From the
¶Department of Pathology and the Center for
Integrative Metabolic and Endocrine Research, Wayne State University School of
Medicine, Detroit, Michigan 48201 and the
Department of Molecular Science, Pfizer Global
Research and Development, Ann Arbor, Michigan 48105
Received for publication, April 18, 2003 , and in revised form, May 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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HNF4 has also been shown recently to play an important role in the
regulation of hepatic glucose output, a key component of the maintenance of
plasma glucose levels. Yoon et al.
(10) recently demonstrated
that the transcriptional regulation of gene for the key gluconeogenic enzyme
phosphoenolpyruvate carboxykinase by cAMP was mediated by the transcriptional
co-activator PGC-1 acting through HNF4
. These findings suggest that
HNF4
may play a role in the transcriptional response of the liver to
metabolic hormones and that factors regulating the activity of HNF4
could have an effect on hepatic glucose output.
AMPK is the mammalian homolog of the yeast SNF1 protein kinase (11). In yeast, the SNF1 complex is a nuclear protein that regulates expression of genes involved in glucose metabolism and is activated by phosphorylation when glucose is removed from the growth medium (12). In mammalian cells, AMPK acts as a fuel sensor that monitors AMP and ATP levels. It is activated by high AMP:ATP ratios during states of low energy charge. Once activated, the enzyme reduces the activity of ATP-consuming anabolic pathways and increases the activity of catabolic ATP-producing pathways, acting to reestablish normal cellular energy balance (for review see Ref. 13). Depending on the tissue, a variety of physiological circumstances can cause AMPK activation including hypoglycemia (14), ischemia (15), heat shock (16), and exercise (1719). AMPK activation has been associated with several key aspects of metabolism including exercise-induced glucose uptake and fatty acid oxidation in muscle (17, 20, 21), reduced lipid (22, 23) and glucose synthesis in liver (12, 24), and altered glucose-stimulated insulin secretion in the endocrine pancreas (14, 25).
AMPK is a heterotrimer consisting of a catalytic subunit and two
non-catalytic
and
subunits
(26,
27) that exist in multiple
isoforms
(2830).
Kinase activity is regulated allosterically by AMP binding and also by direct
phosphorylation mediated by an uncharacterized AMP-sensitive protein kinase. A
potential role of AMPK in the nucleus is suggested by several observations.
The first is the similarity of the mammalian kinase to the yeast SNF1
transcriptional regulator, as described above. Second, AMPK heterotrimers
containing the
2 subunit isoform are preferentially found in the
nucleus (31). In addition,
AMPK has been shown to phosphorylate the transcriptional coactivator p300 and
to regulate its ability to mediate nuclear receptor transcriptional activity
(32).
It has been reported previously
(33) that AMPK activation
reduces HNF4 target gene transcription. These findings, together with
the fact that both proteins participate in the regulation of similar metabolic
pathways, raise the possibility that HNF4
is directly regulated by
AMPK. The experiments described here examine the possibility that AMPK
phosphorylates HNF4
and regulates its transcriptional activity.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Transfection, and Reporter AssayTransient
transfections for in vivo labeling were conducted in a baby hamster
kidney cell line, BHK-21, which was cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum. Chinese hamster ovary (CHO)
cells were used in transfection for transcription assays and nuclear extract
preparation. CHO cells were maintained in Dulbecco's modified Eagle's
medium/F-12 medium supplemented with 10% fetal bovine serum. All transfections
were carried out with LipofectAMINE 2000 (Invitrogen). Transfections for
reporter assays were conducted in 24-well plates in triplicate for each
condition. Cells were harvested at 20 h post-transfection using lysis buffer
(Promega, Madison, WI), and the lysates were analyzed with Tropix Dual Light
Luciferase and -galactosidase assay kit (Tropix, Inc., Bedford, MA)
using an EG&G Berthold Microlumat 96P luminometer.
In Vitro and in Vivo PhosphorylationIn vitro
phosphorylation assays were conducted with in vitro translated
proteins produced using the TNT Quick-Coupled
transcription/translation system (Promega, Madison, WI). TNT
reactions were carried out for 60 min at 30 °C and then immunoprecipitated
by anti-FLAG (Sigma) or anti-T7 (Novagen Inc., Madison, WI) antibodies, and
protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). After
washing five times with HNTG buffer (20 mM HEPES, pH 7.5, 50
mM NaCl, 5% glycerol, 1% Triton, 1.5 mM
MgCl2, 1 mM EDTA), the purified beads were subjected to
an in vitro phosphorylation reaction using 100 milliunits of purified
rat liver AMPK (Upstate Inc., Lake Placid, NY) per reaction. Reactions were
carried out in a buffer containing 5 mM HEPES, pH 7.5, 0.1
mM dithiothreitol, 0.25% Nonidet P-40, 7.5 mM
MgCl2,50 µM ATP, 10 µCi of
[-32P]ATP, and a mixture of protein kinase C/protein kinase
A inhibitors, with or without of 300 µM AMP, and incubated at 30
°C for 30 min. The reactions were stopped by washing the beads three times
with HNTG buffer and adding 30 µl of 2x SDS loading buffer
(Invitrogen). The protein products were separated on SDS-PAGE, transferred to
nitrocellulose membranes, which were then subjected to autoradiography and
Western blot analysis using an anti-FLAG antibody conjugated with horseradish
peroxidase (Sigma), and detected with the SuperSignal West Pico
chemiluminescent system (Pierce).
In vivo phosphorylation labeling was conducted in BHK cells.
Wild-type pCMV.HNF4/C and pCMV.HNF/C.S304A mutant, along with the control
vector, were transfected in 100-mm plates. Each plate was split into two 60-mm
plates 16 h post-transfection. In vivo labeling was conducted with 1
mCi (37 MBq) of [32P]orthophosphate per plate for 1 h at 40 h
post-transfection, with or without of 500 µM
5'-aminoimidazol-4-carboxoxamide 1--D-ribofuranoside
(AICAR). Cells were harvested in 600 µl of HNTG buffer and mixed for 30 min
at 4 °C. The cell lysates were immunoprecipitated with anti-FLAG
monoclonal antibody (Sigma) and 50 µl of protein A/G-agarose beads for 2 h
at 4 °C. The beads were washed five times with same buffer. Bound proteins
were eluted in 30 µl of 2x SDS loading buffer at 95 °C for 5 min,
separated on 412% gradient SDS-PAGE gel, transferred to nitrocellulose
membrane, and detected by autoradiography or Western blot using anti-FLAG
antibody conjugated with horseradish peroxidase as described above.
Gel Mobility Shift and Pull-down AssaysCHO cells
transfected with full-length, wild-type, or mutant HNF4 were harvested
at 20 h post-transfection, with or without 12 h treatment of AICAR. Nuclear
extracts were prepared essentially as described
(34). The recombinant
HNF4
proteins were produced in vitro using TNT
Quik-Coupled transcription/translation system as described by the manufacturer
(Promega, Madison, WI). For dimerization experiments, where different
HNF4
proteins were produced from two plasmids, templates producing
wild-type or mutant HNF4
proteins were used in the co-translation
reactions at ratios determined in pilot translations to produce equal amounts
of protein. For GST pull-down assays, 25 µl of translation reactions were
incubated with 15 µl of glutathione-Sepharose 4B (Amersham Biosciences) in
BC-150 buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 0.01%
Nonidet P-40, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl
fluoride, and 1x protease inhibitor mixture (Roche Diagnostics)) for 30
min at room temperature on a rotary platform. The beads were washed five times
with BC-150 buffer, boiled in 25 µl of SDS sample buffer, and resolved on
420% gradient gels (Invitrogen). Gels were stained with Coomassie Blue,
incubated in Amplify (Amersham Biosciences) for 30 min, dried, and
fluorographed.
The synthetic double-stranded oligonucleotides used in DNA binding reaction
were labeled with [-32P]dCTP using the Rediprime-II random
labeling system (Amersham Biosciences). The double-stranded DNA binding probe
contains the proximal HNF4-binding site from the human apoCIII gene
(35). DNA binding reactions
were carried out in 20 mM HEPES, pH 7.9, 60 mM KCl, 3%
Ficoll, 0.5 mM MgCl2, 0.06% Nonidet P-40, 1
mM dithiothreitol, 1 µg of double-stranded poly(dI-dC), with
20,000 cpm of labeled probe (
0.25 ng), and either with 25 µg of
nuclear extract or 1 µl of TNT reaction. Reactions were
incubated for 20 min at room temperature and then analyzed on precast 6% DNA
retardation gels (Invitrogen) in 0.5x TBE at 125 V for 45 min at room
temperature.
Protein Stability ExperimentsFor the experiments shown in
Fig. 4, B and
C, CHO cells were transfected in 12-well plates as
described above but using LipofectAMINE Plus (Invitrogen) with 1 ng of the
tetracycline-regulated HNF4 expressing plasmids (pH4i-WT, pH4i-304A,
and pH4i-304D) together with 20 ng of pTET-OFF and 40 ng of pEGFP-N1
(Clontech, Palo Alto, CA) per well. 18 h after transfection doxycycline was
added (1 µg/ml) to the culture media to stop transcription of recombinant
HNF4
and cells were harvested 2, 4, 6, and 8 h later by lysis in HNTG
buffer. Gel electrophoresis and Western blot analysis to detect
FLAG-HNF4
and GFP protein were carried out as described above.
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RESULTS |
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Mapping AMPK Phosphorylation Site on
HNF4When the HNF4
amino acid sequence was
screened for the consensus target sequence for AMPK phosphorylation, three
potential sites were found (Fig.
1B) with the best match to a site surrounding serine 304.
To determine whether any of these sites were targets for AMPK phosphorylation,
they were each mutated to alanine and examined in an in vitro
phosphorylation assay. None of the mutants abolished phosphorylation by AMPK
(data not shown), indicating that there are multiple AMPK phosphorylation
sites on HNF4. To examine phosphorylation of individual segments of the
protein independently, four overlapping fragments of HNF4
were
generated from subclones of the HNF4
cDNA
(Fig. 1C). When in
vitro AMPK assays were conducted, all four subfragments were
phosphorylated to some degree (Fig.
1D) and showed the expected enhancement of
phosphorylation in the presence of AMP (an allosteric activator of the
kinase). However, when the results were normalized to the total amount of
protein in the assay, the HNF4/C fragment (containing the Ser-304 site) showed
the highest degree of AMP-dependent phosphorylation (data not shown). A
significant amount of phosphorylation was also observed with the HNF4/A
fragment. The HNF4/A fragment contains one of the three potential consensus
sites for AMPK (Fig.
1B). However, mutation of threonine 15 to alanine in this
sequence did not reduce the phosphorylation of the HNF4/A fragment, suggesting
the presence of one or more cryptic AMPK sites in this part of the
protein.
To determine whether the C fragment is phosphorylated at the Ser-304
residue, the S304A mutant was transferred to the C fragment and examined in an
in vitro phosphorylation assay. As presented in
Fig. 2A, mutation of
serine 304 completely abolished phosphorylation of the C fragment. Although
the Ser-304 site is also present in the D fragment
(Fig 1C), it does not
appear to be a good substrate for the kinase in this context as this fragment
is phosphorylated to a much lower degree than the C fragment
(Fig. 1D). When the
S304A mutation was examined quantitatively in the context of the full-length
protein, it reduced phosphorylation by 50%
(Fig. 2B). Together,
these results suggest that S304A is a phosphorylation site for AMPK in
vitro and that additional sites are present in both the N and C termini
of protein.
In vivo, HNF4 is a phosphoprotein and is a substrate for
multiple kinases (36,
37). To explore the
phosphorylation of HNF4
by AMPK in vivo, wild-type and S304A
versions of the HNF4/C fragment were cloned into a eukaryotic expression
vector and transfected into baby hamster kidney (BHK) cells. In vivo
phosphorylation labeling was carried out with these transfected cells in the
presence or absence of AICAR. AICAR is converted in cells to the AMP analog
ZMP
(5'-aminoimidazole-4-carboxamide-1-E-D-ribofuranotide),
which activates the kinase
(38). The wild-type HNF4/C
fragment was phosphorylated during the labeling reaction, and the treatment of
the cells with AICAR caused an
2-fold increase of phosphorylation
(Fig. 2C). This
phosphorylation was completely absent when the S304A version of the fragment
was used, indicating that Ser-304 is phosphorylated in vivo by AMPK.
In vivo phosphorylation experiments using the full-length HNF4
protein showed the same degree of phosphorylation for the wild-type and
Ser-304 mutant protein (data not shown). This may be due to a combination of
high basal phosphorylation of HNF4
by multiple kinases in vivo
(36,
37) and the likelihood that
there are additional AMPK phosphorylation sites on HNF4
(Fig. 1D).
Functional Effects of HNF4
PhosphorylationTo explore potential functional effects of
phosphorylation of HNF4
at serine 304, an aspartic acid residue was
introduced at this site by in vitro mutagenesis to generate a charge
mimic of the phosphoserine residue. The effect of phosphorylation at Ser-304
on the ability of HNF4
to form functional homodimers and bind to DNA
was examined in a gel mobility shift assay with wild-type and the
phosphomimetic S304D versions of HNF4
proteins synthesized by in
vitro translation. Both the wild-type and S304A version of HNF4
bound to an HNF4
-response element derived from the human apoCIII
promoter (Fig. 3A,
2nd and 3rd lanes). However, the S304D mutant HNF4
showed a dramatically reduced ability to bind to DNA despite similar amounts
of protein present in the reaction (Fig.
3A, 4th lane). Gel mobility shift experiments
using HNF4
protein that was phosphorylated by AMPK in vitro
showed only a mild reduction in DNA binding activity (data not shown). The
effect of phosphorylation on the ability of HNF4
to bind DNA would be
less pronounced than the S304D mutation if less than 100% of the HNF4
protein is phosphorylated in the in vitro kinase reaction. Although
the efficiency of these reactions is unknown, it is likely that only a
fraction of the HNF4
molecules become phosphorylated at Ser-304 leaving
the non-phosphorylated protein free to form dimers and bind DNA. In addition,
it is also possible that dimers composed of one phosphorylated and one
non-phosphorylated HNF4
molecule are competent to bind DNA.
Because HNF4 binds DNA as a homodimer, the defective DNA binding of
the S304D mutant could be due either to reduced dimer formation or to a
diminished affinity of mutant dimers for DNA. To examine directly the effect
of the mutant on dimerization, in the absence of DNA, an in vitro
association assay using 35S-labeled HNF4
monomers of
different sizes was carried out. A fusion protein of GST and wild-type
HNF4
(protein size 78 kDa) was co-translated with either wild-type or
mutant full-length HNF4
protein (52 kDa). The GST-HNF4
fusion
protein was purified out of the reaction with a glutathione resin together
with dimerized 52-kDa wild-type or mutant HNF4
. The results
(Fig. 3B) demonstrated
that wild-type and S304A HNF4
readily formed dimers with the wild-type
GST-HNF4
protein (arrow, lanes 1 and 2). In contrast,
the S304D protein showed a reduced ability to interact with the wild-type
GST-HNF4
(compare lane 3 to lanes 1 and 2).
Densitometry analysis of the Western blot demonstrated that the lower input
amount of S304D protein (lane 6) did not account for the reduction in
the amount of S304D bound to wild-type GST-HNF4
protein (data not
shown).
To examine further the ability of dimers composed of one unphosphorylated
monomer and one phosphorylated monomer to form and bind to DNA, wild-type and
S304D mutant HNF4 proteins of different sizes were mixed together and
the resulting dimers analyzed by gel mobility shift. The results
(Fig. 3C) demonstrate
that dimers composed of one wild-type GFP-HNF4
molecule (81 kDa) and
one S304D molecule (52 kDa) can form and are competent to bind DNA (lane
6) but with less efficiency than dimers of wild-type GFP-HNF4
and
either wild-type or S304A HNF4
proteins
(Fig. 3C, compare
lane 6 to lanes 3 and 9). As was observed in
Fig. 3A, homodimers of
S304D HNF4
failed to form (Fig.
3C, compare lane 4 to lanes 1 and
7). Taken together, these results demonstrate that, in
vitro, a negative charge on serine 304 inhibits dimer formation and that
the effect is more pronounced when both monomers contain the additional
negative charge.
To examine the effects of Ser-304 phosphorylation on dimer formation and
DNA binding in vivo, wild-type, S304A, and S304D expression
constructs were transfected into CHO cells (which have very low amounts of
endogenous HNF4). After transfection, AMPK was activated by AICAR treatment,
and nuclear extracts were prepared for gel mobility shift assays. As observed
in vitro the S304D phosphomimetic mutant showed reduced DNA binding
activity (Fig. 3D,
compare lane 8 to lane 4). In addition there is a mild
reduction in DNA binding of the wild-type protein upon AICAR treatment
(Fig. 3D, compare
lanes 4 and 5) that did not occur in the
phosphorylation-defective S304A protein
(Fig. 3D, compare
lanes 6 and 7). However, much of this reduction in DNA
binding activity correlates with changes in the amount of HNF4 present
in the various transfections (Fig.
3D, lower panel). For example, the amount of
S304D protein is significantly lower than the wild-type protein
(Fig. 3D, compare
lane 8 to lane 4, lower panel). Likewise, the amount of
wild-type HNF4
is reduced after AMPK activation
(Fig. 3D, compare
lanes 4 and 5, lower panel). The reduction of HNF4
amounts after AICAR treatment did not occur with the phosphorylation-defective
S304A mutation (Fig.
3D, compare lanes 6 and 7, lower panel)
suggesting that the effect is due to phosphorylation at serine 304. These
changes in HNF4
protein amounts suggest that addition of a negative
charge to serine 304 reduces the stability of the HNF4
protein in these
cells.
Potential effects of AMPK activation on HNF4 protein stability were
further explored by examining the appearance of proteolytic products of
wild-type and mutant HNF4
proteins in transiently transfected CHO cells
treated with AICAR. Western blot analysis shows that in the absence of AICAR
detectable amounts of degradation fragments appear in only lysates from cells
transfected with the phosphomimetic S304D HNF4
(Fig. 4A). The
activation of AMPK by AICAR addition induced the appearance of degradation
fragments from the wild-type but not the phosphorylation-defective S304A
protein (Fig. 4A).
These findings indicate that phosphorylation of HNF4
on serine 304
increased the proteolytic degradation of the protein. In addition, these
results strongly support the conclusion that AMPK phosphorylates HNF4
on serine 304 in vivo. To measure the rate of degradation of
wild-type and mutant HNF4
proteins, a tetracycline-regulated expression
system was used to transiently express wild-type and 304 mutant HNF4
in
transfected CHO cells. 18 h post-transfection, HNF4
expression was
specifically extinguished by the addition of doxycycline, and the amount of
HNF4
protein remaining in the cells was determined at regular intervals
over the following 8 h. The results (Fig.
4, B and C) demonstrate that the phosphomimetic
S304D mutant protein was degraded more rapidly then either the wild-type or
S304A proteins. Taken together, the in vitro and in vivo
results suggest that phosphorylation of HNF4
at Ser-304 has two effects
on HNF4: to reduce its ability to form functional dimers, and to reduce the
stability of HNF4
in vivo.
The HNF4 dimerization and DNA binding results presented above
predict that the AMPK-mediated phosphorylation of HNF4
on serine 304
would have a negative effect on its transcriptional activity. To determine
whether this is the case, plasmids expressing wild-type and Ser-304 mutant
proteins were transfected into CHO cells together with an
HNF4
-responsive reporter plasmid. As predicted from the DNA binding
studies, the S304D mutant showed significantly less transcriptional activity
than the wild-type HNF4
protein
(Fig. 5). The S304A mutant was
slightly more active than the wild-type protein suggesting the possibility
that there is a basal level of phosphorylation that reduces the activity of
the wild-type protein. These results indicate AMPK-negative regulates
HNF4
activity by phosphorylation of serine 304 and suggest that AMPK
activity could influence the expression of HNF4
target genes.
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DISCUSSION |
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In addition to the effects on HNF4 dimerization and DNA binding,
phosphorylation of HNF4
on Ser-304, or conversion of Ser-304 to an
aspartic acid, also caused an increase in the degradation rate and a reduction
in the amount of HNF4
protein (Fig.
3D and Fig.
4). Our results are consistent with previous findings showing that
activation of AMPK in primary hepatocytes resulted in a reduction in the
amount of endogenous HNF4
(33). Taken together, our
findings suggest a model in which one of the branches of the AMPK signaling
pathway results in the phosphorylation of HNF4
on serine 304, leading
to both a reduction in the amount and activity of HNF4
and a subsequent
reduction in the expression of HNF4
target genes.
Although the work reported here is focused on the phosphorylation of the
serine 304 residue in HNF4, it is clear that this is not the only site
on HNF4 that is phosphorylated by AMPK. The HNF4/A fragment from the
N-terminal half of the protein was strongly phosphorylated in vitro
on a site that we were not able to map. This site (or sites) may be
responsible for the remaining phosphorylation of the full-length protein that
occurs when the Ser-304 residue is mutated to alanine
(Fig. 2B). We are
currently mapping the AMPK site in the N terminus of HNF4
so that we
can determine their functional significance. In addition to the regulation of
HNF4
activity that we describe here, it has been reported previously
that HNF4
DNA binding activity is repressed by protein kinase
A-mediated phosphorylation of residues in the DNA binding domain
(37). It has also been
reported that phosphorylation of HNF4
on tyrosine residues affected its
transcriptional activity and sub-nuclear localization
(39). These findings, together
with those reported here, suggest that that HNF4
is a highly regulated
transcription factor and that multiple signaling pathways influence its
activity.
An interesting question concerning the phosphorylation of HNF4 by
AMPK in vivo is the cellular location in which it occurs. HNF4
is strongly associated with the nucleus, whereas most of the known AMPK
substrates are cytoplasmic enzymes. Although it is possible that HNF4
is also phosphorylated by in the cytoplasm, the most appealing compartment for
the interaction would be the nucleus. Several observations support this
possibility. First, specific isoforms of the kinase have been reported to be
present in the nucleus (31,
40). Second, other
transcriptional components have been reported to be potential substrates of
the kinase; the co-activator p300
(32) and the
carbohydrate-response transcription factor ChREBP
(41). Finally, the yeast
homolog of mammalian AMPK, SNF-1, is present in the nucleus (for review see
Ref. 13) and is associated
with chromatin, possibly in a gene-specific pattern
(42). Together these
observations suggest the intriguing possibility that at least some version of
mammalian AMPK may be a component of the transcriptional complex on specific
genes that are regulated by cellular energy balance.
What would be the physiological outcome of reduced HNF4 target gene
expression? The answer to this question depends on the tissue being
considered. In the endocrine pancreas, reduction of HNF4
activity would
be predicted to have significant effect on the ability of the islet to secrete
the appropriate amount of insulin. This possibility is illustrated by the
phenotype of MODY1 patients who have only one normal allele of the
HNF4
gene and presumably a reduced amount of active
HNF4
protein (3). These
patients have a defective insulin secretion in response that appears to be a
primary effect of reduced HNF4
levels in pancreatic
-cells
(4,
43). Consistent with the
apparent role of HNF4
in pancreatic physiology are the observations
that HNF4
regulates a variety of
-cell genes involved in glucose
sensing and insulin secretion
(68).
Like HNF4, AMPK also clearly plays a role in the physiology of the
endocrine pancreas. It is likely that the
-cell senses glucose
concentration as fluctuations in the amount of intracellular ATP generated by
glucose oxidation. The insulin secretory machinery gauges the amount of
insulin to secrete by sensing these ATP level fluctuations (for review see
Ref. 44). It is possible that
the changes in cellular energy balance in
-cells that occur in normal
physiology have an effect on HNF4
phosphorylation and activity and that
fluctuation in HNF4
activity are part of the normal physiological
regulation of the islet. Consistent with this possibility is the observation
that low glucose levels induce AMPK activity in cell lines derived from
pancreatic
-cells and the suggestion that changes in kinase activity are
involved in insulin secretion
(25). Alternatively, it is
possible that AMPK-mediated changes in HNF4
activity are relevant in
pathological conditions where AMPK is aberrantly activated and HNF4
activity is suppressed below normal levels. This situation could potentially
result in a physiological circumstance analogous to the MODY1 phenotype.
Another tissue where AMPK-mediated changes in HNF4 target gene
expression could have a significant metabolic effect is the liver. It has been
understood for many years that HNF4
plays an important role in the
liver, regulating genes involved in lipid
(45), amino acid
(46,
47), and glucose metabolism
(48,
49) and the coagulation
cascade (50). It has been
reported recently that HNF4
plays a key role in the induction of
hepatic gluconeogenesis by mediating the transcriptional effects of the
co-activator PGC1 (10). On the
other hand, the reduction of HNF4
levels due to haploinsufficiency in
MODY1 patients did not cause major liver abnormalities
(5,
51), although changes in
amounts of some liver-derived serum proteins were observed
(52).
It is clear that an AMPK-mediated phosphorylation of HNF4 could
potentially have important effects on liver and pancreatic islet physiology.
We are currently examining HNF4
phosphorylation in hepatic and
pancreatic cell lines to determine whether AMPK-dependent phosphorylation of
Ser-304 occurs under more physiological conditions and to ascertain the
potential metabolic role of the phosphorylation events described here.
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FOOTNOTES |
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Present address: Dept. of Pediatrics, University of Michigan Medical
School, Ann Arbor, MI 48109.
|| To whom correspondence should be addressed: Dept. of Pathology/CIMER, Wayne State University Medical School, 540 E. Canfield, Detroit, MI 48201. Tel.: 313-577-3006; Fax: 313-577-0057; E-mail: tleff{at}med.wayne.edu.
1 The abbreviations used are: HNF4, hepatic nuclear factor 4
;
AMPK, AMP-activated protein kinase; GST, glutathione S-transferase;
CMV, cytomegalovirus; CHO, Chinese hamster ovary; MODY, maturity onset
diabetes of the young; AICAR, 5'-aminoimidazol-4-carboxoxamide
1-
-D-ribofuranoside; BHK, baby hamster kidney; GFP, green
fluorescent protein; EGFP, enhanced GFP.
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ACKNOWLEDGMENTS |
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REFERENCES |
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