From the Growth and Development Section, Diabetes Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892
Received for publication, December 13, 2002 , and in revised form, April 14, 2003.
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ABSTRACT |
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INTRODUCTION |
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The FOXO1 transcription factors regulate the expression of a wide variety of genes involved in the control of key cellular processes, such as cell survival, apoptosis (1116), cell cycle progression (1722), DNA repair (21), protection against oxidative stress (22, 23), and differentiation (24, 25). Forkhead proteins also influence insulin sensitivity (26) and participate in the transcriptional control of genes regulated by insulin, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase, and insulin-like growth factor-binding protein-1 (IGFBP-1)2 (2631). The FOXO proteins recognize and bind to an insulin response element (IRE) located in the promoter of these genes and activate transcription (27, 29, 32, 33).
Members of the FOXO subfamily share several structural features. In addition to the highly conserved central DNA binding forkhead domain (Fox Box), they contain a C-terminal transactivation domain and three consensus phosphorylation sites for the serine/threonine protein kinase B/Akt (34). For example, in Foxo1 used in the present study, these sites are located at Thr24, Ser253, and Ser316 (35). In vivo, the activity of these transcription factors is tightly regulated by phosphorylation of their PKB/Akt sites in response to insulin, insulin-like growth factor-I (IGF-I), and other growth factors that activate a PI 3-kinase/phosphoinositide-dependent kinase-1 (PDK-1) pathway leading to PKB/Akt activation. PKB/Akt-mediated phosphorylation of the forkhead proteins was shown to result in inhibition of target gene transcription. Phosphorylation of the PKB/Akt sites also caused the redistribution of the FOXO proteins from the nucleus to the cytoplasm (12, 13, 36, 37). The resulting decrease in nuclear FOXO proteins has been proposed as a possible mechanism for the inhibition of FOXO-stimulated transcription (13).
The present study was initiated to understand the mechanism of insulin inhibition of transactivation of the IGFBP-1 promoter by Foxo1 and, in particular, to identify the sites in the forkhead protein that are mediating the insulin effect. We have used a fusion protein in which the yeast Gal4 DNA binding domain (1147) was coupled to a C-terminal fragment of Foxo1 (Foxo1-(208652)) that includes the transactivation domain and corresponds to the FOXO1 fragment (amino acids 211655) originally identified in an alveolar rhabdomyosarcoma (5). The Foxo1-(208652) fusion protein was shown to stimulate transcription in an insulin-inhibitable manner (38). Here, we demonstrate that in rat hepatoma cells, insulin inhibition of Foxo1-(208652)-stimulated promoter activity occurs at the transactivation level rather than by affecting subcellular localization. We show that insulin inhibition is mediated through a PI 3-kinase pathway, but that the two consensus PKB/Akt phosphorylation sites present in the protein are not required. Using mutational and deletion studies, we identify two additional potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364 that are critical for insulin inhibition of transactivation by Foxo1-(208652). Thus, the inhibition of Foxo1 by insulin is complex and involves multiple sites. The direct regulation of the transactivation activity of Foxo1-(208652) by insulin provides an additional mechanism by which insulin promotes Foxo1 inhibition, thereby regulating the expression of genes controlled by this transcription factor.
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EXPERIMENTAL PROCEDURES |
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Cell LinesH4IIE rat hepatoma cells (39) were grown as monolayer cultures in low glucose Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) and antibiotics (50 units/ml of penicillin and 50 µg/ml of streptomycin).
Plasmid ConstructsThe rat IGFBP-1
promoter-luciferase reporter plasmid (p925GL3) and the expression vector
pCMV5/c-Myc-Foxo1, which contains the entire 652 amino acid coding region of
Foxo1 were previously described
(35,
38). pG5E1b, a plasmid
encoding a luciferase gene driven by an E1b promoter and five copies of the
Gal4 binding element, kindly provided by Dr. A. Roberts (NCI, National
Institutes of Health), was used as a reporter in Gal4 promoter assays
(40). C-terminal fragments of
Foxo1 to be tested were expressed as Gal4 fusion proteins by subcloning the
corresponding sequences in a pM2 expression vector (generously provided by Dr.
I. Sadowski (University of British Columbia)) encoding the DNA binding domain
of the yeast transcription factor Gal4
(40,
41). C-terminal fragments of
Foxo1, corresponding to amino acids 208652, 256652,
317652, 332652, 350652, 365652, 412652,
461652, 476652, and 501652 were obtained by PCR using the
sense primers, 5'-ATACGTAGATATCAATTCAATTCGCCACAATCTG-3';
5'-ATACGTAGATATCAACAACAGTAAATTTGCTAAGAGC-3';
5'-ATACGTAGATATCAATGCTAGTACCATCAGTGGG-3';
5'-ATACGTAGATATCCAGGATGACCTGGGAGAT-3';
5'-ATACGTAGATATCGCCAAGATGGCGTCTACG-3';
5'-ATACGTAGATATCCCAGAAAACATGGAGAAC-3';
5'-ATACGTAGATATCCCAAACTACTCAAAGTAC-3';
5'-ATACGTAGATATCGAGTTGTTGACTTCTGAC-3';
5'-ATACGTAGATATCCCGGTTGATCCCGGAGTG-3';
5'-ATACGTAGATATCATGCCAGCGTATGGCAGC-3', respectively, and the
antisense primer, 5'-GCTCTAGATTAGCCTGACACCCAGCTGTG-3'. The PCR
fragments were digested with BamHI and XbaI and sequentially
ligated into the BamHI and XbaI restriction sites of pM2 to
generate pM2-Foxo1-(208652), pM2-Foxo1-(256652),
pM2-Foxo1-(317652), pM2-Foxo1-(332652),
pM2-Foxo1-(350652), pM2-Foxo1-(365652),
pM2-Foxo1-(412652), pM2-Foxo1-(461652),
pM2-Foxo1-(476652), and pM2-Foxo1-(501652). Overlap extension
PCR was used to generate pM2-Foxo1-(208652379460). The
fragments corresponding to amino acids 208378 and 461652
contained a BamHI restriction site at the 5'-end and an
XbaI site at the 3'-end, respectively. Following amplification
of each individual fragment, a second PCR was carried out to generate a single
fragment containing the deletion. This fragment was then subcloned into the
BamHI-XbaI site of pM2.
pM2-Foxo1-(208652
350364) was generated according to the
same procedure. In addition, Foxo1-(208652) and
Gal4DBD-Foxo1-(208652) were ligated into the EcoRI and
XbaI sites of a pCEF expression vector containing a GFP epitope tag.
Expression vectors encoding wild-type p85
(SR
-p85) and a
deletion mutant of p85
(SR
-
p85) that lacks a binding site
for the p110 catalytic subunit of PI 3-kinase, were described previously
(42,
43) and kindly provided by Dr.
K. Yonezawa (Kobe University School of Medicine, Japan). The expression vector
encoding FLAG-p300 was a generous gift from Dr. M. Kawabata (Cancer Institute
of Japanese Foundation for Cancer and Research, Tokyo, Japan). The sequence of
all constructs was confirmed by automated DNA sequencing using a rhodamine
fluorescent terminator sequencing kit (PerkinElmer Life Sciences).
Site-directed MutagenesisMutations were introduced using a
PCR-based method (QuikChangeTM site-directed mutagenesis kit; Stratagene,
La Jolla, CA) following the manufacturer's instructions. The two consensus
PKB/Akt phosphorylation sites (Ser253 and Ser316) in
pM2-Foxo1-(208652) and pM2-Foxo1-(208652379460)
were mutated to alanine. Ser319/Thr320,
Ser322/Ser326/Thr330,
Ser354/Thr355/Ser358/Ser360/Ser363,
Thr464/Ser465/Ser467/Ser475, and
Ser499 were substituted with alanine in
pM2-Foxo1-(208652
379460) and
pM2-Foxo1-(208652/Ser253Ala/Ser316Ala
379460).
Ser319 and Ser499, alone or together, were also mutated
to alanine in pM2-Foxo1-(208652) and pCEF-GFP-Foxo1-(208652).
Mutated oligonucleotides used for DNA amplification are available on request.
The introduction of mutations was confirmed by DNA sequencing.
Transient TransfectionH4IIE cells were transfected as
described in previous publications
(38,
44). Briefly, cells were
seeded in 60-mm tissue culture dishes at a density of 3 x 106
cells/dish 24 h prior to transfection and were 7080% confluent at the
time of transfection. Transient transfection was carried out with a total of
310 µg of DNA using diethylaminoethyl (DEAE)-dextran (Amersham
Biosciences). Plasmid DNA (100 µl) was mixed with 100 µl of DEAE dextran
stock solution (2 mg/ml in 0.15 M NaCl) previously diluted with an
equal volume of Tris-buffered saline (25 mM Tris, pH 7.5, 137
mM NaCl, 5 mM KCl, 0.7 mM CaCl2,
0.5 mM MgCl2, 0.6 mM
Na2HPO4) and incubated at room temperature. After 15
min, the mixture was added to each dish, and, 15 min later, 3 ml of Dulbecco's
modified Eagle's medium supplemented with 10% serum was added, and the
incubation continued overnight. Forty-eight hours after transfection, the
medium was replaced with serum-free Dulbecco's modified Eagle's medium
containing 0.1% bovine serum albumin with or without recombinant human insulin
(0.25 µg/ml), and the incubation was continued for 20 h before harvest. In
some experiments, transfected cells were preincubated for 30 min with LY294002
(50 µM), a selective inhibitor of PI 3-kinase
(45), PD98059 (50
µM), a selective inhibitor of mitogen-activated protein kinase
kinase (46) or SB203580 (20
µM), a selective inhibitor of p38 MAP kinase
(47), before insulin was
added. Transfection efficiency was monitored by cotransfecting
pRSV--galactosidase (40 ng) kindly provided by Dr. P. Yen (NIDDK,
National Institutes of Health). In each experiment, the total amount of DNA
was adjusted by adding empty expression vectors.
Luciferase and -Galactosidase AssaysCell lysates for
luciferase and
-galactosidase assays were prepared according to the
manufacturer's instructions (Tropix, PE Applied Biosystems, Bedford, MA) using
a Lumat LB 9507 luminometer (EG&G Berthold, Germany). Luciferase and
-galactosidase activities were measured in the same tube using the Dual
Light chemiluminescent reporter gene assay kit, allowing 1 h at room
temperature for luciferin fluorescence to decay before measuring
-galactosidase activity. Assays were performed in duplicate.
Immunoprecipitation and Western Blot AnalysisH4IIE cells were transfected with GFP-tagged Foxo1 208652 and Foxo1 208652/Ser253Ala/Ser316Ala using the LipofectAMINETM Plus reagent (Invitrogen) according to the manufacturer's instructions. After 48 h, cells were switched to serum-free medium overnight before incubation with recombinant human insulin (0.25 µg/ml) for the indicated times. Lysis was performed using 0.5 ml of lysis buffer containing 150 mM NaCl, 0.8 mM MgCl2, 5 mM EGTA, 50 mM Hepes, pH 7.5, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysates clarified by centrifugation at 13,000 rpm for 10 min at 4 °C, were either directly separated by SDS-PAGE and transferred onto nitrocellulose membrane, and/or first immunoprecipitated with anti-GFP antibody (Covance). Western blots were detected using anti-FKHR phospho-Ser253 antibody (Cell Signaling Technology, Beverly, MA) and visualized by chemiluminescence (Pierce). The expression level of Foxo1 constructs was examined by immunoblotting analysis using anti-FKHR antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Nuclei from HEK-293T human embryonic kidney cells transfected with GFP-tagged Foxo1-(208652) or Foxo1-(208652/Ser319Ala/Ser499Ala) and FLAG-tagged p300 were prepared using the nuclei EZ prep isolation buffer (Sigma). After immunoprecipitation with anti-GFP antibody, proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-FLAG antibody to detect p300 (Covance).
ImmunofluorescenceH4IIE cells were transiently transfected with the LipofectAMINETM Plus reagent (Invitrogen) using c-Myc-tagged full-length Foxo1, GFP-tagged Foxo1-(208652), Gal4DBD/Foxo1-(208652) or the corresponding empty vector. After 48 h, cells were serum-starved overnight, before incubation with recombinant human insulin (1 µg/ml) for 1 h at 37 °C. Then, cells were washed twice with phosphate-buffered saline, fixed with 2% paraformaldehyde and mounted with mounting medium from Molecular Probes. Cells transfected with c-Myc-tagged full-length Foxo1, were serum-starved overnight, treated with or without insulin, and fixed as described above, before permeabilization with Triton X-100 and detection with anti-c-Myc antibody (clone 9E10) (Covance) followed by incubation with goat anti-mouse FITC-labeled secondary antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA). Cells were visualized using a fluorescence microscope.
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RESULTS |
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Insulin Inhibition of Foxo1-(208652)-stimulated Transactivation
Does Not Require the Two Consensus PKB/Akt Phosphorylation
SitesSeveral studies reported that insulin inhibition of
transcription stimulated by full-length Foxo1 requires the phosphorylation of
three consensus sites (Thr24, Ser253, and
Ser316) by PKB/Akt
(11,
13,
48). In contrast to what has
been shown for full-length Foxo1, we suggested in a previous publication
(38) that PKB/Akt was not
involved in insulin inhibition of Foxo1-(208652)-stimulated
transactivation. To explore further the mechanism by which insulin inhibits
Foxo1 activity, we examined whether a Foxo1-(208652) double mutant
(Foxo1-(208652/Ser253Ala/Ser316Ala)) that cannot
be phosphorylated at its two PKB/Akt phosphorylation sites (Ser253
and Ser316) (Fig.
2A) is inhibited by insulin. A yeast Gal4 promoter system
(40,
41) was used in which the
transactivation domains of wild-type and double mutant Foxo1-(208652)
were fused to the Gal4 DNA binding domain and cotransfected with a reporter
plasmid (pG5E1b) containing a luciferase gene coupled to five copies of the
Gal4 binding elements (Fig.
2B). Basal pG5E1b promoter activity was negligible in
H4IIE cells so that luciferase activity reflected only transactivation by
Foxo1 C-terminal fragments. As shown in
Fig. 2B, insulin
inhibited transactivation of the Gal4 promoter by Foxo1-(208652) by
70%. Moreover, substitution of Ser253 and Ser316
with alanine in Foxo1-(208652) had no effect on insulin inhibition.
These results indicate that Foxo1-(208652) contains the information
necessary for insulin inhibition and that neither of the two consensus PKB/Akt
phosphorylation sites in this fragment is required for insulin inhibition of
Foxo1-(208652)-mediated transactivation.
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Insulin Inhibition of Foxo1-(208652)-stimulated Transactivation Is Mediated by PI 3-KinaseTo determine which signaling pathway is involved in the insulin inhibition of Foxo1-(208652), specific inhibitors of PI 3-kinase (LY294002), MAP kinase kinase (PD98059), and p38 MAP kinase (SB203580) were used (Fig. 3A). Inhibition of PI 3-kinase abolished the ability of insulin to decrease Foxo1-(208652)-stimulated transactivation. By contrast, neither MAP kinase kinase nor p38 MAP kinase had any effect on insulin inhibition of Foxo1-(208652).
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To confirm the involvement of PI 3-kinase in insulin inhibition of
transactivation stimulated by the Foxo1 C-terminal fragment,
Foxo1-(208652) was cotransfected together with the wild-type regulatory
subunit of PI 3-kinase, p85, or a dominant negative mutant of
p85
(
p85), that contains a deletion that prevents it from
binding to and activating the p110 catalytic subunit
(42,
43). When
p85 was
overexpressed, in contrast to wild-type p85, insulin inhibition of
transactivation of the Gal4 promoter by Foxo1(208652) was greatly
decreased (Fig. 3B).
Taken together, these results indicate that insulin inhibition of
Foxo1-(208652)-stimulated transactivation is mediated by PI 3-kinase
but does not require the PKB/Akt phosphorylation sites.
Ser319 and Ser499 Play a Critical Role in Insulin
Inhibition of Transactivation by Foxo1-(208652)To identify
the site(s) responsible for insulin inhibition of transactivation by
Foxo1-(208652), progressive N-terminal deletions of Foxo1 fused to the
Gal4 DNA binding domain were generated
(Fig. 4). The different
C-terminal Foxo1 fragments tested, residues 256652, 317652, and
501652, significantly stimulated Gal4 promoter activity in the absence
of insulin (data not shown). As shown in
Fig. 4, insulin inhibited
transactivation of the Gal4 promoter by Foxo1-(256652) and
-(317652) to the same extent as Foxo1-(208652) (70%). In
contrast, with the shorter C-terminal Foxo1 fragment, 501652, the
insulin inhibitory effect was lost, and instead a slight stimulation of
transactivation (
1.5-fold) was observed. These results suggest that the
site(s) critical for insulin inhibition of transcription activation by
Foxo1-(208652) is (are) located between amino acids 317 and 500.
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The region 317500 of Foxo1 contains numerous potential
phosphorylation sites: 43 Ser/Thr and 7 Tyr. A majority of them, 25 Ser/Thr
and 6 Tyr, are located between amino acids 379 and 460
(Fig. 5A). This
observation prompted us to ask whether insulin inhibition of transactivation
by Foxo1-(208652) is affected when amino acids 379460 were
deleted. As shown in Fig.
5B, transactivation of the Gal4 promoter by
Foxo1-(208652379460) was inhibited by insulin to the same
extent as wild-type Foxo1-(208652). To exclude the possibility that the
two PKB/Akt phosphorylation sites present in the fragment might compensate for
the deletion, we also analyzed the effect of this deletion in the double
mutant
(Foxo1-(208652/Ser253Ala/Ser316Ala
379460))
(Fig. 5B). In either
case, deletion of residues 379460 had no effect on insulin
inhibition.
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We next carried out mutational studies to investigate the role of the
potential phosphorylation sites present in the regions flanking amino acids
379460: residues 317378 and 461500
(Fig. 5A). Five groups
of Ser/Thr-Ala mutations (designated AE) were tested in
Foxo1-(208652/Ser253Ala/Ser316Ala379460).
As shown in Fig. 5C,
insulin inhibition still was observed when groups B, C, and D, which contain a
total of 12 Ser/Thr, were mutated. Insulin inhibition, however, was abolished
when Ser319Ala/Thr320Ala (Group A) or
Ser499Ala (Group E) were mutated. Similar results were obtained
when these point mutations were introduced into
Foxo1-(208652
379460), which possess functional PKB/Akt
sites (data not shown). Of the 2 residues mutated in Group A,
Ser319 is most likely to be involved since it has a high
probability of being phosphorylated
(49).
Furthermore, neither S319A nor S499A alone was sufficient to abolish insulin inhibition, suggesting that the 379460 region could compensate for the loss of one of these two sites (Fig. 5D). However, when S319A and S499A were simultaneously mutated in Foxo1-(208652), insulin no longer inhibited transactivation stimulated by this fragment. Together, these results indicate that Ser319 and Ser499 are important for insulin inhibition of Foxo1-(208652)-stimulated transactivation. The presence of the 379460 region can compensate for mutation of one of these two sites, but insulin inhibition is lost when both sites are mutated even though residues 379460 are present.
Mutation of Ser319 and Ser499 could regulate transactivation by altering the interaction of Foxo1 with coactivators/corepressors. Nasrin (66) previously reported that the coactivator p300/CBP (CREB-binding protein) binds to full-length Daf-16 and FOXO1. As shown in Fig. 6, p300 interacted with Foxo1-(208652). Alanine substitution of Ser319 and Ser499 in the C-terminal transactivation domain of Foxo1, however, did not affect the binding to p300, indicating that these serines are not involved in the interaction with the coactivator.
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Residues 350364 of Foxo1-(208652) Are Required for Insulin Inhibition of TransactivationAs a parallel approach, we also analyzed the ability of insulin to inhibit transactivation stimulated by progressive N-terminal deletions of Foxo1. The different fragments tested originated at different points in the 317500 region but terminated at residue 652. As seen in Fig. 7A, insulin potently inhibited transactivation of the Gal4 promoter by the Foxo1 317652, 332652, and 350652 fragments. When further truncations were tested (Foxo1-(365652), -(412652), -(476652), and -(501652)), insulin inhibition was lost and instead a stimulation of transactivation was observed. These results suggest that the region 350364 of Foxo1-(208652) is necessary for insulin inhibition. Moreover, our results also suggest that insulin stimulation of Foxo1 activity is masked in Foxo1-(208652) and can only be observed in the shortest Foxo1 fragments.
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To confirm the importance of the 350364 region in insulin
inhibition, we examined the effect of insulin on
Foxo1-(208652)-stimulated transactivation when amino acids
350364 were deleted (Foxo1-(208652350364))
(Fig. 7B). Deletion of
these fifteen residues in Foxo1-(208652) was sufficient to abolish
insulin inhibition. Taken together, these results indicate that, in addition
to Ser319 and Ser499, the region 350364 in
Foxo1-(208652) is also critical for insulin inhibition. Moreover,
mutation of the potential phosphorylation sites (4 Ser and 1 Thr) present in
the 350364 region did not affect Foxo1-(208652)-stimulated
transactivation, indicating that phosphorylation of these sites is not
required for insulin inhibition (Fig. 5,
A and C).
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DISCUSSION |
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In this report, we demonstrate that a C-terminal fragment of Foxo1,
residues 208652, is a potent activator of transcription when fused to
the Gal4 DNA binding domain. This is likely to be physiologically relevant, as
a fusion protein of Pax3 with the human counterpart of this fragment
identified in alveolar rhabdomyosarcomas
(5) is also known to enhance
transcription (62). Consistent
with what has been described for full-length Foxo1
(36,
38), we found that insulin
strongly inhibits transactivation by Foxo1-(208652) (70%) in H4IIE
rat hepatoma cells. Our results, however, contrast with the absence of insulin
regulation reported for a comparable C-terminal fragment of FOXO4, another
member of the FOXO subfamily
(52). The discrepancy may
reflect differences between the transactivation domains of the two related
transcription factors or differences in the insulin responsiveness of the cell
lines used (mouse fibroblasts overexpressing insulin receptors
(52) or H4IIE rat hepatoma
cells in the present study).
Surprisingly, our results show that the subcellular localization of Foxo1-(208652) is not significantly affected by insulin, whereas under the same conditions insulin promotes the redistribution of full-length Foxo1 to the cytoplasm. Therefore, changes in the subcellular localization of Foxo1-(208652) are unlikely to account for the inhibition of its transcriptional activity by insulin, and suggest that insulin is directly regulating transcription.
Transcriptional regulation by insulin may reflect an altered binding of the transcription factor to the promoter region or the regulation of transactivation. These possibilities are not mutually exclusive, as described for the regulation of the pancreatic islet homeobox transcription factor PDX-1 by glucose (63). Phosphorylation of members of the FOXO subfamily, Daf-16 (64) and FOXO1 (65), in response to insulin have been shown to inhibit their binding to the IRE in the promoter of target genes. In the present study, using a yeast Gal4 system (40, 41) in which the level of promoter activity is strictly determined by the transactivation domain of Foxo1, we provide the first direct demonstration that the insulin effects can also be mediated by inhibition of transactivation.
Mutation and deletion studies were performed to identify the sites involved in insulin inhibition of Foxo1-(208652)-stimulated transactivation. As we demonstrate in this report, and in contrast to what has been described for full-length Foxo1, the two PKB/Akt consensus phosphorylation sites (Ser253 and Ser316) contained in Foxo1-(208652) are not necessary for inhibition of its transactivation activity by insulin. Indeed, a Foxo1-(208652) mutant in which the two PKB/Akt sites were mutated, and more extensive N-terminal deletions of Foxo1 (for example, Foxo1-(317652)) that do not contain those sites, are still strongly inhibited by insulin.
We have identified three sites in Foxo1-(208652) that are critical for insulin inhibition: Ser319 and Ser499, as well as a 15-amino acid region located within residues 350 to 364 (Fig. 8). Mutation of Ser319 and Ser499 together was sufficient to abolish insulin inhibition. These two serines act in concert and are presumably phosphorylated. The kinase(s) that phosphorylate them remain to be identified. Phosphorylation of Ser319 by casein kinase I (equivalent to Ser322 of FOXO1) can be excluded, however, since it requires prior phosphorylation of Ser316 (equivalent to Ser319 of FOXO1) by PKB/Akt (66). Our results also indicate that the 379460 region of Foxo1 can compensate for the mutation of either Ser319 or Ser499, but not for the mutation of both sites (Fig. 8). We do not know yet whether the same or different sites in Foxo1-(379460) compensate for mutation of the two serines. Both Foxo1-(208652) and Foxo1-(208652/Ser319Ala/Ser499Ala) bind to the coactivator p300, indicating that mutation of these serines does not abolish insulin inhibition of transactivation by disrupting interactions with the coactivator.
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Residues 350364 are also critical for insulin inhibition of Foxo1-(208652) activity. Mutation of the Ser/Thr residues contained in this region does not prevent inhibition of transactivation by insulin, suggesting that phosphorylation of these particular sites is not required for insulin action. Whether insulin acts in Foxo1-(350364) by another type of post-translational modification or by promoting the interaction with a protein (that may or may not be phosphorylated) remains to be determined. In addition, it will be interesting to examine whether Ser319/Ser499 and residues 350364 are sequential components of the same inhibitory pathway or belong to separate inhibitory pathways that mediate insulin action. For example, the 350364 domain may be required for phosphorylation of Ser319 or Ser499. This is reminiscent of the ability of the Foxo1-(256317) domain to inhibit phosphorylation of Thr24 and Ser316 following phosphorylation of Ser253 (36).
Insulin inhibition of Foxo1-(208652)-stimulated transactivation is
dependent on PI 3-kinase, but the kinase(s) downstream of PI 3-kinase that are
involved in the insulin response remain unknown. Several lines of evidence
(the results presented here and Ref.
38) suggest that PKB/Akt is
unlikely to be involved. The possibility that PKB/Akt might act indirectly, by
phosphorylating a coactivator that interacts with Foxo1
(67), is excluded by the fact
that coexpression of constitutively active PKB/Akt with Foxo1-(208652)
does not mimic the effect of insulin
(38). Candidate kinases other
than PKB/Akt downstream from PI 3-kinase known to be involved in insulin
signaling include the serum- and glucocorticoid-induced protein kinase (SGK)
(6870),
the mammalian target of rapamycin (mTOR), and the atypical protein kinase C
variants (PKC- and -
, Ref.
71). Direct phosphorylation of
the C-terminal fragment of Foxo1 by SGK is unlikely to be involved in insulin
inhibition of transactivation since SGK recognizes the same consensus
phosphorylation sites as PKB/Akt
(69,
70,
72) and we have shown that
these sites are not essential for insulin inhibition. mTOR has been reported
to mediate insulin inhibition of FOXO-stimulated transcription in some cell
lines. The mTOR inhibitor, rapamycin, decreased insulin inhibition of IGFBP-1
gene expression in rat hepatocytes
(73), had no effect on insulin
inhibition of IGFBP-1 promoter activity in HepG2 cells
(74), and partially decreased
insulin inhibition of transcription by FOXO1 in H4IIE cells
(75). Finally, the atypical
PKCs have been shown to mediate insulin stimulation of glucose transport in
adipocytes and in muscle
(7678),
but do not induce phosphorylation of Ser253 in FOXO3a
(72). Whether these PKC
variants or an insulin receptor-specific kinase that phosphorylates Foxo1 at
Thr24 in hepatocytes
(36,
79) might phosphorylate other
sites in Foxo1 is presently unknown.
An unexpected result from the mutation and deletion approaches used in the present study, was the demonstration that insulin stimulates transactivation by Foxo1-(365652) and smaller C-terminal Foxo1 fragments (Fig. 9).3 Stimulation was observed with the shortest Foxo1 fragment, 501652, indicating the presence of a stimulatory site (St) in this region. The deletion of an inhibitory site (Inh) located within amino acids 350364 in Foxo1-(208652) abolished insulin inhibition, but was not sufficient to stimulate transcription. Furthermore, in the absence of this inhibitory site, the enhancement of transactivation in response to insulin was masked unless amino acids 208350 were also deleted, suggesting the presence of an additional regulatory site, designated suppressor of stimulation (Sup), in this region.
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In summary, these data provide compelling evidence to demonstrate that insulin regulation of transactivation by Foxo1-(208652) is complex and involves multiple sites as well as different mechanisms. Insulin inhibition of Foxo1-(208652)-stimulated transactivation is mediated by PI 3-kinase, and requires Ser319/Ser499 acting in concert and amino acids 350364. Moreover, our results also indicate that in contrast to full-length Foxo1, the PKB/Akt phosphorylation sites are not necessary for insulin inhibition of Foxo1-(208652) activity. Further N-terminal truncations unmask a stimulatory site located within the C terminus of Foxo1, amino acids 501652, leading to an increase of transcription by Foxo1 in response to insulin. In this study, we demonstrate that insulin can directly regulate transactivation by Foxo1-(208652). Therefore, the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding (64, 65) and nuclear exclusion (13). This model is consistent with the reported multistep regulation of other transcription factors. In yeast, for example, the availability of phosphate regulates the transcription factor Pho4 at the levels of nuclear import, nuclear export and transactivation (80, 81). Thus, the direct regulation by insulin of Foxo1-stimulated transactivation provides an additional mechanism to allow the precise control of transcription of FOXO target genes.
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FOOTNOTES |
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To whom correspondence should be addressed: Oral and Pharyngeal Cancer Branch,
NIDCR, National Institutes of Health, Bldg. 30, Rm. 207, 9000 Rockville Pike,
Bethesda, MD 20892-4340. Tel.: 301-402-7529; Fax: 301-402-0823; E-mail:
vperrot{at}nidcr.nih.gov.
1 The human (FOXO) and mouse (Foxo) members of the FOXO subfamily of forkhead
transcription factors were previously known as FKHR (FOXO1, Foxo1), FKHRL1
(FOXO3a, Foxo3), and AFX (FOXO4, Foxo4).
2 The abbreviations used are: IGFBP, insulin-like growth factor-binding
protein; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B;
GFP, green fluorescent protein; MAP, mitogen-activated protein.
3 Characterization of the enhancement of Foxo1-(501652)-stimulated
transcription by insulin will be reported separately (V. Perrot and M. M.
Rechler, unpublished data).
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ACKNOWLEDGMENTS |
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REFERENCES |
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