Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232
Received for publication, February 14, 2001, and in revised form, March 2, 2001
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ABSTRACT |
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Glucose-6-phosphatase is a multicomponent system
that catalyzes the terminal step in gluconeogenesis. To examine the
effect of the cAMP signal transduction pathway on expression of the
gene encoding the mouse glucose-6-phosphatase catalytic subunit
(G6Pase), the liver-derived HepG2 cell line was transiently
co-transfected with a series of G6Pase-chloramphenicol
acetyltransferase fusion genes and an expression vector encoding the
catalytic subunit of cAMP-dependent protein kinase A (PKA).
PKA markedly stimulated G6Pase-chloramphenicol acetyltransferase fusion
gene expression, and mutational analysis of the G6Pase promoter
revealed that multiple cis-acting elements were required for this
response. One of these elements was mapped to the G6Pase promoter
region between Glucose-6-phosphatase is a multicomponent system located in the
endoplasmic reticulum that catalyzes the terminal step in gluconeogenesis and hepatic glycogenolysis (1, 2). The kinetics of
glucose-6-phosphate hydrolysis by glucose-6-phosphatase are complex,
and several models for the glucose-6-phosphatase system have been
proposed (1, 2). One model has been proposed in which, in adults, in
contrast to the fetus (3), the active site of the catalytic subunit of
glucose-6-phosphatase
(G6Pase)1 is contained within
the lumen of the endoplasmic reticulum (1, 2). In this model the other
components of the glucose-6-phosphatase system act as transport
proteins to shuttle both substrate and product across the endoplasmic
reticulum membrane (1, 2). These include a glucose-6-phosphate
transporter and putative transporters for inorganic phosphate and
glucose (1, 2). Inactivating mutations in the G6Pase and
glucose-6-phosphate transporter genes gives rise to glycogen storage
disease types 1a and 1b, respectively (4). Type 1 glycogen storage
disease is characterized by severe hypoglycemia in the postabsorbtive
state, hyperlipidemia, hyperuricemia, and lactic acidemia (4-6). In
addition, patients are prone to growth retardation, hepatic steatosis
and cirrhosis, hepatic adenoma, and renal failure (4-7). Because of
the wider tissue distribution of the glucose-6-phosphate transporter,
patients afflicted with glycogen storage disease type 1b also suffer
from infectious complications as a result of functional deficiencies in
neutrophils and monocytes, indicating an important role for this
transporter in the immune response (4).
In contrast to glycogen storage disease type 1a, which is caused by
decreased G6Pase activity, increased G6Pase activity contributes to the
pathophysiology of diabetes. In both type 2 and poorly controlled type
1 diabetics, the ability of insulin to stimulate peripheral glucose
utilization and to repress hepatic glucose production is reduced as a
consequence of insulin resistance. Although the causes of insulin
resistance are unclear (8), it is apparent that the elevation in
hepatic glucose production is caused by an increased rate of
gluconeogenesis rather than glycogenolysis in both type 1 (9) and type
2 diabetics (10). Several lines of evidence suggest that increased
expression of key gluconeogenic enzymes including G6Pase contribute to
this increase in hepatic glucose production. Thus, hepatic G6Pase
expression is markedly elevated in various diabetic animal models
(11-15), and overexpression of G6Pase in hepatocytes using recombinant adenovirus was associated with enhanced rates of gluconeogenesis as
well as defects in glycogen metabolism (16). Furthermore, a modest
overexpression of G6Pase in rats, again using recombinant adenovirus,
resulted in approximately a 1.6-3-fold increase in hepatic G6Pase
enzymatic activity that was associated with glucose intolerance,
hyperinsulinemia, decreased hepatic glycogen content, and increased
peripheral triglyceride stores, changes similar to those found in early
stage type 2 diabetic patients (17). These observations (16, 17)
suggest that G6Pase is a major control point in the
glucose-6-phosphatase system and represents a prime therapeutic target
for the treatment of diabetes.
In the liver cAMP, glucocorticoids, glucose, fatty acids, leptin, and
Materials--
[ Plasmid Construction--
The construction of a series of 5'
truncated G6Pase-CAT fusion genes has been described previously (29,
30). A site-directed mutant of the G6Pase HNF-6 motif was generated
within the context of the
Expression vectors encoding the
HNF-6 contains three consensus PKA phosphorylation sites
(serines 309, 411, and 440). The codons in HNF-6 that encode these serine residues were mutated by site-directed mutagenesis to codons that encode alanine residues. Briefly, HNF-6 pGEM1 (34) was digested
with SacI, and the resultant SacI-SacI
fragment was then ligated into SacI-digested pGEM7. This
plasmid contains the carboxyl-terminal third of HNF-6 and was used as
the template in the PCR-based mutagenesis procedures. The codon
encoding serine 309 was mutated using the following
oligonucleotides:
5'-CCGAGCTCAAACGTTACGCCATCCCACAGGCCATC-3' and
5'-CGGGATCCGAGCTCGAATTC-3'. SacI sites and the
mutated bases are underlined. The resulting PCR product was digested
with SacI and ligated into SacI-digested pGEM7 to
generate a plasmid designated HNF-6 site 1 SDM pGEM7. The codons
encoding serines 411 and 440 were mutated individually using the
QuikChangeTM site-directed mutagenesis kit (Stratagene) as directed by
the manufacturer using the following oligonucleotides: serine 411, 5'-GGAAAATAAGCGTCCGGCCAAAGAATTACAAATC-3' and
5'-GATTTGTAATTCTTTGGCCGGACGCTTATTTTCC-3'; and serine 440, 5'-GAATGCCAGAAGGAGGGCTCTGGACAAGTGGCAGG-3' and
5'-CCTGCCACTTGTCCAGAGCCCTCCTTCTGGCATTC-3'. The underlined
nucleotides indicate the mutated bases. The resulting plasmids were
designated HNF-6 site 2 SDM pGEM7 and HNF-6 site 3 SDM pGEM7, respectively.
Convenient restriction enzyme sites flanking the mutated codons were
then utilized to isolate smaller fragments that contained these mutated
codons. Thus, the plasmid encoding the mutated serine 309 codon was
digested with SacI and PstI, and the plasmids
encoding the mutated serine 411 and 440 codons were digested with
PstI and PvuII and PvuII and
SacI, respectively. The fragments generated were then
ligated together into SacI-digested pGEM7, and the resulting plasmid was designated HNF-6 site 1-3 SDM pGEM7. This plasmid was then
digested with SacI and EcoRI, and the fragment
generated that contained the mutated HNF-6 sequence was exchanged for
the equivalent fragment in the wild-type HNF-6 pcDNA3 plasmid
described above to create a plasmid designated HNF-6 SDM pcDNA3. In
addition, this same SacI-EcoRI fragment was
exchanged for the equivalent fragment of HNF-6 in the wild-type HNF-6
pET15b bacterial expression vector, which corresponded to a
SacI-BamHI fragment. The noncompatible ends were
filled in using the Klenow fragment of E. coli DNA
polymerase I and then religated. The resulting plasmid was designated
HNF-6 SDM pET15b. All fragments generated by PCR were completely
sequenced using the USB SequenaseTM kit to verify the absence of
polymerase errors. Plasmid constructs were purified by centrifugation
through cesium chloride gradients (35).
Cell Culture, Transient Transfection, CAT, and Gel Retardation Assays--
IPTG was used to induce the
expression of HNF-6 in the BL21(DE3) pLysS E. coli strain
(Stratagene) transformed with the full-length HNF-6 pET15b plasmid
described above. Bacterial extracts were prepared by sonication in 20 mM HEPES, pH 7.5, 50 mM KCl, 1 mM MgCl2, 0.5 mM EGTA, and 1 mM
phenylmethylsulfonyl fluoride at 4 °C. The soluble fraction was
separated from the particulate fraction via centrifugation.
Complementary oligonucleotides representing the mouse G6Pase HNF-6
motif (Fig. 1B) were synthesized with
HindIII-compatible ends, gel purified, annealed, and then
labeled with [ HNF-6 Purification--
The full-length, wild-type and mutated,
and carboxyl-terminal truncated forms of HNF-6 were expressed in the
BL21-CodonPlusTM(DE3)-RIL E. coli strain (Stratagene)
transformed with the HNF-6 pET15b plasmids described above. Once
bacterial cultures (1 liter) had reached an
A600 of ~0.6, protein expression was induced
with IPTG (1 mM) by incubation for 4 h at 37 °C.
After centrifugation bacterial pellets were stored at Phosphorylation Experiments--
Partially purified
histidine-tagged HNF-6 was phosphorylated at room temperature by PKA
(0.12 µM) in a final reaction volume of 40 µl, pH 8.0, containing 5 mM NaH2PO4, 2.5 mM HEPES, 25 mM imidazole, 50 mM
NaCl, 200 µM [ An Element Located between An HNF-6 Binding Site Located between HNF-6 Binds to the G6Pase HNF-6 Motif--
To provide evidence
that HNF-6 is indeed the factor that is mediating the effect of PKA
through the The G6Pase HNF-6 Motif Can Confer a Direct Stimulatory Effect of
PKA on the Expression of a Heterologous Fusion Gene--
To determine
whether the G6Pase HNF-6 motif was sufficient to mediate a direct
stimulatory effect of PKA on gene transcription, six copies of a
double-stranded oligonucleotide representing the wild-type G6Pase
promoter sequence between HNF-6 Is Phosphorylated by PKA in Vitro--
Rousseau and
co-workers (39) have previously noted that rat HNF-6 contains five
potential PKA phosphorylation sites, but only three of these strongly
match the consensus PKA phosphorylation sequence (40). All five sites
are perfectly conserved among human, rat, and mouse HNF-6. Fig.
3 shows that PKA can phosphorylate a
histidine-tagged form of HNF-6 in a time- and
concentration-dependent manner in vitro. The
kinetics of the phosphorylation were markedly affected by the
concentration of magnesium in the reaction (Fig. 3, compare
A and B). In the presence of low magnesium (0.1 mM), PKA phosphorylates HNF-6 with an apparent
Km of ~0.25 µM (Fig. 3A).
However, in the presence of high magnesium (5 mM), we were
not able to calculate an apparent Km, because we
were unable to add sufficient HNF-6 to the phosphorylation reaction to
reach Vmax (Fig. 3B). In the presence
of either low (0.1 mM) or high (5 mM)
concentrations of magnesium, the time course of HNF-6 phosphorylation
was similar with maximal phosphorylation by ~120 min (Fig. 3,
C and D). However, the maximal incorporation of
phosphate into HNF-6 was much greater when phosphorylation reactions
contained 5 mM magnesium. A maximum stoichiometry of ~1.75 mol of phosphate/mol of HNF-6 was calculated in the presence of
5 mM magnesium. In contrast, in the presence of 0.1 mM magnesium, a maximum stoichiometry of ~0.3 mol of
phosphate/mol of HNF-6 was obtained. Such magnesium-dependent
variations in the kinetics and stoichiometry of phosphorylation by PKA
have been reported for other PKA substrates (41-43).
The three consensus PKA phosphorylation sites in HNF-6 are
located in the carboxyl-terminal region of the protein (39). Therefore,
an expression vector was constructed that encoded a histidine-tagged
carboxyl-terminal truncated form of HNF-6 in which these three putative
PKA phosphorylation sites were deleted. The phosphorylation of this
truncated form of HNF-6 by PKA was markedly reduced compared with the
nontruncated form of the protein (Fig.
4). This result suggests that HNF-6 is
phosphorylated in vitro by PKA predominantly on one or more
of the carboxyl-terminal sites that match the consensus PKA
phosphorylation sequence. Further support for this conclusion was
obtained by constructing an expression vector that encoded a
histidine-tagged full-length form of HNF-6 in which these three
putative PKA serine phosphorylation sites were changed to alanine
residues by site-directed mutagenesis (Fig. 4). The phosphorylation of
this mutated form of HNF-6 by PKA was markedly reduced compared with
the wild-type form of the protein (Fig. 4).
To explore the functional consequence of mutating these three serine
residues on PKA-stimulated G6Pase-CAT fusion gene expression, expression vectors encoding full-length wild-type and mutated HNF-6
were constructed. Fig. 5 shows that the
co-transfection of the wild-type Multiple promoter elements are required for the full stimulatory
effect of the cAMP signal transduction pathway on G6Pase gene
transcription in hepatoma cells that together comprise a CRU (27, 36).
The large induction of G6Pase-CAT fusion gene expression obtained by
using the PKA co-transfection technique was critical for the
delineation of such a multiple component CRU (Fig. 1A; Ref.
27). Thus, in contrast, using cAMP analogs both Chou and co-workers
(25) and Burchell and co-workers (26) reported the involvement of
single elements in the cAMP response. Lin et al. (25) found
that a region of the human G6Pase promoter encompassing the sequence
between HNF-6 is a member of the ONECUT family of transcription factors that is
characterized by a bipartite DNA binding domain consisting of a single
cut domain and an atypical homeodomain (39). Classical homeodomains are
60 amino acids long and contain a conserved tryptophan and histidine at
positions 48 and 50 of the homeodomain as opposed to the homeodomain in
the ONECUT transcription factor family, in which the amino acid
residues located at positions 48 and 50 are phenylalanine and
methionine, respectively (37). The cut domain has been shown to be
required for HNF-6 binding to DNA in all target genes examined, whereas
for a subset of HNF-6 target genes, the homeodomain seems to be
dispensable for DNA binding (37). Both the cut domain and the
homeodomain of HNF-6 are also involved in transcriptional activation by
HNF-6 (37, 44). Activation of HNF-6 target gene transcription on
promoters that do not require the HNF-6 homeodomain for DNA binding
involves the recruitment of the CREB-binding protein (44). The
interaction of CREB-binding protein with rat HNF-6 requires an LXXLL
motif (where L is a leucine residue and X is any amino acid) in the cut
domain and the amino acid residues located at positions 48 and 50 (phenylalanine and methionine, respectively) of the homeodomain (44).
The LXXLL motif has previously been shown to be important for the
interaction of other transcription factors with CREB-binding protein
(45). In contrast, activation of gene transcription by HNF-6 on target
genes that require the homeodomain for DNA binding involves the
recruitment of the coactivator p300/CREB-binding protein-associated
factor through an unidentified domain (44).
Of the three consensus PKA phosphorylation sites in HNF-6, one is
located in the vicinity of the LXXLL motif in the cut domain (serine
residue 309), and the other two are located in the homeodomain (serine
residues 411 and 440). As described above, both the cut domain and the
homeodomain are involved in DNA binding and in recruitment of
coactivators, and thus the phosphorylation of HNF-6 by PKA could
potentially have affected either or both parameters. Because
phosphorylation by PKA has little effect on HNF-6 binding to DNA (data
not shown), we hypothesize that it increases the transactivation
potential of HNF-6. However, this putative effect of
PKA-dependent phosphorylation on HNF-6 transactivation
potential is only apparent under conditions in which the concentration
of HNF-6 is limiting (Fig. 5). Thus, overexpression of either wild-type or mutated HNF-6 stimulates basal G6Pase-CAT fusion gene expression to
the same extent as that achieved by co-transfection with the expression
vector encoding PKA (Fig. 5). It may be possible to prove that
PKA-dependent phosphorylation increases the transactivation potential of HNF-6 by analyzing the effect of mutating the three PKA
phosphorylation sites in the context of an HNF-6 molecule in which the
basal activation domains (37, 44) have been mutated but only if these
same domains are not also required for the PKA response. These
observations are somewhat related to those recently reported by Quinn
and co-workers (46, 47), who investigated the relative contributions of
different domains in CREB to transcription initiation. Of particular
note is the observation that the constitutive activation domain in CREB
mediates recruitment of the polymerase complex, whereas the
kinase-inducible domain mediates later PKA-stimulated steps in
transcription initiation. In the case of HNF-6 we hypothesize that when
the HNF-6 binding site in the G6Pase promoter is fully occupied, the
basal activation domains in HNF-6 are sufficient to mediate a maximal
rate of transcription initiation. Interestingly, protein kinase C and
casein kinase II phosphorylate the Cut/CCAAT displacement
protein, a member of the superclass of cut homeodomain proteins, on residues located in the cut domain and alter the binding
of the Cut/CCAAT displacement protein to DNA (48, 49).
Because overexpression of HNF-6 stimulates G6Pase fusion gene
expression (Fig. 5), this raises the possibility that
hormones/metabolites could regulate G6Pase gene expression indirectly
through an action on HNF-6 gene expression. There is circumstantial
evidence that HNF-6 expression may be regulated by cAMP/PKA. Thus a CRE
is present in the HNF-6 promoter (50) and growth hormone, which
activates PKA in liver (51), stimulates HNF-6 expression (52). However, whether the CRE in the HNF-6 promoter contributes to this stimulation is unknown (50). The available data show that the effect of growth
hormone on HNF-6 expression is mediated at least in part through
increases in signal transducer and activator of transcription-5 and
HNF-4 binding (53) and a decrease in CCAAT/enhancer-binding protein- The phosphorylation of HNF-6 by PKA varied with changes in the
magnesium ion concentration (Fig. 3). Other substrates are also
differentially phosphorylated by PKA when the magnesium ion concentration is altered (41-43). Thus, Singh et al. (41,
43) demonstrated that PKA phosphorylates a single site in the In summary, the data presented in this manuscript demonstrate that an
HNF-6 site located between 114 and
99, and this sequence was shown to bind
hepatocyte nuclear factor (HNF)-6. This HNF-6 binding site was able to
confer a stimulatory effect of PKA on the expression of a heterologous
fusion gene; a mutation that abolished HNF-6 binding also abolished the
stimulatory effect of PKA. Further investigation revealed that PKA
phosphorylated HNF-6 in vitro. Site-directed mutation of
three consensus PKA phosphorylation sites in the HNF-6 carboxyl
terminus markedly reduced this phosphorylation. These results suggest
that the stimulatory effect of PKA on G6Pase fusion gene transcription
in HepG2 cells may be mediated in part by the phosphorylation of
HNF-6.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 adrenergic receptor agonists all stimulate G6Pase gene
expression (11, 14, 18-24), whereas insulin both inhibits basal G6Pase
gene expression and overrides the stimulatory effects of cAMP,
glucocorticoids, glucose, and fatty acids (11, 18-20, 22, 24).
Multiple cis-acting elements in the G6Pase promoter are required for
the full stimulatory effect of the cAMP signal transduction pathway on
G6Pase gene expression (25-27). This paper shows that one of these
elements is a binding site for hepatocyte nuclear factor (HNF)-6 and
demonstrates that HNF-6 is a substrate for the catalytic subunit of
protein kinase A (PKA).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP (>3000 Ci
mmol
1) and [
-32P]ATP (>6000 Ci
mmol
1) were obtained from Amersham Pharmacia Biotech, and
[3H]acetic acid sodium salt (>10 Ci mmol
1)
was obtained from ICN. BL21(DE3) pLysS and
BL21-CodonPlusTM(DE3)-RIL-competent Escherichia coli cells
were obtained from Stratagene, and nickel-nitrilotriacetic acid
agarose was from Qiagen. Purified bovine PKA was a generous gift from
Drs. Jackie Corbin and Sharron Francis (28).
231 to +66 G6Pase promoter fragment using a
previously described three-step PCR strategy (29, 31). The resulting construct was used as the template in a second PCR to create a site-directed mutant of the G6Pase HNF-6 motif within the context of
the
129 to +66 G6Pase promoter fragment (Fig. 1). The heterologous XMB vector contains a minimal Xenopus 68-kDa albumin
promoter ligated to the CAT reporter gene (32). Double-stranded
complementary oligonucleotides representing the wild-type or mutated
HNF-6 motif (Fig. 1B) were synthesized with
HindIII-compatible ends and ligated into
HindIII-cleaved XMB in multiple (5-6) copies. The number of
inserts was determined by restriction enzyme analysis and confirmed by
DNA sequencing.
and
forms of PKA were a
generous gift from Dr. Richard Maurer (33). An empty vector control was
generated by digesting the PKA
plasmid with XhoI and
HindIII to remove the open reading frame, filling in the
noncompatible ends using the Klenow fragment of E. coli DNA
polymerase I, and then religating. A mammalian cell expression vector
encoding the full-length form of HNF-6 was constructed by cloning the
open reading frame of mouse HNF-6, isolated as an
EcoRI-EcoRI fragment from the plasmid HNF-6 pGEM1
(a generous gift from Dr. Robert Costa) (34), into
EcoRI-digested pcDNA3 (Invitrogen). A bacterial cell
expression vector for HNF-6 was constructed by re-isolating the coding
region of HNF-6 from the pcDNA3 plasmid, minus nine amino acids at
the N terminus, as a PvuI-XhoI fragment and
ligating into XhoI-digested pET15b (Novagen). The
noncompatible ends were then filled in using the Klenow fragment of
E. coli DNA polymerase I prior to blunt-end ligation. In the
resulting plasmid designated full-length HNF-6 pET15b, the HNF-6 coding
sequence is in frame with that of a 6x histidine tag. A
carboxyl-terminal truncation of HNF-6 was constructed by digesting this
HNF-6 pET15b plasmid with SacI and BamHI, filling
in the noncompatible ends using the Klenow fragment of E. coli DNA polymerase I, and then religating.
-galactosidase
Assays--
Human HepG2 hepatoma cells were grown and transiently
transfected in suspension using the calcium phosphate DNA
co-precipitation method as described previously (29, 30). CAT and
-galactosidase assays were also performed exactly as described
previously (29, 30). The CAT activity directed by the various fusion
gene constructs was corrected for the
-galactosidase activity in the
same samples, and each construct was analyzed in duplicate in multiple
transfections as specified in the figure legends.
-32P]dATP by using the Klenow fragment
of E. coli DNA polymerase I to a specific activity of ~2.5
µCi/pmol. The labeled HNF-6 oligonucleotide (~7.5 fmol, ~30,000
cpm) was incubated with bacterial extract in a final reaction volume of
20 µl containing 20 mM HEPES, pH 7.5, 50 mM
KCl, 1 mM MgCl2, 0.5 mM EGTA, 1 mM dithiothreitol, 10% glycerol (v/v), and 2 µg of
poly(dI-dC)·poly(dI-dC). After incubation for 10 min at room
temperature, the reactants were loaded onto a 6% polyacrylamide gel
and electrophoresed at room temperature for 120 min at 150 V in 0.25×
TBE buffer (1× TBE is 89 mM Tris, 89 mM boric
acid, and 2 mM EDTA). After electrophoresis, gels were
dried and exposed to Kodak XAR5 film, and binding was analyzed by
autoradiography. For competition experiments, a 100-fold molar excess
of the unlabeled double-stranded wild-type or mutated HNF-6
oligonucleotides (Fig. 1B) was incubated with the labeled oligomer prior to the addition of bacterial extract. Binding was then
analyzed by polyacrylamide gel electrophoresis as described above.
20 °C
overnight. The histidine-tagged HNF-6 protein was then partially
purified using metal affinity chromatography. Briefly, bacterial
pellets were thawed on ice for 15 min and then resuspended in 20 ml of
lysis buffer, pH 8.0, containing 50 mM
NaH2PO4, 500 mM NaCl, 20 mM imidazole, 10 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme.
After incubation on ice for 30 min, bacteria were sonicated at 4 °C
10 times for 30 s each with a 1-min incubation on ice between each
round of sonication. Lysates were then diluted with an additional 20 ml
of lysis buffer, and particulate matter was removed by centrifugation.
The supernatant was incubated with 5 ml of nickel-nitrilotriacetic acid
agarose (Qiagen) for 1 h at 4 °C before the application of the
slurry to a column. After washing twice with 50 ml of a buffer, pH 8.0, comprising 50 mM NaH2PO4, 500 mM NaCl, 60 mM imidazole, 10 mM
-mercaptoethanol, 0.5 mg/ml bovine serum albumin, and 1 mM phenylmethylsulfonyl fluoride, HNF-6 was eluted in a
buffer, pH 7.2, containing 50 mM
NaH2PO4, 500 mM NaCl, 250 mM imidazole, 10 mM
-mercaptoethanol, 0.5 mg/ml bovine serum albumin, and 1 mM phenylmethylsulfonyl fluoride. The purification of HNF-6 was analyzed via SDS-PAGE, and
protein was visualized by Coomassie Brilliant Blue staining (35). The
yield of HNF-6 was estimated by comparison to the staining of a bovine
serum albumin standard.
-32P]ATP (1.25 µCi/nmol), 0.6 mg/ml bovine serum albumin, and 0.5% (v/v) ethylene
glycol. The concentrations of HNF-6 and magnesium acetate as well as
the time course of the reaction were as indicated in the figure
legends. All reactions were terminated by the addition of 20 µl of
3× SDS sample buffer and boiling for 5 min. Samples were analyzed by
SDS-PAGE, and protein was visualized via Coomassie Brilliant Blue
staining (35). Gels were subsequently dried and exposed to Kodak XAR5
film. Bands corresponding to phosphorylated HNF-6 were then cut out of
the dried gels, and the incorporation of phosphate into HNF-6 was
quantified by scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
129 and
85 in the G6Pase Promoter
Contributes to the Full Stimulatory Effect of the cAMP Signal
Transduction Pathway on G6Pase-CAT Fusion Gene Expression--
To
examine the molecular mechanisms by which the cAMP signal transduction
pathway stimulates mouse G6Pase gene expression, the liver-derived
HepG2 cell line was transiently co-transfected with a series of
G6Pase-CAT fusion genes and an expression vector encoding PKA. PKA
markedly stimulated G6Pase-CAT fusion gene expression, and mutational
analysis of the G6Pase promoter revealed that multiple regions were
required for this response (Fig.
1A; Ref. 27). This strategy
led to the identification of a cAMP response element (CRE) in the mouse
G6Pase promoter located between
162 and
155 (27). However, even
when this element was deleted, PKA still induced G6Pase-CAT fusion gene
expression ~4-5-fold (Fig. 1A; Ref. 27). Further deletion
of the G6Pase promoter sequence between
129 and
85 resulted in an
additional reduction in the stimulatory effect of PKA on G6Pase-CAT
fusion gene expression (Fig. 1A). These results indicate
that the cAMP signal transduction pathway stimulates the expression of
G6Pase through a complex cAMP response unit (CRU; Ref. 36) and that an
element located between
129 and
85 in the G6Pase promoter
contributes to the full stimulatory effect of PKA on G6Pase-CAT fusion
gene expression.
View larger version (15K):
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Fig. 1.
Multiple regions of the G6Pase promoter
contribute to the full induction of G6Pase-CAT fusion gene
transcription by PKA in HepG2 cells including an HNF-6 binding site
located between 110 and
101 in the G6Pase promoter.
A and C, HepG2 cells were transiently
co-transfected as described under "Experimental Procedures" with
various G6Pase-CAT fusion genes (15 µg), an expression vector
encoding
-galactosidase (2.5 µg), and either an expression vector
(5 µg) encoding PKA or the same vector (5 µg) with the PKA open
reading frame deleted. The G6Pase-CAT fusion genes contained either
distinct lengths of the wild-type promoter sequence as indicated by the
5' deletion end points or a site-directed mutation of the HNF-6 site
designated
129 HNF-6 SDM. The HNF-6 motif was mutated as shown in
B. After transfection, cells were incubated for 18-20 h in
serum-free medium. The cells were then harvested, and both CAT and
-galactosidase activity were assayed as described previously (29,
30). Results are presented as the ratio of CAT activity, corrected for
-galactosidase activity in the cell lysate, in PKA-transfected
versus empty vector-transfected cells (expressed as -fold
induction) and represent the mean ± S.E. of 5-18 (A)
or 3 (C) experiments in which each construct was assayed in
duplicate. SDM, site-directed mutant. B,
comparison of the mouse G6Pase promoter sequence between
114 and
99
with the equivalent sequence from the rat and human G6Pase promoters.
The putative HNF-6 binding motif is boxed. The consensus
HNF-6 sequence is taken from Ref. 37.
110 and
101 in the G6Pase
Promoter Contributes to the Full Stimulatory Effect of PKA on
G6Pase-CAT Fusion Gene Expression--
Examination of the mouse G6Pase
promoter sequence between
129 and
85 for known transcription factor
binding sites revealed a putative binding site for HNF-6 located
between nucleotides
110 and
101 (Fig. 1B; Ref. 37). The
sequence of this HNF-6 motif is highly conserved among the mouse, rat,
and human G6Pase genes (Fig. 1B); this seems to be the only
HNF-6 motif in the mouse G6Pase promoter. Samadani and Costa (38) have
previously shown that HNF-6 is expressed in HepG2 cells. To determine
whether this putative HNF-6 binding site was required for the
stimulatory effect of PKA on G6Pase-CAT fusion gene expression, this
motif was mutated by site-directed mutagenesis in the context of the
129 to +66 G6Pase promoter fragment. The fusion gene containing this
mutation, designated
129 HNF-6 SDM, was transiently co-transfected into HepG2 cells in combination with the expression vector encoding PKA. Mutation of the G6Pase HNF-6 motif resulted in a decreased stimulation of G6Pase-CAT fusion gene expression by PKA compared with
the wild-type
129 G6Pase-CAT fusion gene construct (Fig. 1C) that was equivalent to that seen when the entire HNF-6
site was deleted, as is the case with the
85 G6Pase-CAT fusion gene (Fig. 1C). This result suggests that HNF-6 could be the
factor that is mediating the stimulatory effect of PKA on G6Pase-CAT fusion gene expression through this region.
129 to
85 promoter sequence, HNF-6 binding to this
region was analyzed using the gel retardation assay. IPTG was used to
induce the expression of a histidine-tagged form of mouse HNF-6 in
bacteria, and then a soluble extract from these cells was incubated
with a labeled double-stranded oligonucleotide representing the G6Pase
HNF-6 motif (Fig. 2A). No
protein binding was detected in the gel retardation assay using lysate
from non-IPTG-treated bacteria (data not shown); however, a single
protein-DNA complex was detected using lysate from IPTG-treated cells
(Fig. 2A). Competition experiments, in which a 100-fold
molar excess of unlabeled DNA was included with the labeled probe, were
used to correlate protein binding with the PKA response. An
oligonucleotide representing the wild-type G6Pase HNF-6 binding site
competed effectively with the labeled probe for protein binding (Fig.
2A). By contrast, an oligonucleotide that contains a
mutation identical to that in the
129 HNF-6 SDM construct (Fig. 1)
failed to compete (Fig. 2A). Thus, the binding of HNF-6 to
the G6Pase promoter (Fig. 2A) correlates with the
stimulatory effect of PKA on G6Pase-CAT fusion gene expression (Fig.
1).
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Fig. 2.
HNF-6 binds to the G6Pase HNF-6 motif and
confers a direct stimulatory effect of PKA on the expression of a
heterologous fusion gene. A, a labeled double-stranded
oligonucleotide representing the wild-type mouse G6Pase HNF-6 binding
site (Fig. 1B) was incubated in the absence ( ) or presence
of a 100-fold molar excess of the unlabeled oligonucleotides shown,
representing either the wild-type (WT) or mutated
(MUT) G6Pase HNF-6 motif (Fig. 1B). Extract from
IPTG-treated E. coli transformed with an expression vector
encoding a 6x histidine-tagged HNF-6 fusion protein was then added, and
protein binding was analyzed using the gel retardation assay as
described under "Experimental Procedures." A representative
autoradiograph is shown. B, HepG2 cells were transiently
co-transfected as described under "Experimental Procedures" with
heterologous XMB fusion genes (15 µg), an expression vector encoding
-galactosidase (2.5 µg), and either an expression vector (5 µg)
encoding PKA or the same vector (5 µg) with the PKA open reading
frame deleted. The heterologous XMB fusion genes were generated by
ligating oligonucleotides representing either the wild-type or mutated
G6Pase HNF-6 motif (Fig. 1B) into the HindIII
site of the XMB vector in multiple (5-6) copies. After transfection,
cells were incubated for 18-20 h in serum-free medium. The cells were
then harvested, and both CAT and
-galactosidase activity were
assayed as described previously (29, 30). Results are presented as the
ratio of CAT activity, corrected for
-galactosidase activity in the
cell lysate, in PKA-transfected versus empty
vector-transfected cells (expressed as -fold induction). Results are
the mean ± S.E. of 3-4 experiments in which each construct was
assayed in duplicate. No reporter gene expression was detected when the
basic XMB vector was transiently transfected into HepG2 cells even in
the presence of the PKA expression vector (data not shown).
114 and
99 (Fig. 1B) were
ligated into the heterologous XMB expression vector (32). Transient
co-transfection of the resulting construct, designated HNF-6 WT XMB,
into HepG2 cells with the expression vector encoding PKA resulted in an
approximately 6-fold stimulation of reporter gene expression (Fig.
2B). Similarly, three copies of a larger double-stranded
oligonucleotide representing the wild-type G6Pase promoter sequence
between
114 and
77 conferred a 4.12 ± 0.19-fold induction
(n = 4) of reporter gene expression by PKA when ligated into the heterologous XMB expression vector (data not shown). To verify
that the stimulatory effect of PKA was mediated through the HNF-6
binding site, five copies of a double-stranded oligonucleotide containing the same mutation (Fig. 1B) that abolished HNF-6
binding in gel retardation assays (Fig. 2A) were ligated
into the XMB vector. No basal reporter gene expression was detected
when the resulting construct, designated HNF-6 MUT XMB, was transiently transfected into HepG2 cells. Furthermore, co-transfection with the
expression vector encoding PKA failed to induce reporter gene expression (Fig. 2B). These results support a model in which
HNF-6 is a target of PKA signaling.
View larger version (25K):
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Fig. 3.
HNF-6 is phosphorylated by PKA in
vitro. The ability of PKA to phosphorylate a partially
purified 6x histidine-tagged HNF-6 fusion protein was assessed as
described under "Experimental Procedures." Phosphorylation
reactions were analyzed by SDS-PAGE, and phosphate incorporation was
quantitated by scintillation counting. A and B
show the relationship between HNF-6 concentration and 32P
incorporation in the presence of 0.1 mM or 5 mM
magnesium acetate, respectively. Phosphorylation reactions were
performed for 5 (A) or 1 min (B) at room
temperature. Under these conditions the rate of 32P
incorporation into HNF-6 was linear at all HNF-6 concentrations tested.
C and D show the relationship between time and
32P incorporation in the presence of 0.1 mM or
5 mM magnesium acetate, respectively. Phosphorylation
reactions were performed using 1 µM HNF-6. Each
panel shows the mean data ± S.E. from three
experiments with a representative autoradiograph shown as an
inset. Plots without error bars indicate
that the S.E. values were smaller than the plot symbol.
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Fig. 4.
Truncation of the carboxyl-terminal of HNF-6
or site-directed mutation of three consensus PKA phosphorylation sites
in the carboxyl terminus of HNF-6 markedly reduces phosphorylation by
PKA in vitro. The ability of PKA to phosphorylate
partially purified 6x histidine-tagged full-length wild-type HNF-6
(lane 2), HNF-6 containing site-directed mutations of three
consensus PKA phosphorylation sites (lane 3), or the
carboxyl-terminal truncated form ( HNF-6; lane
4) was assessed as described under "Experimental Procedures."
Phosphorylation reactions were analyzed by SDS-PAGE. A shows
a Coomassie-stained SDS-PAGE gel, and B shows an
autoradiograph of the same gel. Lane 1 contains molecular
mass markers. An arrow indicates the position of
autophosphorylated PKA in B. A representative experiment is
shown.
129 G6Pase-CAT fusion gene construct
with either of these expression vectors stimulated reporter gene
expression to the same extent as that achieved by co-transfection with
the expression vector encoding the catalytic subunit of PKA. Because phosphorylation by PKA has little effect on HNF-6 binding to DNA (data
not shown), we hypothesize that it increases the transactivation potential of HNF-6 but that this effect is only apparent under conditions in which the concentration of HNF-6 is limiting.
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Fig. 5.
Overexpression of wild-type or mutated HNF-6
stimulates basal G6Pase-CAT fusion gene expression. HepG2 cells
were transiently co-transfected as described under "Experimental
Procedures" with a G6Pase-CAT fusion gene (15 µg) containing the
promoter sequence between 129 and +66, an expression vector encoding
-galactosidase (2.5 µg), and either an expression vector (5 µg)
encoding PKA (P) or the same vector (5 µg) with the PKA
open reading frame deleted (C). Cells were also
co-transfected as indicated with 1 µg of the empty pcDNA3 vector,
the wild-type HNF-6 pcDNA3, the HNF-6 SDM pcDNA3 expression
vectors described under "Experimental Procedures," or no further
addition (None). After transfection, cells were incubated
for 18-20 h in serum-free medium. The cells were then harvested, and
both CAT and
-galactosidase activity were assayed as described
previously (29, 30). Results are presented as a ratio relative
to the CAT activity, corrected for
-galactosidase activity in the
cell lysate, in empty PKA vector, no pcDNA3-transfected cells
(expressed as -fold induction) and represent the mean ± S.E. of
3-5 experiments assayed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
136 and
134 was required for the stimulatory effect of cAMP
on G6Pase fusion gene expression in HepG2 cells, whereas Schmoll
et al. (26) found that the sequence located between
161
and
152 was critical for the combined stimulatory effects of cAMP and
glucocorticoids in H4IIE hepatoma cells. The reason for these disparate
results with the human promoter is unclear, but our data indicate that
both of the equivalent regions in the mouse G6Pase promoter contribute
to the induction of G6Pase-CAT fusion gene expression by PKA (Fig.
1A; Ref. 27). However, even with both of these regions
deleted, mouse G6Pase-CAT fusion gene expression was still induced by
~5-fold in response to PKA (Fig. 1A). Further truncation
of the G6Pase promoter sequence between
129 and
85 resulted in a
reduction in this stimulatory effect of PKA on G6Pase-CAT fusion gene
expression (Fig. 1A). The data presented in this paper
suggest that the stimulatory effect of PKA through the
129 to
85
region of the G6Pase promoter is mediated by the phosphorylation of
HNF-6. Mutation of this HNF-6 motif in the context of an otherwise
intact CRU has little effect on the induction of G6Pase-CAT fusion gene
expression by PKA (data not shown). In contrast, mutation of the CRE,
located between
162 and
155, in the context of an otherwise intact
CRU almost abolishes the induction of G6Pase-CAT fusion gene expression
by PKA in HepG2 cells (data not shown) and LLC-PK cells (27). These results are consistent with the 5' deletion analysis (Fig.
1A) that shows a much greater contribution of this CRE than
the HNF-6 motif to the PKA response. Whether the relative contribution
of the HNF-6 motif to the induction of G6Pase gene transcription by PKA
increases under certain metabolic conditions and whether HNF-6 is
important for the induction of other hepatic genes by PKA remains to be determined.
binding (54) to the HNF-6 promoter. The relative roles of growth
hormone-stimulated PKA and Janus Kinase/signal transducer and activator
of transcription activation in mediating these changes remain to be determined.
subunit of phosphorylase kinase at low magnesium ion concentrations;
however, at high magnesium ion concentrations, PKA phosphorylates the
subunit of phosphorylase kinase on three additional sites.
Furthermore, Berglund et al. (42) showed that the optimal
phosphorylation of L-type pyruvate kinase by PKA was found
within a very narrow range of magnesium ion concentration. However,
when the magnesium ion concentration was increased above 10 mM or decreased below 4 mM, PKA was less active
(42).
110 and
101 in the G6Pase promoter may
contribute to the full stimulatory effect of PKA on G6Pase-CAT fusion
gene expression. In addition, HNF-6 was shown to be phosphorylated by
PKA in vitro. Taken together, these results suggest that the
stimulatory effect of PKA on G6Pase gene transcription may be mediated
in part by the phosphorylation of HNF-6.
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ACKNOWLEDGEMENTS |
---|
We thank Robert Costa and Richard Maurer for providing the HNF-6 and PKA expression vectors, respectively. We also thank Roger Colbran, Sharron Francis, and Jackie Corbin for providing purified PKA catalytic subunit protein and for advice with the phosphorylation analysis and manuscript. Data analysis was performed in part through the use of the Vanderbilt University Medical School Cell Imaging Resource (CA68485 and DK20593).
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK56374 (to R. M. O.) and the Vanderbilt Diabetes Core laboratory Grant P60 DK20593.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.
Supported by the Vanderbilt Molecular Endocrinology Training
Program (5 T 32 DK07563-12).
§ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biophysics, 761 MRB II, Vanderbilt University Medical School, Nashville, TN 37232-0615. Tel.: 615-936-1503; Fax: 615-322-7236; E-mail: richard.obrien@mcmail.vanderbilt.edu
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M101442200
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ABBREVIATIONS |
---|
The abbreviations used are:
G6Pase, glucose-6-phosphatase catalytic subunit;
HNF, hepatocyte nuclear
factor;
PKA, catalytic subunit of protein kinase A;
CAT, chloramphenicol acetyltransferase;
PCR, polymerase chain reaction;
IPTG, isopropyl--D-thiogalactopyranoside;
PAGE, polyacrylamide gel electrophoresis;
CRE, cAMP response element;
CREB, cAMP-response element-binding protein;
CRU, cAMP response unit.
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