nuclear and 50 µg of cytoplasmic (N/C, lanes
1, 4, 7, 10, 13,
14, 17, 20, and 23)
extracts were resolved on SDS-PAGE and assayed for the presence of
DAF-16 by anti-Flag immunoblot.
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Fig. 5.
PI 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 the
32P-labeled IGFBP-IRE as in Fig. 2A. Preimmune
serum (PI, lanes 1 and 4)
or anti-Flag antibody (M2, lanes 2 and
5) 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 2 and 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. 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 µM, lanes 2, 5,
8, and 10) or wortmannin (10 nM,
lanes 3 and 6). Binding to IGFBP·IRE
was assayed as in Fig. 2A. 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-
-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
-galactosidase (Tropix kit) expression according to the
manufacturers instructions. The mean ratios ± S.E. of
luciferase/
-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-
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, and
E) were compared with serum-starved cells stimulated with
LY294002 (bars B, D, and
F). Luciferase activity was normalized for
-galactosidase
gene expression and is presented as the percentage of the serum value
for each plasmid.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Inhibition of DAF-16 DNA binding via 14-3-3-dependent (I)
and -independent (II) pathways
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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.
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. (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 insulin-regulated,
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 SV40-transformed 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.
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ACKNOWLEDGEMENTS |
---|
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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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.
** To whom correspondence should be addressed: 306 Wellman, Diabetes Unit, Dept. of Medicine, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114. Tel.: 617-726-6998; Fax: 617-726-6954; E-mail: alex-bri@helix.mgh.harvard.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M010042200
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ABBREVIATIONS |
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The abbreviations used are: IGF, insulin-like growth factor; FKH, forkhead, PKB, protein kinase B; PI 3-kinase, phosphatidylinositol 3 kinase; IGFBP-1, insulin-like growth factor binding protein-1; IRE, insulin response element; PEPCK, phosphoenolpyruvate carboxykinase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; WT, wild-type.
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