From the
LAP/NF-IL6 is a member of the C/EBP family of transcriptional
activators and has been shown to be involved in the regulation of the
acute-phase response. We have previously shown that phosphorylation of
the liver-enriched transcriptional activator protein (LAP) Ser-105
enhances the activation of LAP-dependent genes. To identify the region
which is important for gene activation, a series of LAP mutants were
constructed, and domain swapping experiments with the DNA-binding
domain of GAL4 were performed. These experiments point to an acidic
region located between amino acids 21 and 105 of LAP/NF-IL6 which
activates genes independent of the DNA-binding domain and the leucine
zipper of LAP/NF-IL6. Computer-assisted predictions reveal two regions,
a helical and a hydrophobic region in the transactivation domain, which
could be important in mediating the direct interaction with the basal
machinery. Site-directed mutagenesis of acidic residues in both regions
demonstrates that the hydrophobic region located between amino acids 85
and 95 is the likely motif for the interaction with the basal
machinery. Our results demonstrate that a hydrophobic region in the
acidic transactivation domain of LAP/NF-IL6 seems to be relevant in
mediating gene activation of LAP-dependent genes.
For many genes cell type-specific expression has been shown to
be regulated at the level of transcription. Most genes are controlled
by several transcription factors which interact with cis-acting
elements in the promoter or in remote enhancer regions. The interaction
of transcription factors with their DNA recognition sites can enhance
the frequency of transcription initiation. The increase in the rate of
transcription is believed to be mediated by direct contact of the
transcription factor with the transcription apparatus (basal machinery)
after DNA binding(1, 2) .
The gene, encoding the
liver-enriched transcriptional activator protein (LAP)
Besides LAP, two other
transcription factors, C/EBP-
LAP/NF-IL6 has been
implicated as a master regulator of the acute-phase response. Earlier
data indicated that it might be involved in the induction of several
cytokine genes such as IL6, IL8, tumor necrosis factor-
In
response to environmental signals, modulation of target gene expression
can be achieved through posttranscriptional modifications of
transcriptional activators(19) . Posttranscriptional mechanisms
have also been postulated to be important in the activation of genes
with LAP recognition sites in their promoter. An IL6-dependent pathway
leads to increased binding and transactivation of LAP (NF-IL6) (5). A
direct pathway which may lead to the phosphorylation of LAP during IL6
induction has not been shown yet; however, LAP is a phosphoprotein in vivo. Different signal transduction pathways can induce
LAP-mediated gene transcription, and cAMP-mediated phosphorylation of
LAP is associated with the nuclear translocation of LAP in rat
pheochromocytoma PC12 cells(20, 21, 22) . We
showed previously that stimulation of a protein kinase C pathway in
HepG2 hepatoma cells increases the phosphorylation of Ser-105 which is
located within the N-terminal region of LAP(21) .
Next to
LAP, a liver inhibitory protein (LIP) also exists in the liver and is
translated from the same LAP mRNA, lacking the 145 N-terminal amino
acids of LAP. The resulting truncated protein is found to bind the
cognate DNA with higher affinity than LAP. As the N-terminal
transactivation domain is deleted in LIP, binding results in a strong
competitional inhibition of LAP mediated gene transcription(9) .
Additional LIP has been shown to antagonize the effect of LAP in
blocking hepatoma cell proliferation(23) . Therefore, besides
activating liver-specific gene transcription, the N-terminal part of
the protein seems to be important in mediating the LAP-dependent cell
cycle arrest in hepatoma cells.
Here we demonstrate that an 84-amino
acid-long domain in the N-terminal domain of LAP is sufficient to
mediate activation of a LAP-dependent reporter gene construct. Deletion
analysis and domain swapping show that the effect on transactivation
mediated by this domain is independent of the basic DNA-binding domain
and of the leucine zipper region.
For LAP
After
introduction of the restriction sites in the ORF of LAP, constructs
were further modified to obtain the deletions, while maintaining the
ORF of LAP. To obtain the internal deletions of LAP, the same
restriction sites were used, and DNA pieces were modified and
religated. All the resulting constructs were sequenced and subcloned
into a CMV-driven mammalian expression vector as described
before(21) .
To construct the GAL4 fusion proteins the NcoI-EcoRI fragment from the pBS LAP
To
introduce the point mutations of the acidic residues in the activation
domain of LAP the following primers were used: mutation codon 56 and/or
58, 5`-GGC GCG CT G/C GTG CT G/C GCC GAT GGC-3`; mutation codon 94
and/or 95, 5`-GCC GTA GT T/C GT T/C GGC GAA GAG-3`.
These 132
N-terminal amino acids were further characterized by the introduction
of restriction sites into the ORF of LAP to create a series of 5`
deletion mutants between LAP amino acid 21 and 153 (Fig. 1A). The mutant sequences were cloned into a
CMV-driven mammalian expression vector. The effect on transactivation
of the different N-terminal deletion mutants was investigated in
cotransfection experiments with a LAP-responsive reporter plasmid
(CRP-CAT). Interestingly, a deletion of the first 20 amino acid in the
LAP construct LAP
In the LAP
As with the 5` deletions, the internal deletions were
also checked for protein expression by Western blot analysis (Fig. 2B) and DNA binding by gel shift experiments,
respectively (data not shown). For all the proteins, the expression in
the Western blot analysis was in agreement with the effect on DNA
binding in the gel shift experiments, except for the LAP
From earlier
results we know that a chimeric LAP/GAL4 construct has the capacity to
activate a GAL4-responsive reporter construct(21) . To further
support our results in the mapping of the transactivation domain, we
performed domain swapping experiments and tested whether the same
effects on transactivation shown for the whole protein could be
observed for the deletion series using only the N-terminal domain of
LAP. In the following series of experiments the LAP DNA binding and
leucine zipper domain corresponding to the coding sequences of the LAP
protein were swapped versus the GAL4 DNA-binding domain
(GAL4(1-147)).
The resulting 5` deletions of the chimeric
proteins are demonstrated in Fig. 3A. As shown for the LAP
protein, the chimeric LAP/GAL4 5` deletions when cotransfected with a
GAL4 reporter construct also showed a dramatic drop in LUC activity
when the first 20 amino acids were deleted and no increase over
background activity was observed when a further 22 amino acids were
deleted (Fig. 3A). As demonstrated by gel shift
experiments, all the proteins had a comparable affinity in DNA binding (Fig. 3B).
The mutant LAP sequences were cloned into the CMV
expression vector. Subsequently the plasmids were cotransfected with a
LAP-responsive reporter construct into HepG2 cells. The results of four
independent transfection experiments are shown in Fig. 6A. The introduction of a glutamine at amino acid
56 and/or 58 resulted in a slight increase on the activation of the
reporter plasmid. However when the asparagine mutation at amino acid 94
was transfected, a decrease to 16% was observed compared with when the
wild type LAP protein was transfected with the LAP-responsive reporter
construct. The introduction of a double mutation or a glutamine at
position 95 alone showed no further decrease on the CAT activity (Fig. 6A). No dramatic change in DNA binding and
expression of all the mutant proteins compared with the wild type LAP
protein was monitored by gel shift assays (Fig. 6B).
These experiments suggest that the hydrophobic area in the
transactivation domain of LAP is important in mediating the activation
of LAP-dependent genes.
Activation of liver-specific genes is achieved by a set of
transcription factors which are highly expressed in the
hepatocyte(10, 34, 35) . These factors bind to
the promoter of different genes and activate the gene transcription
probably by interacting with parts of the RNA polymerase machinery in
direct or indirect protein-protein interaction as already shown for
other non-liver-specific transcription
factors(27, 28, 36) .
The LAP ORF includes
three ATG codons that could initiate translation. These ATG codons have
different efficiencies concerning the initiation of translation. In the
liver the main products consist of LAP
From our results we do not know which part of the
basal machinery is important for the direct or indirect interaction
with the transactivation domain of LAP. However, other acidic
activators have been shown to bind to
TFIIB(29, 30, 31) . Transfection experiments
with a LAP-dependent reporter construct and CMV expression vectors for
LAP and/or LIP show that the transfection of LAP results in a stronger
activation than cotransfection of LAP and LIP(9) . This result
implies that the binding of a LAP homodimer leads to stronger
interaction with the basal machinery than a LAP/LIP heterodimer. This
increase in transcription could either be achieved by a high on/off
rate in the specific interaction between the transactivation domain of
LAP and a certain site of the basal machinery. A second LAP
transactivation domain in the complex would have an additional effect
on gene transcription. The second possibility consists in an
interaction of the transactivation domain of LAP with more than one
site of the basal machinery, leading to a stronger activation of
LAP-specific genes. The interaction with several partners of the basal
machinery has been reported for the transcription factor
VP16(36) . The interaction with different sites of the basal
machinery can certainly be expected when heterodimers of LAP and
C/EBP-
Extensive mutational analysis of acidic amino acids in the
transactivation domain of the viral transcription factor VP16
demonstrates that the acidic residues are less important for the direct
interaction with the basal machinery(37) . However it has been
predicted that acidic amino acids in the transactivation domain of VP16
could be important in attracting the region in close proximity to the
target sequences(37) . Hydrophobic amino acids in the
transactivation domain are then important in mediating direct
protein-protein interaction with parts of the basal transcription
machinery(37) . Van der Waals forces between the activation
domain of VP16 and the positively charged amphipathic
The activation of LAP-specific genes has been shown to be
regulated by the phosphorylation of LAP at different sites in the
protein(15, 22) . LAP Ser-105 is phosphorylated in
vivo and in vitro(21, 24) .
Phosphorylation in vivo leads to a severalfold activation of
LAP-specific genes, and this activation can at least in part be
mimicked by an aspartic acidic residue at LAP codon 105(21) .
This is in contrast to the phosphorylation of CREB Ser-133, where the
aspartic acid mutation cannot substitute for the phosphorylation of
CREB Ser-133(38) . CREB activates the basal machinery by
interaction with the so-called Q2 region with TAF
Activation of a Ca
Our
data provide evidence that the transactivation domain of LAP is located
in the 84 N-terminal amino acids of LAP. Acidic amino acids and a
hydrophobic region in the transactivation domain are especially
important in mediating LAP-dependent gene transcription. Further
studies will provide insight into the direct interaction between the
transactivation domain of LAP and the target motif of the basal
machinery.
This paper is dedicated to Prof. Dr. K.-H. Meyer zum
Büschenfelde on the occasion of his 65th birthday.
We gratefully acknowledge E. Ziff for providing
anti-LAP antibodies. We thank M. Karin for suggestions of this work. We
thank B. Luescher, P. Straub, and A. Nordheim for critical comments on
the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)has been cloned recently(3) . The same or a
related protein has also been named NF-IL6(4) ,
IL6-DBP(5) , AGP/EBP(6) , C/EBP
(7) , and
CRP2(8) . LAP, a member of the C/EBP family, interacts with
cis-acting elements in the promoter region of the albumin gene and
several other liver-specific genes(9) . The albumin promoter
consists of six cis-acting elements (A-F). The B- and D-sites are
especially important as binding sites for transcription factors which
determine hepatocyte-specific expression of the albumin
gene(10) . LAP shows strong affinity for the D-site of the
albumin promoter and leads to stimulation of transcription in vivo and in vitro(3) .
and DBP, bind to the D-site of the
albumin promoter. All three proteins belong to the bZIP family of
transcriptional activators(3, 11, 12) . LAP and
C/EBP-
have similar affinities toward the cognate DNA and show
more than 71% homology in their DNA-binding domain and the adjacent
helix in the C-terminal part of the protein. The C-terminal part
of the protein is responsible for dimerization in vivo and in vitro of the two proteins. Descombes et al.(3) postulated a model in which the interaction of different
proteins at promoter sites may determine distinct functions in target
gene transcription. According to this model the functional differences
would be encoded in the different N-terminal region of the two
transcription factors LAP and C/EBP-
.
, and
granulocyte-colony-stimulating factor, which include LAP recognition
sites in their critical cis-acting
elements(4, 13, 14) . Recent data of Tanaka et al.(15) , in NF-IL6 deficient mice, raise doubt
about the importance of NF-IL6 for induction of some of these genes.
Further studies have to show whether the results of Tanaka et al.(15) observed in the transgenic mouse model do account for
the in vivo situation. Homozygous LAP/NF-IL6-deficient
transgenic mice might develop mechanisms during embryonal development,
for example by other members of the C/EBP family, which may circumvent
the loss of LAP/NF-IL6. Genes of the acute-phase response that share
LAP/NF-IL6 recognition sites in their promoters have been named the
class I of the acute phase genes(16) , in contrast to the
acute-phase class II genes, where binding sites for acute phase
response factor have been described(17, 18) .
Cell Culture, Transfection Experiments, and CAT
Assays
HepG2 cells (ATCC) were cultured in minimal essential
medium supplemented with 10% fetal calf serum. DNA transfection into
HepG2 cells was carried out as described previously(2) , with
the exception of a calcium phosphate precipitation for only 5 h,
followed by a 15% glycerol shock for 1 min. In experiments with the LAP
deletion proteins, LAP-responsive CAT-reporter constructs, the D- or
CRP-CAT vectors, were cotransfected. CAT assays were performed 48 h
after transfection using thin layer chromatography for the separation
of the reaction products(21) . Quantification of the CAT results
was carried out with a Fuji Imager. In transfection experiments with
the LAP/GAL4 fusion proteins the GAL4-responsive reporter 5
GAL4-Luc was cotransfected. For luciferase activity cell extracts were
prepared and measured as described before(21) .
Plasmid Construction and Site-directed
Mutagenesis
The pBS-LAP 21 construct (24) was used
for plasmid constructions. For site-directed mutagenesis the cDNA of
pBS LAP
21 was used to prepare single stranded DNA in the Escherichia coli strain JM 109 using the helper phage M13 KO
7, according to Sambrook et al.(25) . Site-directed
mutagenesis was performed by using the mutagenesis kit from Amersham,
according to the supplier's instructions. To obtain the 5`
deletion of LAP, restriction sites were introduced into the open
reading frame (ORF) of LAP: LAP
1-41, 5`- CGC GGC GCG GAC
CGC CTT GGC CC -3` (RsrII site); LAP
1-63, 5`-CAG
GTA GGG GCT CAT GAC GAT GGC GCG-3` (BspHI site); LAP
1-75, 5`-GCG GCG AAG TCC TAG GCG GCG GG-3` (AvrII
site); LAP
1-104, 5`-CCG TAG TCG GAC CGC TTC TTG CTC G-3` (RsrII site); LAP
1-121, 5`-GGG AAG CAG GCC TGC GGT
GCG GC-3` (StuI site); LAP
1-189, 5`-GGC GTC GGC
GGG ACC CGG CGT CCC GGG-3`.
1-139 and LAP
1-152 natural sites BsiCI and NcoI,
respectively, in the ORF were used to obtain the deletions.
21
construct containing the basic domain and the leucine zipper region of
LAP was replaced by the BspHI-EcoRI fragment
containing the GAL4 DNA-binding domain (amino acids 1-147) of a
modified version of the pSG424 vector as described before(21) .
The chimeric LAP-GAL4 reading frames were excised by HindIlI-EcoRI digestion and were cloned into the
pSG424 vector from which the GAL4 DNA-binding domain was removed.
Nuclear Extracts and Gel Retardation
Assays
Nuclear extracts were prepared from HepG2 hepatoma cells
using the Dignam C method as described previously(21) . For the
gel retardation assays 2.5 µg of nuclear extracts were used. The
binding reaction was performed for 20 min on ice. For binding assays
where the LAP DNA-binding region was maintained an oligonucleotide
spanning the D-site of the albumin promoter was used as a P-labeled probe. For gel shift experiments using the
chimeric LAP-GAL4 proteins an oligonucleotide spanning the GAL4-binding
site was used as a
P-labeled probe. Free DNA and
DNA
protein complexes were resolved on a 6% polyacrylamide gel as
described previously (24) with the difference that the gel was
run for 4 h at 300 V to get a better resolution of the different
deletion mutants.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis
Nuclear extracts were separated on a 10%
SDS-polyacrylamide gel (26) and blotted onto a nitrocellulose
membrane (Schleicher and Schuell) in 1% SDS, 20% methanol, 400 mM glycine, 50 mM Tris-HCl, pH 8.3, at 4 °C for 2 h at
200 mA. LAP 21 and the LAP deletions were analyzed by using
polyclonal LAP antibodies(21) . The antigen-antibody complexes
were visualized using the ECL detection system as recommended by the
manufacturer (Amersham).
The N-terminal Boundary of the Activation Domain Is
Located Close to the Second ATG in the LAP ORF
Three different
ATGs exist in the ORF of LAP. Only the second and the third ATG are
efficiently used to translate the two different forms of LAP in the
liver; LAP 21 and LIP (LAP
1-152). In contrast, the
full-length LAP is not efficiently translated and has only a minor
effect on transactivation of LAP-responsive reporter constructs
compared with the LAP
21 protein(9) . Therefore LAP
21
was used for deletion analysis to identify the transactivation domain
of LAP. LIP has been shown to be a repressor of LAP, as it blocks
transactivation of a LAP dependent reporter construct(9) . From
these data we concluded that the transactivation domain should be in
the 131 N-terminal amino acids (21-152) of LAP.
1-41 had a tremendous effect on the
activation of the reporter plasmid. Only around 10% of the CAT activity
was detected compared with LAP
1-21 construct (Fig. 1A). A deletion of further 22 amino acids in LAP
1-63, and the other consecutive 5` deletions, showed no
increased activation of the reporter plasmid compared to the
transfection of the reporter construct alone (Fig. 1A).
Figure 1:
Mapping of the N-terminal boundary of
the transactivation domain of LAP. A, transcriptional
activities of the N-terminal LAP deletions. The different N-terminal
deletion mutants are shown. HepG2 cells were cotransfected with the
LAP-responsive reporter construct (CRP-CAT) and either the CMV-0
expression vector, wild type LAP 2, or the N-terminal deletion
mutants as indicated. The CAT activity of CMV-0 cotransfected with the
LAP-responsive reporter construct was set as a reference to 1 (not
shown). Activation compared with the reference are marked as -fold
activation. Additionally the activity of the wt LAP cotransfected with
the LAP-responsive reporter construct was set to 100%. B,
Western blot analysis. Nuclear extracts from cells transfected with one
of the N-terminal deletion plasmids were prepared as described under
``Materials and Methods.'' Nuclear proteins were separated
with a 10% SDS gel and blotted onto nitrocellulose. Mutant LAP proteins
were analyzed by Western blot with anti-LAP
antibody.
The nuclear expression of all the N-terminal LAP deletion mutants
was monitored by Western blot analysis of nuclear extracts (Fig. 1B). All the constructs were well expressed in the
nucleus. As deletion mutants could have an effect on the tertiary
structure of the artificial protein and resulting in a reduction of DNA
binding, gel shift experiments were performed with the same nuclear
extracts as shown in the Western blot experiments. All the deletion
proteins were found to bind the cognate DNA (data not shown). To
exclude that differences in the expression of the proteins may
influence the results shown in Fig. 1A, and because we
showed that LAP has a strong squelching effect on gene
activation(21) , increasing amounts of the LAP expression
constructs were cotransfected with a constant amount of the reporter
plasmid. However only minor effects on the activation of the reporter
plasmid could be observed, when increasing amounts of the LAP deletions
LAP 1-63, LAP
1-75, LAP
1-104, LAP
1-121, LAP
1-139, and LAP
1-152 were
used as these proteins did not activate the reporter plasmid over
background level (data not shown). Like already shown for the LAP
1-21 protein(21) , the first LAP
1-41
mutation showed a similar kinetic on activation of the reporter
construct as found for the wild type protein (data not shown).
The C-terminal Boundary of the Activation Domain Is
Located Close to the Second ATG in the LAP ORF
After we
determined the N-terminal boundary of the activation domain of LAP, the
following experiments were directed to find the C-terminal boundary of
the activation domain. We created a series of internal LAP deletion
mutants. Increasingly larger pieces were excised from the internal part
of the protein beginning at the third ATG in the ORF. The 145
C-terminal amino acids, which code for the LIP protein and which
contain the basic domain and the leucine zipper important for mediating
DNA binding, were left unchanged.
140-152, LAP
105-152, and LAP
64-152, 13, 47, and 90 amino
acids, respectively, were deleted (Fig. 2A). The
modified LAP proteins were cotransfected with the LAP-responsive
reporter plasmid (CRP-CAT). Only a minor decrease in CAT activity was
observed when the smallest deletion LAP
140-152 was
cotransfected with the specific reporter plasmid. The deletion of
further 34 amino acids in the mutant LAP
105-152 resulted in
a reduction of nearly 40% activity of the wild type. Further excision
of 41 amino acids in the LAP
64-152 construct resulted in a
decline to background activity, detected when the reporter construct
was used alone (Fig. 2A). In order to determine the
significance of amino acids 105-152, we deleted amino acids
64-104 in the construct LAP
64-104 and cotransfected
the CMV expression vector with the LAP-responsive reporter plasmid. As
shown in Fig. 2A no increase over background activity was
observed when the LAP
64-104 construct was expressed.
Therefore we concluded that the LAP amino acids 64-104 contain
sequences which are important for mediating the transactivation of LAP.
From our results obtained with the N-terminal and internal LAP deletion
mutants we were confident that an important part of the LAP
transactivation domain was located between the LAP amino acids
21-104. We speculate that the difference in activity between LAP
140-152 and LAP
105-152 could be due to changes
in the tertiary structure of the mutant proteins, alternatively the
amino acids between 105 and 140 are important to stabilize the region
between amino acids 21 and 104.
Figure 2:
Mapping of the C-terminal boundary of the
transactivation domain of LAP. A, transcriptional activities
of the internal LAP deletions. The figure shows the different deletion
mutants in comparison with the LAP 21 protein. HepG2 cells were
cotransfected with the LAP-responsive reporter construct and either the
CMV-0 expression vector, wild type LAP
21 or the internal deletion
mutants as indicated. The CAT activity of CMV-0 cotransfected with the
LAP-responsive reporter construct was set as a reference to 1 (not
shown). Activation compared with the reference are marked as -fold
activation. Additionally the activity of the wild type LAP
cotransfected with the LAP-responsive reporter construct was set to
100%. B, Western blot analysis. Nuclear extracts from cells
transfected with one of the internal deletion plasmids were prepared as
described under ``Materials and Methods.'' Nuclear proteins
were separated with a 10% SDS gel, blotted onto nitrocellulose. Mutants
LAP proteins were analyzed by Western blot with anti-LAP
antibody.
To exclude that an additional
transactivation domain which may be repressed by other sequences is
further 3` of the third ATG in the LAP ORF, an additional deletion was
constructed which leaves only 107 amino acids of the LAP molecule
containing the important region for DNA binding (LAP 1-189).
Like for the LAP
1-152 protein, no change in CAT activity
was observed.
1-189 construct. A dramatic difference was observed between
the nuclear expression of the protein and its strong shift in the gel
shift experiment when compared with the other deletion proteins in the
same experiment. At the present time we have two possible explanations
for this discrepancy in the results. Either the polyclonal antibody
directed toward the LAP protein shows a much lower affinity for these
107 C-terminal amino acids of the protein, or as shown for the LIP
protein, which has a stronger affinity toward the cognate
DNA(9) , further 3` deletions have an even more dramatic effect
on the DNA binding affinity of the protein.
The LAP Activation Domain Acts Independently of the LAP
DNA-binding Domain
For many other transcription factors it has
been shown that the different functional domains could be dissected and
that these domains work independently from each other. The GAL4
DNA-binding domain of GAL4 is an ideal candidate; it has been mapped
within the first 147 amino acids of the wild type protein. The
truncated protein has been shown to translocate to the nucleus and is
incapable of activating transcription in vivo.
Figure 3:
Transactivation experiments of the
N-terminal LAP deletion/GAL4-DNA fusion proteins. A,
transcriptional activities of the N-terminal LAP deletions. The figure
shows the different deletion mutants in comparison to the chimeric LAP
21/GAL4 protein. HepG2 cells were cotransfected with a
GAL4-responsive reporter construct and either the RSV-GAL4(1-147)
expression vector, the RSV-LAP 21-152/GAL4, or one of the
N-terminal RSV-LAP deletion/GAL4-DNA constructs was cotransfected. The
LUC activity of the RSV-GAL4(1-147) expression vector
cotransfected with the GAL4-responsive reporter construct was set as a
reference to 1 (not shown). Activation compared with the reference is
marked as FOLD ACTIVATION. Additionally the activity of the
LAP
21/GAL4 vector cotransfected with the GAL4-responsive reporter
construct was set to 100%. B, gel shift experiments of the
N-terminal LAP deletions/GAL4 fusion proteins. Nuclear extracts from
cells transfected with one of the N-terminal LAP deletions/GAL4 fusion
proteins were used for gel shift experiments. The nuclear proteins were
incubated for 15 min with the
P-labeled DNA of the cognate
GAL4 sequence (25 pg). The DNA
protein complexes were resolved on
a 6% nondenatured polyacrylamide gel. To get a better resolution of the
different mutant LAP/GAL4 DNA
protein complexes, the gel was run
at 300 V for 5 h. Therefore the free probe was run off the bottom of
the gel. The position of DNA-bound LAP(21-152)/GAL4(1-147)
and GAL4(1-147) are indicated. A lane of each of the different
proteins is indicated.
Also the internal LAP deletion mutants
were used to construct and express the chimeric LAP/GAL4 proteins (Fig. 4A). As shown for the internal LAP deletion
mutants also, the chimeric LAP/GAL4 proteins point to a region between
amino acids 22 and 104 which is important for transactivation (Fig. 4A). The gel shift experiment showed only minor
differences in DNA binding between the different chimeric proteins (Fig. 4B).
Figure 4:
Transactivation experiments of the
internal LAP deletion/GAL4-DNA fusion proteins. A, schematic
diagram and transcriptional activity of the internal LAP
deletion/GAL4-DNA fusion proteins. The different internal
deletion/GAL4-DNA fusion proteins were constructed as described under
``Materials and Methods.'' The figure shows the different
deletion mutants in comparison with the
LAP(21-152)/GAL4(1-147) fusion protein. The LUC activity of
the RSV-GAL4(1-147) expression vector cotransfected with the
GAL4-responsive reporter construct was set as a reference to 1.
Activation compared with the reference is marked as FOLD ACTIVATION after each of the constructs. Additionally the activity of the LAP
21/GAL4 vector cotransfected with the GAL4-responsive reporter
construct was set to 100%. B, gel shift experiments of the
internal LAP deletions/GAL4 fusion proteins. Nuclear extracts from
cells transfected with one of the internal LAP deletions/GAL4 fusion
proteins were used for gel shift experiments. The nuclear proteins were
incubated for 15 min with the
P-labeled DNA of the cognate
GAL4 sequence (25 pg). The DNA
protein complexes were resolved on
a 6% nondenatured polyacrylamide gel. To get a better resolution of the
different mutant LAP/GAL4 DNA
protein complexes, the gel was run
at 300 V for 5 h. Therefore the free probe was run off the bottom of
the gel. The positions of DNA-bound LAP(21-152)/GAL4(1-147)
and GAL4(1-147) are indicated. A lane of each of the different
proteins is indicated.
Interestingly a severalfold increase in
activation of the GAL4 LUC reporter construct was observed when the LAP
105-152/GAL4 expression vector was cotransfected instead of
the LAP
1-21/GAL4 expression vector (Fig. 4A). As both proteins have a comparable affinity
in gel shift experiments toward the cognate DNA (Fig. 4B), this difference is very likely to be directly
mediated by the transactivation domain of the LAP
105-152/GAL4 chimeric protein. These findings with the
chimeric LAP/GAL4 proteins support our findings that the N-terminal 84
amino acids are responsible in mediating the effect on the expression
of LAP-dependent genes.
A Hydrophobic Region in the Acidic Transactivation Domain
of LAP Mediates the Activation of LAP-dependent Genes
Distinct
regions in the transactivation domains of transcription factors are
known to mediate the activation of the dependent
genes(27, 28, 29) . A characteristic feature of
the transactivation domain of LAP is its acidic nature. It was shown
for other transcription factors that acidic regions are important to
interact with parts of the general RNA-polymerase machinery and to
regulate gene transcription(30, 31) . Therefore we
analyzed the transactivation domain of LAP between amino acids 21 and
105 for regions which could be important in mediating these
protein-protein interactions. Computer-assisted prediction by Kyte and
Doolittle (32) and by Deléage (33) for protein
structure was performed to determine the possible regions, which could
be important for the LAP transactivation domain. Two regions were found
in the transactivation domain of LAP which could be interesting in
mediating the activation of LAP-dependent genes (Fig. 5). The
region between amino acids 38 and 63 was predicted by the algorithms to
be helical, whereas the region between amino acids 85 and 95 contains
several hydrophobic amino acids.
Figure 5:
Hydrophobic and helical regions in the
transactivation domain of LAP are possible candidates for mediating
protein-protein contacts with the basal machinery. Computer-assisted
analysis of the transactivation domain was performed, showing a
hydrophobic region between amino acids 85 and 95 and a helical region
between amino acids 38 and 63 as possible candidates for the
interaction with the basal machinery. The hydrophobic region is marked
with hatched bars, and the helical region is marked with circles. Acidic amino acids in the hydrophobic and helical
region were chosen. Single or double mutations of the residues were
performed as described under ``Materials and Methods.'' The
positions of the different point mutations are
marked.
From these computer-assisted
predictions we wondered whether acidic amino acids in these two regions
are important in mediating the activation of LAP-dependent genes.
Therefore acidic amino acids in both regions were selected. Mutations
were introduced replacing the negatively charged amino acid by its
corresponding neutral amino acid, e.g. glutamic acid was
changed into glutamine and aspartic acid into asparagine. The mutation
introduced in the helical part of the transactivation domain are
glutamic acids 56 and 58 and in the hydrophobic part aspartic acids 94
and 95 (Fig. 5). The changes were introduced either as single or
double mutations.
Figure 6:
Transactivation experiments of the point
mutations introduced into the LAP transactivation domain. A,
transcriptional activities of the single and double mutations in the
LAP transactivation domain. HepG2 cells were cotransfected with the
LAP-responsive reporter construct and either the CMV-0 expression
vector, wild type LAP 21, or one of the point mutations described
in the legend to Fig. 5. The CAT activity of CMV-0 cotransfected with
the LAP-responsive reporter construct was set as a reference to 1 (lane 1). Activation compared with the reference are marked as FOLD ACTIVATION. The lanes of the mutations transfected is
indicated. B, gel shift experiments. Nuclear extracts from
cells transfected with one of the constructs shown under A were prepared as described under ``Materials and
Methods.'' The nuclear proteins were incubated for 15 min with the
P-labeled DNA of the cognate LAP sequence (25 pg) at 4
°C. The DNA
protein complexes were resolved on a 6%
nondenatured polyacrylamide gel. The position of the bound DNA-LAP
complexes and the free (f) probe are marked. The lanes of the
mutations transfected are indicated.
21 and LIP. LIP is a
repressor of LAP-specific gene transcription. It has a higher affinity
to the cognate DNA, but not the potential in activating gene
transcription(9) . Therefore we concentrated our studies on the
131 N-terminal amino acids of LAP
21. Deletion of the first 19
amino acids reduced the activation of LAP-dependent genes. These
results suggest that the N terminus is important for gene activation.
Further experiments revealed a region of 84 amino acids of LAP, which
is important for the activity of LAP. This is the likely region to
communicate in one way or another with components of the basal
transcription machinery. Computer-assisted predictions showed two areas
in this region which could be important for this interaction.
Site-directed mutagenesis of acidic amino acids in each of the two
areas gives indirect evidence for the importance of a hydrophobic
region between amino acid 85 and 95. It has been shown that
posttranscriptional mechanisms, which modulate LAP/NF-IL6, are
important in leading to a higher activation of the acute phase class I
genes(4, 5) . The HepG2 cells line we used in this study
is insensitive to IL6. Further studies with other cell lines have to
show, whether the here described transactivation domain of LAP is also
important in mediating IL6-dependent gene activation or whether other
regions of the protein might be involved in IL6-dependent effects on
gene regulation.
bind to the cognate DNA. The two proteins have a striking
difference in the N terminus, and therefore, different protein-protein
interactions will be important in mediating liver-specific gene
transcription by members of the C/EBP
family(3, 7, 8) . As more of the factors which
assemble in the basal machinery are cloned, further research will
answer the question of whether TFIIB or one of the other factors is
important in interacting with the transactivation domain of LAP.
-helix of
TFIIB between amino acid 178 and 201 lead to stable protein-protein
interaction and the activation of the RNA polymerase II(29) . In
our experiments the mutation of aspartic acid 94 into an asparagine
leads to a dramatic drop in LAP-specific gene activation. The double
mutation of aspartic acids 94 and 95 has no additional effect on the
activation of LAP and the mutation of aspartic acid 95 alone leads only
to a minor change of LAP-specific gene activation. The mutation of
other acidic amino acids in the predicted helical motif of the
transactivation domain of LAP leads to an even stronger activation of
LAP-specific genes than the wild type protein. These data indicate that
aspartic acid 94 which is in close proximity to a hydrophobic motif in
the transactivation domain of LAP could play a key role in attracting
LAP versus its target sequences in the basal transcription
machinery, or that, alternatively, the loss of this acidic residue
could have a dramatic effect on the tertiary structure of LAP which may
lead to a change in the folding of the protein and render the
hydrophobic motif unable to interact with proteins of the basic
machinery.
110(27) . A hydrophobic region of CREB between amino acids
204 and 208 has been shown to be especially important in mediating the
protein-protein interaction(27) , and a highly homologous region
of Sp1 has also been shown to be important for the interaction with
TAF
110(28) . In contrast to the acidic LAP
activation domain the transactivation domain of CREB has been shown to
be glutamine-rich and the phosphorylation of CREB Ser-133 leads to a
change in protein folding(39) . Therefore in the case of CREB
the addition of a negatively charged amino acid alone has no effect on
its activation. In the case of LAP the transactivation domain is acidic
and the kinase domain of LAP Ser-105 is in close proximity to its
activation domain. Compared with other transactivation domains, as for
example the one of VP16, LAP has less acidic amino residues. Therefore
we postulate that phosphorylation of LAP Ser-105 has more of an effect
on the stabilization of the transactivation domain than on the
interaction of two different parts of the protein, as shown for CREB,
which leads to the activation of LAP-specific genes. A clue which of
the two discussed mechanisms will be more important after
phosphorylation of LAP Ser-105 in mediating gene activation may be the
search of a direct interaction of CBP and LAP. CBP seems to play a role
in the activation and the interaction with certain phosphorylated
transcription factors, like for example c-Jun and CREB(40) . If
the phosphorylation of LAP Ser-105 has more an effect on the
stabilization of the transactivation domain, CBP is less likely to
interact with LAP.
-dependent
pathway leads to the phosphorylation of LAP Ser-276 located in the
leucine zipper(22) . This specific phosphorylation correlates
with the activation of LAP-specific genes. The leucine zipper is far
away from the identified transactivation domain and the stability of
the leucine zipper is important for homo- or heterodimerization with
other members of the leucine zipper family. Therefore a direct
interaction with the transactivation domain is difficult. It seems more
likely that phosphorylation of LAP Ser-276 in the leucine zipper could
facilitate protein-protein interaction with other transcriptional
activators. For example, a protein-protein interaction between the Rel
homology domain and the leucine zipper domain of LAP has been
demonstrated(41) . This leads to the activation of genes with a
binding site for C/EBP in the promoter and to an inhibition of genes
with a
B site in their promoters(41) . Therefore
phosphorylation of Ser-276 could facilitate the protein-protein
interaction without affecting the transactivation domain of LAP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.