From the Developmental Endocrinology Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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In the nematode Caenorhabditis
elegans, mutations of the insulin/insulin-like growth factor-1
receptor homologue Daf-2 gene cause developmental arrest at the dauer
stage. The effect of Daf-2 mutations is counteracted by mutations in
the Daf-16 gene, suggesting that Daf-16 is required for signaling by
Daf-2. Daf-16 encodes a forkhead transcription factor. Based on
sequence similarity, the FKHR genes are the likeliest mammalian Daf-16
homologues. FKHR proteins contain potential sites for phosphorylation
by the serine/threonine kinase Akt. Because Akt is phosphorylated in response to insulin and has been implicated in a variety of insulin effects, we investigated whether insulin affects phosphorylation of
FKHR. Insulin stimulated phosphorylation of endogenous FKHR and of a
recombinant c-Myc/FKHR fusion protein transiently expressed in murine
SV40-transformed hepatocytes. The effect of insulin was inhibited by
wortmannin treatment, suggesting that PI 3-kinase activity is required
for FKHR phosphorylation. Mutation of serine 253, located in a
consensus Akt phosphorylation site at the carboxyl-terminal end of the
forkhead domain, abolished the effect of insulin on FKHR
phosphorylation. In contrast, mutation of two additional Akt
phosphorylation sites, at amino acids threonine 24 or serine 316, did
not abolish insulin-induced phosphorylation. These data indicate that
FKHR may represent a distal effector of insulin action.
Insulin promotes a wide range of metabolic and
growth-promoting functions in multiple target cells (1). The diverse
actions of insulin are mediated by its cell surface receptor. The
insulin receptor is tyrosine-phosphorylated in response to insulin
binding. Autophosphorylation activates the receptor as a
kinase that is able to phosphorylate a host of intracellular protein
substrates. The proximal effectors of insulin action include insulin
receptor substrate proteins and several others (2). Insulin receptor substrate proteins serve an important function as "docking"
molecules to promote the assembly of protein-protein complexes and the
generation of intracellular signals. Among the distal mediators of
insulin signaling, pathways based on the generation of
3-phosphoinositides through the lipid kinase activity of
PI1 3-kinase are thought to
play a prominent role (3). For example, the serine/threonine kinase Akt
and the atypical protein kinase C isoforms The serine/threonine kinase Akt is an important target of PI 3-kinase.
Recent studies showed that Akt is involved in insulin regulation of
gene expression. It is not clear, however, whether transcription
factors are direct targets for the kinase activity of Akt or whether
Akt regulates gene expression by activating other kinases, such as
mitogen-activated protein kinase, p70 S6 kinase, protein kinase C
isoforms, and others (5-8).
Studies of the nematode Caenorhabditis elegans have provided
insights into the conservation of signaling pathways relevant to
mammalian metabolism and reproduction. Under optimal growth conditions,
C. elegans grows rapidly to fertile adult hermaphrodites through four stages (L1-L4). However, when grown at high density or
with high levels of pheromone, larvae enter a reversible arrest of
development characterized by reduced metabolic activity, the dauer
stage. Interestingly, mutations in the Daf-2, Age-1, Akt-1, and Akt-2
genes cause a constitutive dauer phenotype (11-15). The Daf-2 gene
encodes the C. elegans homologue of the insulin/IGF-1 receptor (14); the Age-1 gene is the homologue of PI 3-kinase (13), and
the two Akt genes are homologous to mammalian Akt (11). Thus, the dauer
alleles define a signaling cascade homologous to the insulin and IGF-1
signaling cascade in mammals as important for metabolic regulation and
reproduction in C. elegans. Interestingly, mutations of the
Daf-16 gene in C. elegans prevent Daf-2, Age-1, and Akt
mutants from entering the dauer stage. This observation suggests that
Daf-16 is a negative regulator of the Daf-2/Age-1/Akt signaling
pathway. Positional cloning experiments identified Daf-16 as a member
of the HNF-3/forkhead family of transcription factors, with a unique
forkhead DNA-binding domain consisting of about 100 amino acids (15,
16). Interestingly, Daf-16 has four consensus Akt phosphorylation sites
(17), raising the possibility that Daf-16 may be a direct substrate of Akt.
The closest mammalian homologues of Daf-16 are three members of the
FKHR family: FKHR, FKHRL1, and AFX (18). These genes were originally
identified in chromosomal translocations associated with human
rhabdomyosarcomas (hence the acronym forkhead
in human rhabdomyosarcoma) as fusion proteins
composed of the paired box and homeodomain regions of the transcription
factors Pax-3 or Pax-7 and the transactivation domain of FKHR
(19). Of the three genes, FKHR shares the highest identity (76%) with
Daf-16 in the forkhead DNA-binding domain.
We report that FKHR is the principle member of this family of proteins
in murine SV40-transformed hepatocytes and that insulin stimulates its
phosphorylation. Phosphorylation of FKHR is inhibited by wortmannin,
suggesting that it requires PI 3-kinase activity. The effect of insulin
is abolished by mutation of serine 253 in an Akt phosphorylation
consensus sequence in the forkhead domain of FKHR but not by mutations
of two additional Akt consensus sequences at threonine 24 and serine
316. These data indicate that FKHR may be a substrate of Akt. FKHR
represents a candidate transcription factor that regulates gene
expression in response to insulin.
Antibodies--
Anti-human FKHR antibodies were obtained from
Dr. Barr (20). Anti c-Myc antibodies (clone 9E10) were from Sigma.
Anti-insulin receptor antibodies were from Oncogene Science (Manhasset,
NY) (clone Ab 3) and from Santa Cruz Biotechnology (Santa Cruz, CA) (antiserum C-19).
Cell Culture and Labeling--
SV40-transformed mouse
hepatocytes were cultured at 33 °C in cDNA Cloning, Site-directed Mutagenesis, and Northern
Analysis of Mouse FKHR--
The 3' end of FKHR, including the forkhead
and transactivation domains, was amplified from mouse liver Marathon
cDNA (CLONTECH) using primers from
expressed sequence tag AA254887 (5'-GTCCTGGGCCAAAATGTAATG-3' and
5'-AGCCTGACACCCAGCTGTGTG-3'). The 5' end of the cDNA, encoded by
exon 1 of the FKHR gene, was amplified from genomic DNA using upstream
primer 5'-GGGGGGTCTAGAGTCACCATGGCCGAGGCG-3' (the
XbaI site for subcloning is underlined), and downstream
primer 5'-GACAGGTTGCCCCACGCGTTGCGGCGCGAC-3' (nucleotides
482-453). Overlap exten- sion PCR was used to generate the T24A,
S253A, and S316A mutant FKHR expression vectors (22). The sequences of
the mutagenic oligonucleotides and other oligonucleotides employed for
PCR mutagenesis are available from the authors on request. All
fragments were subcloned and sequenced to rule out the presence of
additional mutations. mRNA was isolated by acid phenol-guanidinium
extraction, followed by affinity chromatography on oligo(dT). Northern
blotting was performed as described (23). The FKHR (nucleotides
1468-1963) and FKHRL1 (nucleotides 603-2866) probes were obtained by
reverse PCR using mouse liver as an RNA source. Standard PCR
amplification conditions were employed.
FKHR Is the Principle Member of the FKHR Family in Murine
SV40-transformed Hepatocytes--
Insulin affects the liver-specific
expression of several genes important for metabolic regulation. To test
which member of the FKHR family was the likeliest candidate as an
effector of insulin action in liver, we performed Northern analysis on
mRNA from murine SV40-transformed hepatocytes (21). The results are shown in Fig. 1A, and indicate
that FKHR is ~10-fold more abundant than FKHRL1. AFX mRNA was not
detected in the same blot (not shown).
Cloning of Mouse FKHR cDNA--
Using available partial
sequence information (GenBankTM accession number AA254887),
cDNA clones encoding the 3' end of murine FKHR were amplified from
a Marathon cDNA library. The 5' end was obtained from mouse genomic
DNA. Murine FKHR shares 92% identity at the amino acid level with
human FKHR (data not shown). The forkhead DNA-binding domain is
identical in human and mouse FKHR and is highly conserved among mouse
FKHR and the other two members of the FKHR family, sharing 82 and 86%
identity with human FKHRL1 and AFX cDNA, respectively (18). The
open reading frame encodes a 652-amino acid polypeptide, with a
predicted molecular mass of 69.497 kDa. Inspection of the amino acid
sequence reveals three consensus phosphorylation sites for the Akt
kinase
(RXRXX(S/T)) around threonine 24 (PRQRSCTW),
serine 253 (PRRRAASM), and serine 316 (FRPRTSSN).
In Vivo Phosphorylation of FKHR in Response to Insulin and
IGF-1 in SV40-transformed Hepatocytes--
To test the hypothesis that
insulin stimulates FKHR phosphorylation, SV40-transformed hepatocytes
from normal mice (WT) or from insulin receptor-deficient mice ( Phosphorylation of FKHR in Response to Insulin Is Inhibited
by Wortmannin--
To investigate further whether phosphorylation of
FKHR is mediated by a PI 3-kinase-dependent pathway, we
performed phosphorylation experiments in the presence of the selective
inhibitors wortmannin, which blocks the activity of PI 3-kinase (24,
25), and rapamycin, which blocks the activity of p70 S6 kinase (26).
For these experiments, epitope-tagged FKHR was transiently transfected
in WT cells using plasmid pCMV5/c-Myc. In 10 experiments, insulin
increased 32P content of the recombinant protein ~2-fold
(Fig. 2, lanes 1 and
2), consistent with the effect on the endogenous protein. The insulin-dependent increase in FKHR phosphorylation was
abolished by treatment of cells with wortmannin (Fig. 2, lanes
5 and 6). In contrast, rapamycin caused an increase in
the basal phosphorylation of FKHR without affecting the amount of
32P in FKHR recovered from insulin-treated cells (Fig. 2,
lanes 3 and 4). Consistent with the
32P loading experiments, 35S labeling
experiments revealed that wortmannin, but not rapamycin, inhibited the
insulin-induced shift in molecular mass of FKHR (Fig. 2, lower
panel). These data indicate that FKHR is phosphorylated by
insulin receptors through a PI 3-kinase-dependent
pathway.
Identification of the Site of Insulin-dependent
Phosphorylation--
To investigate which one of the consensus Akt
phosphorylation sites is phosphorylated in vivo by insulin,
WT hepatocytes were transfected with expression vectors for wild type
and mutant FKHR (pCMV5/c-Myc WT, T24A, S253A, and S316A). Mutation of
Ser253 completely abolished the effect of insulin on FKHR
phosphorylation (Fig. 3, lanes
5 and 6), whereas mutations of T24A and S316A decreased insulin-induced FKHR phosphorylation by ~30% but did not abolish the
effect of insulin (Fig. 3, lanes 3, 4,
7, and 8). However, all three mutants inhibited
the mobility shift induced by insulin stimulation of WT FKHR.
The present studies support the identification of FKHR as a target
of insulin-stimulated, PI-dependent kinases (possibly Akt) and as a distal effector of the insulin receptor signaling cascade. Site-directed mutagenesis data are consistent with a model in which
Ser253 is required for the effect of insulin on FKHR
phosphorylation, whereas Thr24 and Ser316 are
not. The slight decrease of insulin-induced phosphorylation in the T24A
and S316A mutants, in addition to the inhibition of the insulin-induced
mobility shift, indicate that these sites may be phosphorylated
in vivo but that they are not necessary for the effect of
insulin. Thus, Ser253 appears to act as a gatekeeper site
for insulin-induced FKHR phosphorylation in SV40-transformed
hepatocytes. Recently, it has been reported that phosphorylation of the
related molecule FKHRL1 in response to IGF-1 in 293 and Jurkat cells
occurs at three Akt sites: Thr32, Ser253, and
Ser315, corresponding to Thr24,
Ser253, and Ser316 of FKHR (27). However, the
S253A mutant FKHRL1 still retained the ability to be phosphorylated in
response to IGF-1, unlike the corresponding mutant of FKHR, which lost
its ability to be phosphorylated in response to insulin. Further
experiments will be required to determine whether this difference is
simply a reflection of the experimental design or indicates a potential
mechanism of signaling diversity among different members of the FKHR family.
FKHR phosphorylation is exquisitely sensitive to wortmannin, consistent
with a role of PI-dependent kinases in this process (11,
27). Interestingly, basal phosphorylation of FKHR is increased in the
presence of rapamycin. At present, we cannot provide an explanation for
this finding. However, the insulin-dependent mobility shift
is not affected by rapamycin treatment, suggesting that the
insulin-dependent kinases that regulate FKHR
phosphorylation are not rapamycin-inhibitable. Because FKHR has the
potential to participate in cell cycle control and oncogenesis and
because phosphorylation of FKHR presumably inhibits its transcriptional activity, the observation of increased FKHR phosphorylation in the
presence of rapamycin is consistent with antiproliferative effect
of rapamycin (26).
Our observations raise two important questions: how does
insulin-induced phosphorylation affect the function of FKHR and what are the insulin-regulated target genes of FKHR? Based on the data of
Brunet et al. (27), the primary hypothesis to be tested is whether insulin affects the intracellular distribution of FKHR, in a
similar fashion to the effect of IGF-1 on FKHRL1. In preliminary experiments, we have been able to show that phosphorylation of serine
253 affects FKHR binding to DNA in gel shift
assays,2 consistent with the
possibility that insulin may regulate FKHR function at different levels.
With respect to the question of which genes are subject to regulation
by FKHR in an insulin-dependent manner, we have shown that
FKHR mediates insulin inhibition of IGFBP-1 transcription in a
phosphorylation-dependent manner (28). The identification of the exact role of FKHR phosphorylation in insulin and IGF-1 signaling will provide important new insight into the mechanism by
which insulin and IGF-1 regulate gene expression.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
are among the
kinases that are activated in a PI 3-kinase-dependent
manner to regulate glucose transport, glycogen synthesis, cell
survival, and gene expression in response to insulin (4-10).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified minimum essential
medium supplemented with 4% fetal calf serum, 2 mM
glutamine, and 10 nM dexamethasone as described previously
(21). For 32P labeling experiments, cells were sequentially
incubated in serum-free DMEM for 8 h, serum- and phosphate-free
DMEM for 1 h, and DMEM supplemented with 0.05 mCi/ml
[32P]orthophosphate (NEN Life Science Products) for
4 h. Insulin stimulation was carried out for 5-15 min and was
preceded in some experiments by a 30-min incubation in the presence of
wortmannin or rapamycin (Sigma). At the end of the incubation, cells
were solubilized and processed for immunoprecipitation as described (21). For 35S labeling experiments, cells were incubated
overnight in DMEM supplemented with 0.05 mCi/ml
[35S]methionine (NEN Life Science Products) and then
stimulated with insulin. Transfections were carried out with
LipofectAMINE (Life Technologies, Inc.).
RESULTS
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ABSTRACT
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Fig. 1.
A, Northern blot analysis of FKHR
expression in murine SV40 hepatocytes. mRNA was blotted onto nylon
membranes that were then hybridized with 32P-labeled
cDNA probes for FKHR, FKHRL1, and actin. Autoradiography was
performed for 16 h (FKHR and FKHRL1) or 2 h (actin). A
representative experiment is shown. B, phosphorylation of
FKHR by insulin in SV40-transformed mouse hepatocytes. Confluent
monolayers of SV40-transformed hepatocytes from normal mice (WT cells)
or insulin receptor-deficient mice ( /
cells) were incubated in
serum-free medium and labeled with [32P]orthophosphate
for 4 h. Insulin was added for the indicated periods of time, and
proteins were immunoprecipitated using anti-FKHR antibody.
IB, immunoblot. C, time course of insulin-induced
phosphorylation of insulin receptors (IR). The upper
panel shows 32P labeling of immunoprecipitated insulin
receptors. In the lower panel, the filter was blotted with
an anti-insulin receptor antibody to normalize the amount of protein
loaded onto each lane. D, control immunoprecipitation with
nonimmune rabbit serum. Three independent experiments were performed
with identical results.
/
)
were labeled with [32P]orthophosphate, stimulated with
insulin for 5-15 min, and FKHR was immunoprecipitated using an
antibody against the human protein (20). In WT cells, insulin rapidly
induced a 2-fold increase in 32P content of FKHR. This
increase was associated with an electrophoretic shift of the band
corresponding to FKHR on the gel. In
/
cells, which lack insulin
receptors, phosphorylation of FKHR in response to insulin was slower
and increased by about 50% over basal after 15 min of stimulation
(Fig. 1B). The response observed in
/
cells is
presumably due to IGF-1 receptors. As a control, phosphorylation of
insulin receptors in response to insulin is shown in Fig.
1C. These data indicate that FKHR is phosphorylated in an
insulin- and IGF-1 receptor-dependent manner in mouse
hepatocytes. Based on the Northern blot shown in Fig. 1A, we
conclude that FKHR is the main immunoreactive species recognized by the antibody.
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Fig. 2.
Phosphorylation of transfected mouse FKHR in
response to insulin is inhibited by wortmannin. SV40-transformed
mouse hepatocytes (WT cells) were transiently transfected with a
plasmid encoding wild type mouse FKHR (pCMV5/c-Myc mFKHR WT). 48 h
after transfection, cells were labeled with
[32P]orthophosphate as indicated in Fig. 1C
(upper panel) or with [35S]methionine
(lower panel). Rapamycin (50 nM) or wortmannin
(100 nM) was added 30 min prior to the addition of insulin.
Insulin stimulation was carried out for 15 min. Thereafter, cells were
lysed and immunoprecipitated with a monoclonal anti-c-Myc antibody as
indicated under "Experimental Procedures." The experiment was
repeated three times with identical results.
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Fig. 3.
Phosphorylation of site-directed mutants of
FKHR in response to insulin in vivo. Transient
transfections and [32P]orthophosphate labeling of cells
were performed as indicated under "Experimental Procedures." To
normalize the amount of 32P incorporated into FKHR for the
amount of expressed protein, duplicate dishes were labeled with
[35S]methionine and immunoprecipitated with a monoclonal
anti-c-Myc antibody. Each transient transfection was repeated between
three and ten times with similar results. Lanes 1 and
2, WT FKHR; lanes 3 and 4, S316A
mutant; lanes 5 and 6, S253A mutant; lanes
7 and 8, T24A mutant; lanes 9 and
10, untransfected cells. The upper panel shows
the results of 32P labeling experiments; the lower
panel shows the results of 35S labeling
experiments.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Dr. Gary Ruvkun (Harvard Medical School) and members of the Accili lab for helpful discussions and Drs. Jeannette L. Bennicelli and Frederic G. Barr (University of Pennsylvania) for the gift of anti-human FKHR antibody.
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FOOTNOTES |
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* This work was supported in part by a research grant from the American Diabetes Association and by a generous gift of Sigma Tau Pharmaceuticals (to D. A.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF126056.
To whom correspondence should be addressed: Bldg. 10, Rm. 10D18,
NIH, Bethesda, MD 20892. Tel.: 301-496-9595; Fax: 301-435-4358; E-mail:
accilid{at}mail.nih.gov.
2 J. Nakae and D. Accili, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PCR, polymerase chain reaction; IGF, insulin-like growth factor; DMEM, Dulbecco's modified Eagle's medium; WT, wild type.
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