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INTRODUCTION |
The CCAAT/enhancer-binding protein
(C/EBP)1 family consists of
six members (1), including the highly related C/EBP
(2), C/EBP
(3), C/EBP
(4) and C/EBP
(5, 6), and the less related C/EBP
(7) and C/EBP
(8). Like other leucine zipper transcription factors,
they contain a basic amino acid domain and a leucine zipper region
allowing the formation of homo- and heterodimers when binding to DNA
(9). They share similar DNA binding specificities (9). The C/EBPs serve
as critical regulators in the differentiation processes of a variety of
mammalian cells including adipocytes, hepatocytes, and myeloid cells
(10, 11). In the hematopoietic system, early myeloid progenitors have
elevated levels of C/EBP
that decrease during granulocytic differentiation, whereas expression of C/EBP
and C/EBP
is low in
early myeloid stem cells and increase during granulocytic
differentiation (11). Mice with genetic disruption of C/EBP genes have
confirmed the importance of these proteins in myelopoiesis. These
C/EBPs, together with other factors such as Myb, AML1, or PU.1 can
induce the expression of myeloid-specific target genes (12-17).
C/EBP
is expressed almost exclusively in the myeloid lineage of the hematopoietic system and functions during terminal differentiation of
neutrophils and to a lesser extent macrophages; it also is involved in
the regulation of cytokine gene expression in macrophages and T
lymphocytes (5, 6, 18-20). A neutrophil-specific, secondary granule
deficiency syndrome in two individuals with repeated infections with
germline mutations of C/EBP
gene has recently been reported (21,
22).
Structural/functional studies of the C/EBPs have been only partially
explored using molecular genetic methods (23-30). The conserved basic
amino acid-rich region and leucine zipper domain at the carboxyl end
probably adopt a helical configuration upon binding with DNA (31,
32); the structures of the activation and repression regions are
unknown. Several regulatory sites that can be phosphorylated have been
identified (30, 33, 34). The C-terminal conserved cysteine of C/EBP
has been demonstrated to form a disulfide bond after dimer formation
in vitro (9). In this report, we provide evidence that no
disulfide bonds exist in C/EBP
and conjecture that the same probably
pertains to C/EBP
and C/EBP
. Additionally, no disulfide linkage
occurs between monomers after dimer formation in vivo.
Alanine scanning studies of the activating domain 1 (ADM1) and ADM2 of
C/EBP
indicate that negatively charged amino acid residues in these
two regions are critical for its transactivational activity.
Translation analysis of these mutants suggests that formation of
a special secondary structure of C/EBP
mRNA results in the
translation of the two major C/EBP
isoforms.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
The GST fusion protein vectors were
constructed using the Escherichia coli expression vector,
pGEX-5X-1 (Amersham Pharmacia Biotech). Full-length human C/EBP
gene
was obtained by PCR using pcDNA3 epsilon as template utilizing two
primers, JT32N and NDELC (primer sequences listed in Table I).
EcoRI and SalI sites were introduced into each
C/EBP
gene at the beginning and end of the constructs, respectively.
Pfu DNA polymerase (Stratagene, La Jolla, CA) was employed,
and the PCR was carried out under standard conditions. The PCR product
was digested by EcoRI and SalI and inserted into the corresponding sites of pGEX-5X-1, downstream of the GST gene.
For promoter-reporter assays in mammalian cells, a CMV promoter
expression vector, pCMV-
-gal (GIBCO), was cut by EcoRI
and XhoI, and the CMV promoter fragment was purified and
ligated with the C/EBP
gene with EcoRI and
SalI cohesive sites. The wild-type C/EBP
gene placed in
the CMV-C/EBP
plasmid (p32) was prepared from the GST-C/EBP
(GST-
) construct. C280S mutant C/EBP
gene (p32C280S) was obtained
by PCR with primers JT32N and C280SA (Table II). Both C34S and C148S
mutant genes were obtained by two rounds of PCR. Equal amounts of the
two purified PCR products from primers JT32N and C34SR, and from
primers NDELC and C34SF, were mixed as template for the full-length
C34S mutant gene by a second round of PCR using primers JT32N and
NDELC. The C148S mutant gene was prepared in the same manner as C34S
mutant by using for the first reaction, primers JT32N and C148SR and
primers NDELC and C148SF, and then primers JT32N and NDELC were used
for the second round of PCR. All three of the PCR mutant genes were
cleaved with EcoRI and SalI and inserted into the
CMV promoter expression vector at EcoRI and XhoI
sites, separately. Other mutant constructs were prepared in a similar
manner. The mutant genes p32Y6A, p32Y7A, p32E8A, p32C9A, p32E10A,
p32P11A, p32R12A, p32E8/10A, p32Y6/7A, p32S2E, p32S2ET5D,
p32,
p32, and
p32 used the wild-type C/EBP
gene as template, and
with the respective mutant primer and NDELC primer in the PCR
amplification (Table I). The p32Y6/7AR12A
was obtained with p32R12A as template. The mutant genes (p30, p30E3A, p30E5A, p30D9A, p30Y13A,
p30,
p30,
p30, and
p30R6A) were
obtained also with the wild-type C/EBP
gene as template, and with
the respective mutant primer and NDELC primer in one PCR. Mutant gene p32M33I was obtained just like p32C148S and p32C34S. Human C/EBP
gene (generous gift from Dr. Daniel G. Tenen, Harvard Medical School,
Cambridge, MA) was used to subclone the activation domain of C/EBP
.
A second PCR linked this fragment to the N-terminal 32-amino acid
truncated C/EBP
isoform with the N-terminal 32 amino acids deleted
to get
Ap32. All recombinant constructs were confirmed by sequencing
the DNA.
Expression and Purification of GST Fusion Proteins--
The GST
fusion gene constructs were used to transform E. coli strain
BL21. Single colonies of BL21 bearing GST-C/EBP
gene were inoculated
into 3 ml of 2× YT medium containing 50 µg/ml ampicillin. The cells
grew at 37 °C overnight with shaking. After 10-fold dilution into
fresh 2× YT medium, the cells were incubated with shaking for about
2 h until an A600 of 0.5-2.0 was
reached. IPTG was added to a final concentration of 0.5 mM,
and the cells were cultured for another 6 h. Cells were spun down
and kept at
20 °C. For purification of the GST fusion proteins,
the cells from 20 ml of culture medium were suspended in 1 ml of
ice-cold PBS buffer (140 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.3) and sonicated in short bursts
on ice. The supernatant was loaded onto a spin column (Centri-Spin-10,
Princeton Separations) containing 20 µl of PBS buffer and
glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The elute
from the column was reloaded 3-5 times to increase the recovery of the
GST fusion protein. The beads containing the GST fusion protein were
washed with 400 µl of PBS buffer to purify partially the GST fusion
protein. The yield for the GST-C/EBP
fusion protein was about 1.5 µg/ml cell culture, while the yield for GST was about 50 µg/ml cell
culture. 12% PAGE Ready Gels (Bio-Rad ) were used for SDS-PAGE
analysis of the proteins. We tried to use factor Xa (751 units/mg,
Amersham Pharmacia Biotech) to cleave C/EBP
from the bound fusion
protein. Only GST was released from the bound beads, but a negligible
C/EBP
band was detected in the fraction that was subjected to factor Xa cleavage, probably as a result of C/EBP
degradation by
contaminating proteases (data not shown).
Analysis of GST-C/EBP
--
Unlike many purified GST proteins,
the isolated GST-C/EBP
contained a high content of nucleic acid
probably as a result of its DNA binding capacity. For thiol group
determination of the C/EBP
protein, the Ellman's method was used
with a minor modification (35). Protein samples were placed in 6 M guanidine HCl and PBS buffer.
5,5'-Dithio-bis-2-nitrobenzonic acid (Sigma) was prepared in the same
buffer to 2 mM. DTT was used as a thiol group standard. 20 µl of sample was mixed with 50 µl of
5,5'-dithio-bis-2-nitrobenzonic acid solution at room temperature.
A412 of the mixture was monitored in a
microcuvette. Protein concentration was determined by the experimental
equation: [protein mg/ml] = 1.5 × A280
0.75 × A260 (36). Fresh
GST-C/EBPC/EBP
or GST protein samples were washed from the beads by
6 M guanidine HCl in PBS buffer and used for thiol group
determination. For reduced preparation of the GST proteins, the protein
was washed off of the beads with 6 M guanidine HCl in PBS
buffer, and 100 mM DTT was added to a final concentration of 10 mM. The reduction reaction was allowed to proceed at
37 °C for 40 min (37, 38). Spin column with gel filtration
medium was used to remove excess reductant and denaturant. The thiol group content of the protein was immediately measured.
Western Blot of C/EBP
--
Either reduced (0.1 M
DTT) or non-reduced samples were employed for detection of dimers,
which were possibly linked by disulfide bond(s), using 12% SDS-PAGE.
For GST-C/EBP
analysis, the proteins on the gel were transferred to
a nitrocellulose membrane overnight in a cold room in Towbin transfer
buffer (25 mM Tris, 192 mM glycine, 20%
methanol, 0.1% SDS, pH 8.4) at a voltage of 22 V. The transferred membrane was exposed to 5% milk powder in TPBS buffer (10 mM sodium phosphate, 0.9% NaCl, 0.1% Tween 20) to block
nonspecific protein binding sites. Rabbit polyclonal IgG against the
rat C/EBP
C-terminal epitope (C-22, LRNLFRQIPEAASLIKGVGGCS, Santa
Cruz Biotechnology, 100 µg/ml) was used as the first antibody (1 µg
of antibody/ml) in the TPBS buffer containing 5% milk powder for
1.5 h. After washing twice with TPBS buffer, 15 µl of second
antibody, anti-rabbit IgG (horseradish peroxidase-linked whole antibody
from donkey, Amersham Pharmacia Biotech), was added to 8 ml of 5% milk
powder TPBS buffer and incubated for 1 h. After washing twice with
TPBS, the membrane was subjected to a 1.5-ml mixture of equal volume of
luminal/enhancer solution and stable peroxide solution (Pierce) for 5 min, then exposed to a x-ray film for several seconds.
Western blot analysis of C/EBP
expressed from mammalian cells was
performed in a similar manner as described above for bacterial expressed GST-C/EBP
. Much more sensitive rabbit polyclonal
antibodies raised against the N-terminal C/EBP
peptide were utilized
as first antibody for this assay using a protein concentration of 1 µg/ml (6).
Mammalian Cell Transfections and Protein Activity
Analysis--
For promoter-reporter assays in Jurkat cells, 3 × 106 cells in 0.8 ml of serum-free RPMI 1640 medium were
transfected with ~3 µg of DNA (0.4 µg of pCMV-C/EBP
or mutant
vectors, 2.5 µg of pMim-luc, and 0.05 µg of pRLSV40) in 20 µl of
Lipofectin and 200 µl of Opti-MEM I reduced serum medium in 35-mm
tissue culture plates as described by the manufacturer (Life
Technologies, Inc.). The plasmid pRLSV40 (Promega) was included as a
monitor of transfection efficiency and pMim-luc (a gift from Dr. Achim
Leutz, Max Delbruck-Centrum for Molekulare Medizin, Berlin, Germany)
containing a C/EBP consensus DNA sequences as a reporter of
transactivation. After 6 h of exposure to the DNA/liposome
complexes, 4 ml of RPMI 1640 containing 10% fetal bovine serum was
added. The cells were activated with 10 ng/ml
12-O-tetradecanoylphorbol-13-acetate and 125 ng/ml calcium ionophore (A23187, Sigma) at 24 h after transfection. At 60 h
after transfection, 1.5 ml of cell culture was centrifuged, and the
cells were washed with 200 µl of PBS buffer. Dual-luciferase assay
kit (Promega) was used for analysis of activity of both firefly and
Renilla luciferases. The washed cells were lysed in 30 µl
of passive lysis buffer at room temperature for 15 min., and 20 µl of
supernatant was used for assay of enzymatic activity. For experiments
utilizing Myb, 0.4 µg of plasmid containing c-myb gene
under the control of the CMV promoter was mixed with the above 3 µg
of DNA. Either reduced or non-reduced total proteins from 100 µl of
cell culture were loaded onto a 12% SDS-polyacryamide gel for Western
blot analysis to examine for disulfide bond formation.
C/EBP
mRNA Secondary Structure Analysis--
Possible
mRNA structure of C/EBP
was simulated with a RNA folding program
(mfold version 3.0; Ref. 51). The folding temperature was fixed at
37 °C. No other constraint information was entered during our
simulation. N-terminal C/EBP
mRNA sequences were used for this
simulation, and structures having the lowest free energy were obtained.
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RESULTS |
C/EBP
Contains No Disulfide Bonds--
Analysis of DNA coded
protein sequences showed that C/EBP
contained five cysteine residues
(Fig. 1) (5). Analysis to determine if
this protein has disulfide bonds was pursued to understand its
structure. As the intact C/EBP
protein is not easily available, the
GST-C/EBP
protein was used. The GST contains four free cysteines without a single disulfide bond and can serve as a control (39, 40).
The GST-C/EBP
protein was removed from the glutathione beads by 6 M guanidine HCl in PBS buffer, and used directly for thiol
group determination. Both freshly prepared GST-C/EBP
and the reduced
form gave ~10 free thiol groups, which was very close to the expected
number of 9 (Table II). Both fresh GST
and reduced GST gave ~4 thiol groups. RNase A was employed as another
control. The data in Table II suggest that the active C/EBP
protein
contains no disulfide bonds. The partially purified GST or GST-C/EBP
protein showed simultaneously oxidation at neutral pH (pH 7.3). In the presence of 6 M guanidine HCl, 70% of the thiol groups in
the GST protein were observed to be oxidized in 1 week at 4 °C,
neutral pH. During this long period of storage of the GST-C/EBP
protein on beads at 4 °C, 0.1 mM DTT was added to the
PBS buffer to prevent oxidation of the protein. Nevertheless, the
content of thiol groups dropped from 10.0 to 8.9 in 6 days under these
conditions.

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Fig. 1.
Protein sequence analysis and chimeric
construction of C/EBP . Panel
A, conserved cysteine containing regions in human C/EBP
(5, 6). Conserved cysteines are underlined, and amino acid
residue numbers are shown below the sequence. Homologies of
other human C/EBPs (2-4) in the basic/leucine zipper domain to this
region of C/EBP are indicated as a percentage, which is shown in
brackets. Panel B, ADM1 (amino acids
1-12) and ADM2 (amino acids 33-48) sequences of C/EBP are aligned
with those of C/EBP , C/EBP , and C/EBP . The identical amino
acids are represented by vertical lines, and
conservative substitutions with colons. Panel
C, schematic representation of the C/EBP chimeric
constructs. The black bars show the ADM1
replacements and gray bars the ADM2
replacements. The names of the established constructs are on
their right. Ap32 represents the
ADM1 of C/EBP and the connecting sequence between ADM1 and ADM2 of
C/EBP linked to the ADM2 and the remaining carboxyl sequences of
C/EBP .
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Effects of Mutations of Conserved Cysteines of C/EBP
on Its
Transactivation Activity--
Among five cysteines in C/EBP
, three
of them are relatively conserved within the C/EBP family members:
Cys-34, Cys-148, and Cys-280 (Fig. 1A). In order to
understand the role of these conserved cysteines in transactivation by
C/EBP
, site-directed DNA mutagenesis was employed to change the
corresponding cysteines to serines. The mutant genes were cloned into a
eukaryotic expression vector and transfected into Jurkat cells (human T
lymphocytes). No distinguishable changes in ability to transactivate
were observed between wild-type and single site cysteine to serine
mutants (Fig. 2). The expression levels
of these mutants were almost the same as that of the wild-type C/EBP
as evidenced by Western blot assay (Fig. 2). These results indicate
that the three conserved cysteines are not critical for the
transactivation of C/EBP
and may not be involved in disulfide bond
formation between two C/EBP
monomers. Data have suggested previously
that C/EBP
may interact with Myb (41), therefore, the
transactivation experiments were repeated with a c-myb
expression vector included in the assays. Again, no notable changes in
activity of the mutants as compared with the wild-type C/EBP
were
noted (data not shown).

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Fig. 2.
Transactivation activity of
C/EBP bearing mutations of the conserved
cysteines. Promoter-reporter assays were performed in Jurkat
cells. These cells were transiently transfected with either
pCMV-C/EBP or the cysteine mutants of C/EBP , as well as the
pMim-luc as reporter and the pRLSV40 in order to measure transfection
efficiency. Dual-luciferase assay was used to measure activity of both
firefly and Renilla luciferases. The level of
transactivation was quantitated and normalized to the activity mediated
by wild-type C/EBP , which was set at 100%; values represent
mean ± S.D. of three independent transfections. A representative
Western blot of expressed C/EBP and its mutant proteins harvested
from Jurkat cells are shown on the top. Affinity column
purified C/EBP polyclonal antibodies raised in rabbits were employed
as first antibody. Two C/EBP isoforms are indicated by
arrows. The band above the C/EBP protein represents a
nonspecific band.
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Reducing and Non-reducing Gel Analysis of C/EBP
Proteins--
In order to know whether a disulfide bond was involved
in dimer formation, C/EBP
proteins expressed from either bacterial or mammalian cells were subjected to either reducing or non-reducing SDS-polyacrylamide gel analysis. Analysis of total bacterial expressed proteins in both the presence and absence of DTT showed that the vast
majority of GST-C/EBP
protein was in its monomer form (58 kDa; Fig.
3A, lanes
3, 4, 7, and 8). No obvious
dimer band (about 120 kDa) could be seen from the corresponding
non-reducing lanes (lanes 4 and 8).
Small amounts of high molecular weight proteins were detected because
of the absence of DTT, but not those bands that would correspond to the
dimers. A 50-kDa band below the GST-C/EBP
most likely represents the
N-terminal region of the C/EBP
-GST fusion product. A putative
basic/leucine zipper domain was observed in lane
7. The proteins expressed from mammalian cells were also studied (Fig. 3B). The C280S mutant was included in the
analysis, as Cys-280 was proposed to be involved in disulfide bond
formation between C/EBP
monomers in vitro (9). Under both
reducing and non-reducing conditions, the expressed C/EBP
proteins
were in their monomer form with no obvious dimer bands.

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Fig. 3.
No disulfide bond linkage within
C/EBP dimers in vivo.
Panel A, 12% SDS-polyacrylamide gel analysis of
total bacterial expressed proteins. Lanes 1 and
2 represent the expressed GST protein in the presence and
absence of DTT, respectively. Lanes 3 and
4 are expressed GST-C/EBP protein in the presence and
absence of DTT, respectively. Lanes 5-8 are
Western blot analysis of the gel shown in lanes
1-4, respectively. C/EBP C-terminal specific polyclonal
antibodies were used. Panel B, Western blot
analysis of Jurkat cell transiently transfected with C/EBP
expression vectors probed with affinity-purified polyclonal C/EBP
antibodies raised from rabbits. Lanes 1-3 are
samples in non-reducing conditions, and lanes
4-6 are those in reducing conditions. Lanes
1 and 5 are wild-type C/EBP , lanes
2 and 6 are C280S mutant C/EBP ;
lanes 3 and 4 are control cells
containing no C/EBP expression vector.
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Mutational Studies of the ADM1 of C/EBP
--
The activation and
repression domains of C/EBP
have been roughly defined by reporter
gene analysis of various segments of C/EBP
(23-30). The amino acid
sequences of the ADM1 and ADM2 of the C/EBP
activation domain have
been aligned with those of C/EBP
, -
, and -
(Fig.
1B). Full-length C/EBP
contains both the ADM1 and ADM2
regions, while an N-terminal truncated C/EBP
isoform (p30, with the
first 32 amino acids deleted) retains only ADM2 (17, 29). In order to
get a clear understanding of the structure/function relationship of the
activation domain of C/EBP
, both alanine scanning mutagenesis and
domain swapping with the ADM1 of other C/EBP genes were used to
construct a series of mutants in either the ADM1 or ADM2 region. If the
transactivation activity of p32 transfected cells was taken as 100%,
the activity for p30 was about 30%; but the ratio for expressed p32
compared with p30 isoform proteins was about 3 to 1 (Fig.
4A), indicating that the p32
and p30 isoforms gave nearly comparable transactivation activity in our
assay system when adjusting for protein expression. Mutation of two
tyrosines, p32Y6A and p32Y7A, produced a C/EBP
that showed a
slightly higher transactivation activity than the wild-type molecule.
Combining both mutations (p32Y6/7A) gave the same activity as either
alone (Fig. 4A). Further mutations explored the importance of the negative charge at the carboxyl end of the molecule. For mutant
p32E8A and p32E10A, each gave slight activity, while p32R12A demonstrated a higher activity than the wild-type C/EBP
. The negative to positive charge change at both p32E8/10R nearly
extinguished transactivational activity (Fig. 4A).

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Fig. 4.
Effect of ADM1 and ADM2 mutations on
transactivation activity of C/EBP .
Promoter-reporter assays were employed for these mutational studies.
Jurkat cells were transiently transfected with either pCMV-C/EBP
(p32) or its mutants, together with the pMim-luc as reporter and the
pRLSV40 as a measurement of transfection efficiency. The level of
transactivation was quantitated and normalized to the activity of the
wild-type C/EBP , which was set at 100%. Values represent the
mean ± S.D. of three independent transfections. A representative
Western blot of expressed C/EBP and its mutant proteins harvested
from Jurkat cells is shown on the top panel.
C/EBP affinity-purified, rabbit polyclonal antibodies were employed
as first antibody. Two C/EBP isoforms are indicated by
arrows as p32 and p30. The band above the C/EBP protein
represents a nonspecific band. Panel A shows the
data for ADM1 studies. Panel B displays the data
for ADM2.
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Sequence analysis of the ADM1 regions of C/EBP-
, -
, and -
indicates that, unlike C/EBP
, each has a glutamic acid at position 2 (Figs. 1B). A glutamic acid at codon 2 of C/EBP
was
substituted for its serine (p32S2E), which resulted in an 1.8-fold
enhanced activity of p32 (Fig. 4A). This emphasizes that the
negative charge of glutamic acid is important for transactivation
activity. We also constructed a double mutant of C/EBP
(p32S2ET5D)
in order to mimic C/EBP
at these two positions; these changes almost
doubled the transcriptional activity compared with wild-type C/EBP
(Fig. 4A).
We substituted the entire ADM1 (codons 1-12) of C/EBP
(
p32),
C/EBP
(
p32), and C/EBP
(
p32) for the existing ADM1 of
C/EBP
32, and examined the ability of these chimerics to
transactivate a myeloid promoter (Figs. 1C and
4A). Both the
p32 and
p32 enhanced activity nearly
2-fold as compared with the wild-type C/EBP
. In contrast, the
chimeric containing the ADM1 of C/EBP
conferred about the same
activity as C/EBP
. In further experiments, the ADM1 and the
connecting sequences between ADM1 and ADM2 of C/EBP
were linked to
the rest of the C/EBP
, beginning at ADM2. This chimeric tripled the
transactivating activity as compared with the wild-type p32 isoform of
C/EBP
(Fig. 5A).

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Fig. 5.
Effect of partial C/EBP
mRNA secondary structure on its translation. The
possible mRNA structure of C/EBP was simulated with a RNA
folding program (mfold, version 3.0; Ref. 51). The folding
temperature was fixed at 37 °C. No other constraint information was
entered during the simulation. Structure having lowest free energy was
obtained. A 152-nucleotide sequence is shown above. This sequence
includes the initial codon for full-length C/EBP and the
initial codon for the truncated p30 C/EBP as marked by
arrows. An 8-base pair double-helical structure was also
indicated, with one strand sequence including the initial codon
for the full-length C/EBP and the complementary strand representing
the initial codon for the p30 truncated C/EBP .
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Sequence analysis of ADM1 also indicates that C/EBP
has one more
tyrosine residue between positions 7 and 8 as compared with the other
C/EBPs (Fig. 1B). This may account for its lower activity, as mutant p32Y6A and p32Y7A both gave higher transactivating activity (Fig. 4A). A deletion mutant
p32
Y7 was constructed,
and demonstrated nearly a 3-fold greater activity compared with either
p32 or
p32. In summary, mutational studies showed that several
negative charges of the first 32 amino acid peptides enhanced
transcriptional activity of C/EPB
, while hydrophobic replacements in
its ADM1 destroyed its activity. Furthermore, substitution of ADM1 of
either C/EBP
or C/EBP
for the ADM1 of C/EBP
enhanced the
activity of the molecule.
Mutational Studies of the ADM2 of C/EBP
--
Both alanine
scanning mutagenesis and C/EBP fragment swapping were also used to
explore transactivational activity of the ADM2 of C/EBP
. Negative
charge appears also to be important for its activity. Mutants p30E3A,
p30E5A, and p30D9A (p30 sequence numbering of ADM2, Fig. 1B)
all gave lower activities as compared with p30 C/EBP
(Fig.
4B). ADM2 domain swapping showed that both
p30 and
p30 gave an equivalent activity as p30 C/EBP
, while
p30 showed
lower activity (Fig. 4B). Sequence analysis of the ADM2
region indicates that unlike C/EBP-
, -
, and -
, C/EBP-
has a
basic arginine residue at position 6 (Fig. 1B). This
probably results in the lower activity of
p30, because a positive to
neutral charge mutant
p30R6A resulted in a chimeric with higher
activity (Fig. 4B). Unlike ADM1, the hydrophobic
environment is not favorable for transactivation activity as the mutant
p30Y13A gave a little lower activity than p30 (Fig. 4B).
Mobility differences of the C/EBP
mutants as related to charge were
noted on Western blot analysis. Those proteins with a more positive
charge (p32E8/10R and
p32) or a less negative charge (p32E8A,
p32E10A, p30E3A, p30E5A, p30D9A) migrated more quickly on the gel (Fig.
4, A and B).
Effect of mRNA Structure on Translation of Two C/EBP
Isoforms--
The possible mRNA structure of C/EBP
was
simulated with a RNA folding program (mfold, version 3.0; Ref. 51). The
sequences within 500 bp of the start of transcription were input and
analyzed. A stable RNA structure was always identified at the 5'
terminus of C/EBP
after various sizes of the transcript were
analyzed (Fig. 5). The coding amino acid for full-length C/EBP
(p32)
was marked in bold and italic numbers.
This sequence includes the initial codon for full-length C/EBP
and
the initial codon for the truncated p30 C/EBP
missing the N-terminal
32 amino acids. An 8-base pair double-helical structure could be
observed, with one strand sequence including the initial codon for the
full-length C/EBP
and the complementary strand representing the
initial codon for the p30 truncated C/EBP
. The very close contact of
these two initiation codons perhaps allows ribosomal co-translation of
these two C/EBP
isoforms. Mutational destruction of this putative structure resulted in a marked diminution of translation of the p30
isoform as shown in Fig. 4A. The mutants, p32S2E, p32S2ET5D,
p32,
p32,
p32,
Ap32, and
p32
Y7, showed almost no
expression of the p30 isoform as a result of destruction of this
helical structure. Furthermore, the mutation of the initial codon of
p30 C/EBP
(p32M33I) resulted in p32 expression activity nearly
equivalent to the wild-type p32, and no p30 expression occurred (Fig.
4A). In contrast, mutations at other positions (amino acids
6-12; p32Y6A, p32Y7A, p32E8A, p32C9A, p32E10A, p32P11A, p32R12A,
p32E8/10R, p32Y6/7A, and p32Y6/7AR12A), which did not intrude
structurally on the 8-base pair helical structure, gave the expected 3 to 1 ratio of p32 and p30 C/EBP
expression (Fig. 4A).
 |
DISCUSSION |
The majority of our knowledge about the structure and function of
the C/EBPs derives largely from analysis of reporter systems defining
activation and repression regions of this protein family (23-30).
Structural knowledge about conserved amino acid residues in the
activation regions of the C/EBPs is limited. The C/EBP
protein
during preparation of nuclear extract has been reported to be degraded
rapidly by an endogenous protease with the basic/leucine zipper domain
being the major remaining product C/EBP
(41). Likewise, during
preparation of C/EBP
protein by factor Xa cleavage of the
GST-C/EBP
fusion protein, we found that the C/EBP
protein is very
unstable, making the recovery of intact C/EBP
protein difficult. As
GST contains four free thiol groups, we could gleam the status of the
thiol groups of C/EBP
by studying the GST-C/EBP
fusion protein.
With partially purified GST-C/EBP
protein (Fig. 1), we determined
that no disulfide bonds were present in C/EBP
(Table II). Other
C/EBPs, such as C/EBP
and -
, may also possibly contain no
disulfide bonds, as judged from their protein sequence homologies to
that of the C/EBP
(Fig. 1B). The transactivation analysis
of C/EBP
after mutation of each of the three conserved cysteines is
also consistent with our hypothesis. Each of these mutants gave almost
the same potency of transactivation as the wild-type protein,
indicating that those three cysteines may not be involved in disulfide
bond formation (Fig. 2). Additionally, these cysteines probably are not
involved in binding to metal ions, as metal binding cysteines often
play a key role in protein function. We have shown previously that Myb
interacts and enhances the transactivation by C/EBP
(17). In the
presence of Myb, the three cysteine mutations of C/EBP
showed no
distinguishable change of activity as compared with co-transfection of
c-Myb and the wild-type C/EBP
, suggesting that these cysteines may
not participate in the interaction with Myb (data not shown).
The C/EBP
, -
, -
, and -
proteins as well as several other
leucine zipper proteins such as Fra-1, Jun-B, and Fos-B (42-44) contain a conserved cysteine at their C-terminal region (Fig. 1A). A study has reported that this conserved cysteine could
allow efficient disulfide cross-linking between paired leucine zipper helices, and all pairwise combination of dimer interactions among those
family members were possible (9). Using non-reducing SDS-polyacrylamide
gel analysis, we tested newly expressed C/EBP
proteins within cells
of both bacterial and mammalian origins. In both situations, we could
not detect a notable dimer band on the gel, nearly all of the protein
was in monomer form (Fig. 3). The mutant C280S, which would result in
the loss of a putative covalent linkage between two monomers, gave a
nearly identical gel band to that of the wild-type C/EBP
(Fig.
3B). Meanwhile, the mutant C280S protein displayed the same
transactivation activity as that of the wild-type C/EBP
(Fig. 2).
This strongly suggests that this conserved cysteine is not involved in
disulfide bond formation in vivo. Studies have shown that
oxidized old protein of a mutant C/EBP(L12V), which should be incapable
of forming a dimer, could indeed form a dimer with wild-type C/EBP (9). Two cysteines can form a disulfide bond in vitro by random
oxidation. We also observed the oxidation of both GST and GST-C/EBP
proteins in vitro. Another study demonstrated that
redox changes affect the in vitro DNA binding capacity of
some leucine zipper proteins (45). The bacterially expressed DNA
binding domains of Fos, Jun, and BZLF1 were unable to bind DNA under
non-reducing conditions, whereas the binding of the C/EBP
DNA
binding region was unaffected. Sensitivity to redox state is due to the
presence of a conserved cysteine residue in the basic DNA binding motif
of the Fos, Jun, and BZLF1 proteins but not C/EBP
. Under
non-reducing conditions, an intermolecular disulfide bridge was formed
between the cysteine residues of each basic motif within a dimer, which
prevented binding to DNA. C/EBP
could bind DNA in either the absence
or presence of DTT. Furthermore, the nuclear extracts contained a
moderately heat-stable factor, other than reduced glutathione, that
could activate the DNA binding ability of FBGZ
(FB = Fos basic motif; GZ = GCN4 leucine
zipper) (44). This suggests that stronger reducing conditions exist in
the nucleus than in vitro.
We propose that formation of a dimer disulfide bond at the conserved
C-terminal cysteine of the C/EBP proteins may not exist in
vivo. This cysteine in other homologous proteins such as Fra-1, Jun-B, and Fos-B may also not be involved in disulfide bond formation. In fact, many other leucine zipper proteins, including GCN4, can form
dimers without the corresponding cysteine (32). Furthermore, a chimeric
protein, in which the leucine zipper of C/EBP
was replaced by the
analogous region of GCN4, showed similar DNA binding and
transactivation activities as that of C/EBP
itself (22).
The DNA binding domain of C/EBPs is an
-helical structure. The
configuration of the activation and repression regions of this family
of proteins is still unknown, probably because of their flexible
conformation. The negative charged residues in the poorly structured
activation regions has been proposed to be responsible for
stimulating the formation and activity of transcriptional preinitiation
complexes, and these regions have been called "acidic blobs"
(46-48). Hydrophobic amino acid residues are also important for the
potency of some transcriptional activators (49, 50). In order to obtain
a clearer understanding of the importance of the conserved residues in
the activation region of C/EBP
, alanine scanning mutagenesis of the
ADM1 and ADM2 regions of C/EBP
was performed; furthermore,
interchange of the ADM1 and ADM2 domains of C/EBP
for those of other
C/EBP family members (domain swapping) was examined.
Negative charge was critical for transactivational activity in both of
the ADM1 and ADM2 regions. The negative to neutral charge mutants
(p32E8A, p32E10A, p30E3A, p30E5A, and p30D9A) resulted in each case in
lower activity compared with wild-type C/EBP
(Fig. 4, A
and B). Additionally, the negative to positive charge mutations (p32E8/10R) markedly diminished activity. The result of
hydrophobic residue mutation (p30Y13A) in ADM2 agreed with the
established theory that hydrophobic amino acid residues are important
for the activity of some transcriptional activators (49, 50).
The domain swapping analyses indicated that when ADM 1 and/or ADM2 of
C/EBP
was removed and replaced with either the ADM 1 and/or ADM2 of
either C/EBP
(
p32) or C/EBP
(
p32), transactivational activity nearly doubled compared with wild-type p32 C/EBP
. This could be as a result of the presence of a glutamic acid at position 2 in C/EBP
and -
. This concept was fostered because, when a glutamic acid was substituted at position 2 of C/EBP
(p32S2E), it
had nearly double the activity of the wild-type protein. Additionally, the chimeric of the ADM2 of either C/EBP
(
p30) or C/EBP
(
p30) with the C/EBP
p30 molecule showed an equivalent activity
to p30 of C/EBP
, whereas the ADM2 of C/EBP
fused to p30 C/EBP
(
p30) gave a lower activity (Fig. 4B). This latter
finding probably is a consequence of the presence of an arginine
residue at position 6 in
p30. In contrast, the positive to neutral
charge mutant
p30R6A resulted in an equivalent activity as p30
C/EBP
.
Although the first 32-amino acid peptide did not have a major influence
on the transactivation activity of the p32 C/EBP
isoform compared
with p30 lacking ADM1, our mutational studies showed that changes of
this peptide could alter transactivation activity. Accumulation of
negative charges in this peptide enhanced the activity of full-length
C/EPB
, while a hydrophobic environment destroyed its activity. The
Ap32 chimeric protein had about triple the activity as compared with
p32. This indicated that the ADM1 and connecting sequence of C/EBP
are much more effective than the corresponding N-terminal 32-amino acid
peptide of C/EPB
.
The functional relevancy of isofoms of C/EBP
is unclear. Some
analogies perhaps can be gleaned from C/EBP
, which has three distinct isoforms: LAP* (full-length protein), LAP (truncation of the
N-terminal 21-amino acid peptide), and LIP (only C-terminal 145-amino
acid peptide). The LAP* and LAP are qualitatively and quantitatively
comparable as transcriptional activators. Nevertheless, the N-terminal
21-amino acid peptide has been implicated in recruiting the SWI/SNF
complex to activate myeloid genes (50). C/EBP
has four isoforms:
p32, p30, p27, and p14 (17). The first two are expressed at the highest
levels and are analogous to LAP* and LAP. Perhaps the N-terminal
32-amino acid peptide of C/EBP
has a role similar to this segment in
C/EBP
.
Based on our mutational data and possible mRNA structure, we
predict a stable RNA structure located at the 5' terminus of C/EBP
(Fig. 5), which includes the initial codon for full-length C/EBP
and
the initial codon for p30 N-terminal 32-amino acid truncation. This
predicted, very stable 8-base pair double-helical structure would
incorporate one strand sequence having the initial codon for
full-length C/EBP
and a complementary strand including the
initiation codon for the p30, N-terminal truncated C/EBP
. The
contact of these two initiation codons enhances the possibilities of
their equivalent co-translation from the ribosomes. Destruction of this
8-base pair helical structure would be expected to no longer
juxtaposition the two translational start sites, resulting in a marked
decrease of translation of the p30 form of C/EBP
as shown by the
following constructs: p32S2E, p32S2ET5D,
p32,
p32,
p32,
Ap32, and
p32
Y7 (Fig. 4A). Mutations at other
positions (amino acids 6-12), which did not affect the 8-base pair
helical structure, did not disrupt the co-translation of the p30 and
p32 isoforms of C/EBP
. Taken together, these data suggest that the equivalent co-translation of the two major C/EBP
isoforms is due to
the formation of a 8-base pair helical structure, pulling the two
corresponding initiation codons together to allow the ribosomes to
start translating from both sites.