From the University of Illinois at Chicago College of
Medicine and Veterans Affairs Chicago Health Care System, Chicago,
Illinois 60612, the ¶ Research Genetics, Huntsville, Alabama
35801, and the ** Eukaryotic Transcriptional Regulation Section,
Regulation of Cell Growth Laboratory, NCI, Frederick Cancer Research
and Development Center, Frederick, Maryland 20892-0822
Received for publication, September 19, 2000, and in revised form, December 8, 2000
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ABSTRACT |
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CAAT/enhancer-binding proteins
(C/EBPs) play an important role in the regulation of gene expression in
insulin-responsive tissues. We have found that a complex containing
C/EBP CAAT/enhancer-binding proteins
(C/EBPs)1 play an important
role in the regulation of gene expression in insulin-responsive tissues
(1, 2). Studies in cell culture and with knockout mice indicate that
C/EBP Previous studies have shown that insulin inhibits basal activity of the
IGFBP-1 promoter via an IRS, and that this effect of insulin is
mediated by protein kinase B (PKB) downstream from phosphatidylinositol
3'-kinase (PI 3-kinase) (12). Activated PKB is translocated to the
nucleus where it may interact with nuclear targets directly involved in
the regulation of gene expression (13). PKB has been shown to
phosphorylate and suppress transactivation by a subset of
forkhead transcription factors, including FKHR, FKHRL1, and AFX
(14-16). Since a nucleoprotein complex containing C/EBP Mink et al. (17) have reported that N-terminal
transactivation domains in C/EBP Plasmid Constructs--
The SauI/HgaI
fragment of the IGFBP-1 promoter which extends 320 base pairs 5'
from the RNA cap site was cloned into pGL2 (Promega) (BP1.Luc) and
modified to create the Site-directed Mutagenesis of p300 and Creation of Fusion
Proteins--
The full-length p300 cDNA was provided by Dr. David
Livingston (37). The region coding for amino acids 1752-1859 region was amplified by polymerase chain reaction with one primer which contains an XhoI site
(5'-CAGCTCGAGACCATGGGGATCCTCAATTGCTCACTGCCATCCTGC-3') and a
second primer (5'-GGCTCTAGACTAAGTTGGTGTCGTTGGAGTGGCAGGAG-3') which
contains a stop codon and an XbaI site. This fragment was cloned into the XhoI/XbaI site in pAlter.Max
vector (Promega) and single stranded DNA was prepared with helper phage
for site-directed mutagenesis. A BamHI site was introduced
at the 5' end of this fragment and Ser1834 was replaced
with alanine or aspartate by site-directed mutagenesis using the
following oligonucleotides: p300.BamHI,
5'-P-CAGCGGCGGCGGGGATCCCCGGCGGGTGCTGC-3'; S1834A,
5'-P-CACACCAGTCCGCTGCATGGCGGCCATCCTCCTGCGAAG-3'; S1834D, 5'-P-CACACCAGTCCGCTGCATGTCGGCCATCCTCCTGCGAAG- 3'. The
sequences of the polymerase chain reaction product and its mutations
were confirmed by DNA sequencing in the University of Illinois at
Chicago DNA Sequencing Center.
BamHI-XbaI fragments coding for amino acids
1752-1859 of p300 with/without mutation of Ser1834 were
subcloned in-frame with the VP16 activation domain in the pVP16
eukaryotic expression vector (CLONTECH). A copy of
the SV40 nuclear localization signal has been introduced in-frame with the VP16 activation domain in this vector by the manufacturer to ensure
that expressed proteins are transported to the nucleus. Using a
NotI site in the pAlter polylinker,
BamHI-NotI fragments also were excised and cloned
in-frame with glutathione S-transferase (GST) in pGEX4T-3
(Amersham Pharmacia Biotech). pGEX4T-3 vectors were introduced into
pBL21RIL bacteria (Stratagene) and fusion proteins were induced at
30 °C with isopropyl-1-thio- In Vitro Kinase Studies--
For kinase studies with synthetic
peptides, a peptide containing residues 1826-1842 of p300
(QMLRRRMASMQRTGVVG) was synthesized, purified by high performance
liquid chromatography, and analyzed by mass spectroscopy at the UIC
Protein Synthesis Center. A peptide (RPRAATF) derived from a known PKB
phosphorylation site in glycogen-synthase kinase-3 was purchased from
Upstate Biotechnology, Inc. HepG2 cells were transfected with
expression vectors for HA-tagged Myr-PKB and
Myr-Lys179-PKB, and tagged proteins were immunoprecipitated
with monoclonal anti-HA antibodies (Calbiochem) and protein G-Sepharose
(Amersham Pharmacia Biotech), as before (12). Beads were washed and
then incubated with target peptides for 10 min at 30 °C with
[
For phosphorylation studies with GST-p300 fusion proteins, bacterial
lysates containing ~3 µg of fusion protein were incubated with 25 µl of a 50% slurry of washed glutathione-agarose beads (Amersham
Pharmacia Biotech). Washed beads were equilibrated with 50 µl of
kinase buffer containing 0.25 units of active PKB Cell Culture, Transfection, and Reporter Gene
Analysis--
HepG2 cells in 60-mm dishes were transfected in
triplicate with calcium phosphate precipitates containing reporter gene
and expression vectors together with appropriate amounts of empty vector, as previously reported (12, 14). Cells were refed with
Dulbecco's modified Eagle's medium plus 1 g/liter fatty acid-free bovine serum albumin (Sigma) with/without 100 nM
recombinant human insulin (Sigma) and/or 50 µM LY294002
(Calbiochem) or 200 nM rapamycin (Sigma) 18 h prior to
the preparation of lysates and analysis of luciferase activity
(12).
Insulin inhibits promoter activity in luciferase reporter gene
constructs containing an IRS (CAAAACA; interacts with an insulin response sequence in the
insulin-like growth factor-binding protein-1 (IGFBP-1) gene and
that a C/EBP-binding site can mediate effects of insulin on promoter
activity. Here, we examined mechanisms mediating this effect of
insulin. The ability of insulin to suppress promoter activity via a
C/EBP-binding site is blocked by LY294002, a phosphatidylinositol
3-kinase inhibitor, but not by rapamycin, which blocks
activation of p70S6 kinase. Dominant negative
phosphatidylinositol 3-kinase and protein kinase B (PKB) block the
effect of insulin, while activated PKB suppresses promoter function via
a C/EBP-binding site, mimicking the effect of insulin. Coexpression
studies indicate that insulin and PKB suppress transactivation by
C/EBP
, but not C/EBP
, and that N-terminal transactivation domains
in C/EBP
are required. Studies with Gal4 fusion proteins reveal that
insulin and PKB suppress transactivation by the major activation domain
in C/EBP
(AD II), located between amino acids 31 and 83. Studies
with E1A protein indicate that interaction with p300/CBP is required
for transactivation by AD II and the effect of insulin and PKB. Based on a consensus sequence, we identified a PKB phosphorylation site (Ser1834) within the region of p300/CBP known to
bind C/EBP
. Mammalian two-hybrid studies indicate that insulin and
PKB disrupt interactions between this region of p300 and AD II and that
Ser1834 is critical for this effect. Signaling by PKB and
phosphorylation of Ser1834 may play an important role in
modulating interactions between p300/CBP and transcription factors and
mediate effects of insulin and related growth factors on gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and C/EBP
both contribute to the regulation of hepatic
glucose production (3-5), including the multihormonal regulation of
phosphoenolpyruvate carboxykinase (PEPCK) (6-10), a rate-limiting
enzyme for gluconeogenesis. We have reported that a nucleoprotein
complex containing C/EBP
interacts with oligonucleotide probes
containing insulin response sequences (IRSs) from the insulin-like growth factor-binding protein-1 (IGFBP-1) and PEPCK genes (11). This
complex interacts with the IRS in a sequence-specific fashion that
correlates with the ability of the IRS to mediate effects of insulin on
promoter activity, and replacing the IRS with a consensus C/EBP-binding
site in reporter gene constructs maintains the effect of insulin (11).
Together, these results suggest that signaling to C/EBP proteins may
contribute to the effects of insulin on gene expression via an IRS.
Based on these observations, we examined specific mechanisms by which
insulin may suppress promoter activity via C/EBP proteins.
interacts
with IRSs in the IGFBP-1 and PEPCK genes, we considered the possibility
that C/EBP proteins also might contribute to the ability of insulin to
regulate promoter activity downstream from PI 3-kinase and PKB.
interact with p300/CBP coactivator
proteins, and that this interaction is critical for transactivation by
C/EBP
. GST pull-down and two-hybrid studies revealed that C/EBP
interacts with residues 1752-1859 of p300 (17). This region of
p300/CBP also interacts with a number of other factors known to be
important in the regulation of gene expression, including E1A (18),
PCAF (19, 20), TFIIB (21), cyclin E-Cdk2 kinase (22),
pp90Rsk (23), SV40 large T antigen (24), c-Jun (25),
c-Fos (26), MyoD (27), YY1 (28), Ets-1 (29), the
glucocorticoid receptor (30), and forkhead proteins (31). Since
p300/CBP proteins are important for transactivation by C/EBP
, we
considered the possibility that insulin signaling via PKB may modify
interactions between this region of p300/CBP and C/EBP
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IRS.1,
IRS.1M, and
C/EBP reporter gene
constructs, as previously reported (11, 12). The pAlb(DEI)4
reporter gene construct (32) contains 4 copies of a naturally occurring
C/EBP-binding site placed immediately upstream from the albumin minimal
promoter, and the pG5e1b construct consists of 5 Gal4-binding sites
introduced immediately upstream of the e1b minimal promoter. Expression
vectors for C/EBP proteins and fusion proteins containing the Gal4
DNA-binding domain have been described (32). Expression vectors for 12S
E1A and
E1A, where amino acids 2-36 are deleted, were provided by
Dr. Pradip Raychaudhuri (33). The
p85 expression vector was provided
by Dr. M. Kasuga (34). Vectors expressing kinase-defective
(Lys179-PKB) and constitutively active, myristoylated PKB
(Myr-PKB) were provided by Dr. Thomas Franke (35). Mammalian expression
vectors for CHOP and CHOP-LZ
were provided by Dr. David
Ron (36).
-D-galactopyranoside. Cells were harvested by centrifugation and lysed by sonication, and
lysates were cleared by centrifugation.
-32P]ATP (Amersham Pharmacia Biotech) using buffers
and peptide inhibitors of PKA and calcium-dependent kinase
provided with the Akt kinase kit (Upstate Biotechnology, Inc.). Kinase
reactions were terminated by heating at 100 °C for 5 min, and
phosphorylation was measured by scintillation counting after
spotting onto p81 phosphocellulose discs and washing with phosphoric
acid, as before (12).
, provided by Dr.
P. Cohen. Phosphorylation reactions were initiated at 30 °C by the
addition of [
-32P]ATP and terminated 20 min later by
addition of 5 × Laemmli sample buffer and heating at 100 °C.
Samples were cleared by centrifugation and loaded for 4-20% gradient
SDS-polyacrylamide gel electrophoresis. Gels were stained with
Coomassie Blue, then dried and phosphoproteins identified by
autoradiography at
70 °C with enhancing screens.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IRS.1) or when the IRS is
replaced by a consensus binding site for C/EBP proteins (TTGCGCAA;
C/EBP), but not when the IRS is replaced by an unrelated sequence
(
IRS.1M) (Fig. 1). This indicates that
the ability of insulin to suppress promoter activity is
sequence-specific and can be mediated by either an IRS or C/EBP
site.
View larger version (36K):
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Fig. 1.
Effect of insulin and PKB on promoter
activity. HepG2 cells were transfected with calcium phosphate
precipitates of plasmid DNA (15 µg/dish), including 1 µg of
luciferase reporter gene constructs containing a single IRS (CAAAACA;
IRS.1), a consensus C/EBP-binding site (TTGCGCAA;
C/EBP), or an
unrelated sequence (
IRS.1M), with/without vectors expressing
dominant negative PI 3-kinase (
p85) (5 µg), kinase-defective
Lys179-PKB (10 µg), or constitutively active PKB
(Myr-PKB) (1 µg) plus appropriate amounts of empty vector. Cells were
refed with serum-free medium with/without 100 nM insulin,
50 µM LY294002, and/or 200 nM rapamycin and
lysates prepared 18 h later. Luciferase activity is expressed
relative to control and presented as the mean ± S.E. for at least
three experiments performed in triplicate.
Previous studies have shown that the ability of insulin to inhibit
promoter activity via an IRS is mediated through the PI 3-kinase/PKB
signaling pathway (12), and we asked whether signaling to C/EBP
proteins also might occur through this pathway. As shown in Fig. 1,
treatment with LY294002, a specific inhibitor of PI 3-kinase, or
coexpression of a dominant negative form of PI 3-kinase (p85) blocks
the ability of insulin to suppress promoter activity via either an IRS
or C/EBP-binding site (Fig. 1), indicating that this effect of insulin
also is PI 3-kinase-dependent. Treatment with rapamycin,
which blocks the activation of p70S6 kinase downstream from
PI 3-kinase, does not disrupt the ability of insulin to inhibit
promoter activity. Coexpression of a kinase-deficient form of PKB
(Lys179-PKB), which blocks the activation of PKB in HepG2
cells (12), disrupts the ability of insulin to inhibit promoter
activity. Expression of constitutively active PKB (Myr-PKB) inhibits
promoter activity of reporter gene constructs containing either an IRS (
IRS.1) or a C/EBP-binding site (
C/EBP), but not an unrelated sequence (
IRS.1M), mimicking the effect of insulin. Together, these
results indicate that signaling via PKB can mediate effects of insulin
on promoter activity via either an IRS or C/EBP-binding site.
To determine whether endogenous C/EBP proteins are required for the
ability of insulin to inhibit promoter activity via a C/EBP-binding
site, we performed coexpression studies with CHOP. CHOP, a C/EBP family
member which contains a leucine zipper dimerization domain but no
functional DNA-binding domain, disrupts the ability of endogenous C/EBP
proteins to form hetero- or homodimers that are capable of binding to
canonical C/EBP sites (11, 36). In contrast, CHOP does not prevent
C/EBP from interacting with other nuclear proteins and forming a
complex with an IRS (11). As shown in Fig.
2, overexpression of CHOP blocks the
ability of insulin and Myr-PKB to inhibit promoter activity via a
C/EBP-binding site in the
C/EBP construct (solid bars).
This effect of CHOP is specific, since CHOP does not disrupt other
effects of insulin and Myr-PKB in these cells, including the ability to
suppress promoter function via an IRS in the
IRS.1 construct
(open bars). CHOP-LZ
, which is missing the
leucine zipper dimerization domain, does not block the ability of
insulin and Myr-PKB to suppress promoter function via a C/EBP-binding
site, indicating that this effect of CHOP is mediated through
interaction with endogenous proteins via its leucine zipper domain.
Together, these results indicate that interaction with hetero- or
homodimers of C/EBP proteins is necessary for the ability of insulin
and PKB to suppress promoter activity via this C/EBP-binding site.
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To determine whether insulin and PKB suppress promoter activity when
specific C/EBP proteins occupy this C/EBP-binding site, we performed
co-transfection studies with expression vectors for C/EBP and
C/EBP
. As shown in the left panel of Fig.
3A, C/EBP
and full-length
C/EBP
(C/EBP
1-276) effectively stimulate
promoter activity in the
C/EBP construct. This effect is disrupted
when the C/EBP site is replaced by an unrelated sequence (
IRS.1M),
indicating that it is mediated through the C/EBP site. Truncated
C/EBP
(C/EBP
132-276), which is missing two
N-terminal transactivation domains, is less effective in stimulating
promoter activity and this residual effect is not disrupted when the
C/EBP-binding site is replaced by an unrelated sequence (
IRS.1M).
These results confirm that C/EBP
and full-length C/EBP
stimulate
promoter activity via the C/EBP-binding site and that N-terminal
activation domains are required for sequence-specific transactivation
by C/EBP
.
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As shown in the right panel of Fig. 3A,
coexpression of C/EBP disrupts the ability of insulin and PKB to
suppress promoter activity in the
C/EBP construct. In contrast,
insulin and PKB suppress the ability of full-length C/EBP
(C/EBP
1-276) to stimulate promoter activity. This
effect of insulin and PKB is disrupted when the N-terminal region of
C/EBP
is deleted (C/EBP
132-276). These results
indicate that insulin and PKB suppress transactivation by C/EBP
, but
not C/EBP
, and that the N-terminal region of C/EBP
is required
for this effect.
We performed similar studies using a reporter gene construct where an
array of 4 naturally occurring C/EBP-binding sites has been placed
immediately upstream of the minimal albumin promoter (pAlb[DEI]4). In contrast to the C/EBP construct,
insulin modestly stimulates basal activity of the
pAlb(DEI)4 promoter, possibly reflecting effects which are
mediated via other transcription factors that can interact with this
naturally occurring C/EBP-binding site or with other parts of this
construct. As shown in Fig. 3B, overexpression of C/EBP
or C/EBP
stimulates the activity of this artificial promoter
construct and truncated C/EBP
is less effective. Insulin and PKB
suppress the ability of C/EBP
to stimulate the activity of this
artificial construct. In contrast, neither C/EBP
nor truncated
C/EBP
confers this effect of insulin and PKB, confirming that
insulin and PKB suppress transactivation by C/EBP
, but not C/EBP
,
and that the N-terminal region of C/EBP
is required for this effect.
We next examined which regions of C/EBP are involved in mediating
the effect of insulin and PKB on transactivation. For these experiments, we expressed fusion proteins containing the Gal4 DNA-binding domain (which contains its own nuclear localization signal)
in-frame with portions of C/EBP
, or the activation domain of VP16 as
a control. Transactivation was measured using the pG5e1b luciferase
reporter gene construct, which contains 5 Gal4-binding sites
immediately 5' to the e1b minimal promoter and has negligible basal
activity in HepG2 cells.
Fig. 4A indicates the location
of the leucine zipper dimerization domain (LZ), DNA-binding domain, and
the two N-terminal transactivation domains previously identified in
C/EBP (AD I and II) (32). As shown in Fig. 4B, the Gal 4 fusion protein containing full-length C/EBP
stimulates the pG5e1b
promoter. Transactivation is enhanced when the C-terminal region
containing the leucine zipper and DNA-binding domain is removed from
C/EBP
(Gal4.
1-132), and transactivation is increased still
further when an additional 39 amino acids are removed (Gal4.
1-83),
consistent with previous studies (32). The N-terminal region of
C/EBP
contains two activation domains, which are located between
amino acids 1-31 (AD I) and 31-83 (AD II). The Gal4.
31-83 fusion
protein is more potent in stimulating promoter activity than the
Gal4.
1-31 protein, consistent with previous studies indicating that
the major activation domain in C/EBP
is located between amino acids 31 and 83 (32).
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As shown in Fig. 4C, insulin and PKB suppress
transactivation by Gal4.1-276 but not Gal4.VP16, indicating that
this effect of insulin and PKB is specific. Insulin and PKB also
suppress transactivation by Gal4.
1-132 and Gal4.
1-83,
indicating that regions outside the N-terminal transactivation domains
of C/EBP
are not required for this effect of insulin and PKB.
Insulin and PKB suppress transactivation by Gal4.
31-83 (AD II) but
do not suppress transactivation by Gal4.
1-31 (AD I), indicating
that this effect is specific for ADII.
Previous studies have shown that p300/CBP coactivator proteins interact
directly with the N-terminal region of C/EBP (17). As shown in the
left panel of Fig.
5A, coexpression of E1A, an adenovirus protein that binds and sequesters p300/CBP, disrupts transactivation by the Gal4.
1-132 fusion protein in a
dose-dependent fashion. In contrast, coexpression of
E1A, which does not interact with p300/CBP but still binds
retinoblastoma protein and related proteins (33, 38), does not suppress
transactivation by the Gal4.
1-132 fusion protein but actually
enhances this effect. Together, these results indicate that interaction
with p300/CBP is critical for transactivation by this region of
C/EBP
, and that other cellular proteins that can interact with
E1A may repress this function, similar to results obtained with
other transcription factors
(39).2
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As shown in the right panel of Fig. 5A,
coexpression of E1A (but not E1A) also disrupts the ability of
insulin and PKB to suppress transactivation by the Gal4.
1-132
fusion protein. Together, these results suggest that interaction with
p300/CBP is required for effective transactivation by the N-terminal
region of C/EBP
and for the inhibitory effect of insulin and PKB.
As shown in the left panel of Fig. 5B, E1A (but
not E1A) also suppresses transactivation by AD II of C/EBP
(Gal4.
31-83), but not AD I (Gal4.
1-31), indicating that
interaction with p300/CBP is required for transactivation by AD II, but
not AD I. As shown in the right panel of Fig. 5B,
E1A (but not
E1A) disrupts the ability of insulin and PKB to
suppress transactivation by Gal4.
31-83, indicating that interaction
with p300/CBP is required for insulin and PKB to suppress
transactivation by AD II.
Previous studies have shown that C/EBP interacts directly with
residues 1752-1859 of p300 and that this interaction involves the
N-terminal region of C/EBP
that contains ADII (17). As shown in Fig.
6A, this region of p300/CBP,
which includes a portion of a cysteine/histidine-rich domain (CH3) and
the adjacent glutamine-rich domain (Q), also interacts with a number of
other trans-acting factors that contribute to the regulation
of gene expression (18-31).
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Based on a consensus sequence for PKB phosphorylation sites
(Arg-Xaa-Arg-Xaa-Xaa-Ser/Thr) (40), we identified a putative PKB
phosphorylation site in this region of human p300
(Ser1834). This PKB site is conserved in both human and
mouse CBP (Fig. 6B), suggesting that it may be
physiologically important. The sequence for this region of mouse p300
has not been reported. We did not identify other PKB sites in p300,
CBP, or C/EBP.
Based on this observation, we asked whether this site is phosphorylated
by PKB and is required to disrupt interactions between this region of
p300 and AD II. As shown in Fig.
7A, activated PKB (but not
kinase-defective PKB) phosphorylates a synthetic peptide containing
amino acids 1826 through 1842 of human p300 and this phosphorylation is
similar to the level achieved with a peptide derived from
glycogen-synthase kinase-3, a known substrate for PKB. As shown in Fig.
7B, PKB also phosphorylates a bacterially expressed
recombinant fusion protein containing amino acids 1752-1859 of p300
in-frame with GST (GST-p300), but not GST alone. This phosphorylation
is blocked when Ser1834 is replaced by either alanine
(GST.p300(S/A)) or aspartate (GST.p300(S/D)), confirming that
Ser1834 is a target for phosphorylation by PKB in
vitro.
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To examine interactions between this portion of p300 and C/EBP, we
next performed mammalian two-hybrid studies in HepG2 cells. As shown in
Fig. 8A, coexpression of a
fusion protein containing amino acids 1752-1859 of p300 in-frame with
the VP16 activation domain enhances the ability of the Gal4.
1-132
fusion protein to stimulate luciferase activity in HepG2 cells
transfected with the pG5e1b reporter gene construct in a
dose-dependent fashion. No effect is seen when the VP16
activation domain is expressed alone, indicating that this effect is
mediated through interaction with this region of p300. The VP16.p300
fusion protein also enhances transactivation by the Gal4.
32-83
fusion protein (Fig. 8B), but does not enhance
transactivation by Gal4.
1-31 (data not shown), indicating that this
region of p300 interacts with AD II, but not AD I in cells.
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We next examined whether insulin and PKB disrupt the interaction
between this region of p300 and C/EBP, and whether the
phosphorylation of Ser1834 is required for this effect of
insulin and PKB. As shown in Fig. 8B, coexpression of
VP16.p300 enhances transactivation by the Gal4.
1-132 fusion protein
(left panel) and Gal4.
31-83 (right panel)
without disrupting the ability of insulin and PKB to suppress transactivation. This result indicates that signaling via insulin and
PKB disrupts interaction between this region and ADII of C/EBP
in cells.
Replacing Ser1834 with aspartate, a negatively charged
amino acid (VP16.p300(S/D)) blocks the ability of insulin and PKB to
suppress interaction between this region of p300 and ADII in cells, but does not impair the ability of the fusion protein with interact with
ADII in two-hybrid studies. This result supports the concept that
phosphorylation of Ser1834 is critical for the effect of
insulin and PKB, and suggests that this effect of insulin and PKB is
not mediated simply by introducing a negative charge at this site.
Replacing Ser1834 with alanine, a neutral amino acid that
is not susceptible to phosphorylation, also blocks the ability of
insulin and PKB to disrupt interaction between this region of p300 and
ADII, supporting the concept that phosphorylation at this site is
required for the effect of insulin and PKB. Replacing
Ser1834 with alanine partially reduces the ability of this
fusion protein to interact with ADII in the two-hybrid assay (Fig.
8B), supporting the concept that Ser1834 is
important for interaction between this region of p300 and ADII.
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DISCUSSION |
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C/EBP plays an important role in the regulation of gene
expression in the liver, mammary gland, and adipose, hematopoietic and
reproductive tissues (41-45). We recently found that a nucleoprotein complex containing C/EBP
interacts with insulin response sequences in the IGFBP-1 and PEPCK genes and that a consensus C/EBP-binding site
confers effects of insulin on promoter activity, similar to an IRS
(11). Based on these findings, we asked whether insulin may suppress
transactivation by specific C/EBP family members and examined
mechanisms mediating this effect of insulin.
Studies with pharmacological inhibitors and with constitutively active
and dominant negative forms of PI 3-kinase and PKB revealed that PI
3-kinase plays a critical role in mediating effects of insulin on
promoter activity via a C/EBP-binding site and that signaling via PKB
can mediate this effect of insulin. Previous studies have shown that
signaling via PI 3-kinase-dependent pathways mediates
effects of insulin on hepatic expression of IGFBP-1, PEPCK, and
glucose-6-phosphatase genes (12, 46-48) and we have reported that
insulin suppresses basal IGFBP-1 promoter activity through an IRS via
events that are mediated by PKB downstream from PI 3-kinase (12).
Recent studies indicate that PKB can mediate effects of insulin on
promoter activity via the phosphorylation of FKHR, FKHRL1, or AFX, a
subgroup of the forkhead/winged-helix family of transcription
factors which contain several PKB phosphorylation sites (14, 15, 16,
49). The results of the present study indicate that signaling via PI
3-kinase and PKB also can disrupt transactivation by C/EBP and,
thereby, suppress promoter activity by a mechanism that is independent
of forkhead transcription factors.
This effect of insulin appears to be selective, since insulin and PKB
suppressed transactivation by C/EBP, but not C/EBP
. We found that
insulin and PKB suppress transactivation by the major activation domain
in C/EBP
, located between residues 31 and 83. It is interesting to
note that the corresponding region of C/EBP
contains an insertion of
12 amino acids that are not present in AD II of C/EBP
(50). It is
possible that these additional residues may alter the function of this
activation domain, including the ability of insulin and PKB to regulate
interactions with coactivator proteins required for transactivation.
Also, C/EBP
contains another activation domain which is not present
in C/EBP
(50) and which may not be susceptible to regulation by
insulin and PKB. In addition, regions outside the activation domains
have been found to contribute to functional differences between
C/EBP
and C/EBP
(32, 51) and also might contribute to differences
in the effects of insulin and PKB on transactivation by full-length
C/EBP
and C/EBP
.
Previous studies have shown that extracellular stimuli can alter the
phosphorylation of several sites in C/EBP, and modify their effects
on promoter activity. Calcium-regulated phosphorylation of
Ser276 in the leucine zipper domain of C/EBP
results in
enhanced transactivation (52), as does Ras-dependent
phosphorylation of Thr235, and the phosphorylation of
Ser105 by protein kinase A (53, 54) or pp90Rsk
(55). Phosphorylation by protein kinase C of Ser240 in the
DNA-binding domain of C/EBP
reduces transactivation by interfering
with DNA binding (54). Our studies with Gal4 fusion proteins indicate
that phosphorylation at these sites is not required for the ability of
insulin and PKB to suppress transactivation by C/EBP
. Instead, we
find that insulin and PKB suppress the function of the major C/EBP
activation domain by disrupting interactions with p300/CBP proteins. To
our knowledge, this report provides the first evidence for an effect of
insulin or PKB that is mediated through the modification of a nuclear
coactivator, in this case p300/CBP.
In this context, it is important to note that other kinases also may
contribute to this effect of insulin. PKB, like other members of the
AGC kinase family, is activated by the phosphorylation of a conserved
hydrophobic activation loop by phosphatidylinositoldependent kinase-1 (56, 57). We found that overexpression of a kinase-deficient form of PKB, which blocks the activation of PKB in HepG2 cells (12),
disrupts the ability of insulin to suppress transactivation by
C/EBP. It is possible that overexpression of kinase-deficient PKB,
which may interfere with the ability of
phosphatidylinositol-dependent kinase-1 to phosphorylate
and activate PKB, also may block the activation of other AGC kinases
downstream from phosphatidylinositoldependent kinase-1. As
recently reviewed, several of these kinases can phosphorylate sites
that are similar to the consensus motif for PKB, including serum- and
glucocorticoid-inducible kinase (glycogen-synthase kinase-3),
pp90Rsk, and p70S6 kinase (56, 58). Preliminary
studies indicate that pp90Rsk and p70S6 kinase
also can phosphorylate Ser1834 in
vitro, but not as efficiently as
PKB.3 Additional studies will
be required to determine whether other AGC kinases also contribute to
signaling to Ser1834 in vivo.
To explore the possibility that phosphorylation of Ser1834
is required to disrupt interactions between p300 and C/EBP, we
performed mammalian two-hybrid studies with VP16.p300 fusion proteins
where this residue was altered. Replacing Ser1834 with
either aspartate or alanine disrupts the ability of insulin and PKB to
suppress interaction between this domain and ADII in cells, indicating
that this site is critical for the effect of insulin and PKB.
Interestingly, replacing Ser1834 with alanine, a neutral
amino acid, reduces the ability of VP16.p300 fusion proteins to
interact with ADII in two-hybrid studies, while introducing aspartate,
a negatively charged amino acid, is neutral in this assay. Preliminary
studies indicate that replacing Ser1834 with alanine also
partially reduces the ability of GST fusion proteins containing this
region of p300 to bind C/EBP
in in vitro pull-down
binding assays, while placing an aspartate residue at this location
does not alter C/EBP
binding either positively or
negatively,3 similar to the results of two-hybrid assays.
These results support the concept that Ser1834 is important
for the ability of this region of p300 to interact with C/EBP
, and
indicate that this interaction is not altered simply by introducing a
negative charge at this site.
Previous studies have shown that phosphorylation can modify protein
function by multiple mechanisms, and the introduction of a negative
charge is not always sufficient to mimic the effect of phosphorylation.
For example, phosphorylation of Ser133 in CREB results in
the recruitment of p300/CBP, and replacing this residue with aspartate
does not reproduce that effect (59). Based on the results of the
present study, it is possible that phosphorylation of
Ser1834 may recruit other proteins to this site in p300
and, thereby, block interactions with ADII. Nakajima et al.
(23) have reported that insulin treatment causes pp90Rsk to
associate with this region of p300/CBP. Additional studies will be
required to determine whether phosphorylation of Ser1834 is
sufficient to alter interactions between p300/CBP and AD II of
C/EBP, or whether the recruitment of other proteins (including pp90Rsk) is required for this effect.
p300/CBP can interact with multiple proteins at the same time via
distinct domains and, thereby, mediate cooperative effects of
transcription factors on gene expression (60, 61). Cooperative interactions involving C/EBP are important for the ability of thyroid hormone, glucocorticoids, and cAMP to stimulate the PEPCK promoter (8, 10), and a complex containing C/EBP
can interact with
IRSs which function cooperatively to enhance effects of glucocorticoids on activity in the IGFBP-1 and PEPCK promoters (11, 62, 63). C/EBP
proteins appear to play an important role in mediating effects of
insulin on stimulated PEPCK promoter activity that are independent of
an IRS (9), and overexpressing a fragment of p300/CBP that contains the
C/EBP
-binding domain disrupts the ability of insulin to suppress
PEPCK gene expression in cAMP-stimulated hepatoma cells (23). Based on
the present study, it is interesting to speculate that signaling to
this region of p300/CBP may contribute to the ability of insulin or
related growth factors to suppress promoter activity by disrupting
cooperative interactions involving C/EBP
.
In this context, it is important to note that this region of p300 (1752-1859) also interacts with other factors that are known to be important in the regulation of gene expression. Nasrin et al. (31) recently reported that DAF-16, a forkhead protein in Caenorhabditis elegans, interacts with this region and the KIX (CREB binding) domain of p300/CBP (31), and pull-down studies indicate that FKHR, a mammalian homologue of DAF-16, also interacts with these regions of p300/CBP.3 It will be important to determine whether signaling to p300/CBP may contribute to the ability of insulin to suppress the function of the C-terminal transactivation domain of these forkhead proteins (64).
As recently reviewed, several lines of evidence suggest that p300/CBP
proteins play an important role in the regulation of the cell cycle and
cell survival (65). C/EBP proteins have important effects on the
proliferation of hepatocytes (55), and the region of p300/CBP that
binds C/EBP (17) also interacts with other factors that are involved
in the regulation of cellular growth and differentiation, including E1A
(18), cyclin E-Cdk2 kinase (22), pp90Rsk (23), SV40 large T
antigen (24), c-Jun (25), c-Fos (26), MyoD (27), YY1 (28), and Ets-1
(29). Based on these observations and the results of the present study,
it will be important to examine the role that signaling through PI
3-kinase and PKB to this region of p300/CBP plays in mediating effects
of insulin and related growth factors on cellular proliferation and
differentiation, and metabolism.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Andrew
Paterson, University of Dundee, for expression and activation of
PKB, and Drs. Philip Cohen and Graham Rena for helpful suggestions
and advice.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health, NIDDK, Grant DK41430 and the Department of Veterans Affairs Merit Review Program.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.
§ Present address: Lexicon Genetics, 4000 Research Forest Dr., The Woodlands, TX 77381-4287.
Present address: Section of Rheumatology (M/C 733), Dept. of
Medicine, University of Illinois at Chicago, 900 South Ashland Ave.,
Chicago, IL 60607.
To whom all correspondence should be addressed: Rm. 5A122A
Research (MP 151), Veterans Affairs Chicago Health Care System (West
Side), 820 South Damen Ave., Chicago, IL 60612. Tel.:
312-666-6500 (ext. 57427); Fax: 312-455-5877; E-mail:
unterman@uic.edu.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M008542200
2 A. K. Ghosh, unpublished observations.
3 S. Guo and T. Unterman, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: C/EBP, CAAT/enhancer-binding protein; CRE, cAMP response element; GST, glutathione S-transferase; IGFBP-1, insulin-like growth factor-binding protein-1; IRS, insulin response sequence; PEPCK, phosphoenolpyruvate carboxykinase; PKB, protein kinase B; PI 3-kinase, phosphatidylinositol 3'-kinase; CBP, CREB-binding protein; Myr, myristoylated; LZ, leucine zipper.
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