©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Transactivation of LAP/NF-IL6 Is Mediated by an Acidic Domain in the N-terminal Part of the Protein (*)

Christian Trautwein , Diana L. Walker , Jörg Plümpe , Michael P. Manns (§)

From the (1)Abteilung Gastroenterologie und Hepatologie, Medizinische Hochschule, 30625 Hannover, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)()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) .

Besides LAP, two other transcription factors, C/EBP- 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-.

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-, 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) .

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.


MATERIALS AND METHODS

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`.

For LAP 1-139 and LAP 1-152 natural sites BsiCI and NcoI, respectively, in the ORF were used to obtain the deletions.

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 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.

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`.

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 DNAprotein 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).


RESULTS

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.

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 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.

In the LAP 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.

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 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.

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).


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 DNAprotein complexes were resolved on a 6% nondenatured polyacrylamide gel. To get a better resolution of the different mutant LAP/GAL4 DNAprotein 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 DNAprotein complexes were resolved on a 6% nondenatured polyacrylamide gel. To get a better resolution of the different mutant LAP/GAL4 DNAprotein 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.

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.


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 DNAprotein 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.




DISCUSSION

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 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.

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- 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.

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 -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.

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 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.

Activation of a Ca-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.

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.


FOOTNOTES

*
This work was supported by Grant Tr 28513-1 from the Deutsche Forschungsgemeinschaft. Part of this work was presented as an oral presentation at the 45th Annual Meeting of the American Association for the Study of the Liver, November 11-15, 1994, Chicago, IL. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This paper is dedicated to Prof. Dr. K.-H. Meyer zum Büschenfelde on the occasion of his 65th birthday.

§
To whom correspondence should be addressed: Abteilung Gastroenterologie und Hepatologie, Zentrum Innere Medizin und Dermatologie, 30625 Hannover, Germany. Tel.: 49-511-532-3865; Fax: 49-511-532-4896.

The abbreviations used are: LAP, liver-enriched transcriptional activator protein; LIP, liver inhibitory protein; IL, interleukin; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; CREB, cAMP-responsive element-binding protein; C/EBP, CCAAT/enhancer-binding protein; ORF, open reading frame; CBP, phospho-CREB-binding protein.


ACKNOWLEDGEMENTS

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.


REFERENCES
  1. Johnson, P. F., and McKnight, S. L.(1989) Annu. Rev. Biochem.58, 799-839 [CrossRef][Medline] [Order article via Infotrieve]
  2. Mitchell, P. J., and Tjian, R.(1989) Science245, 371-378 [Medline] [Order article via Infotrieve]
  3. Descombes, P., Chojkier, M., Lichtsteiner, S., Falvey, E., and Schibler, U.(1990) Genes & Dev.4, 1541-1551
  4. Akira, S., Isshiki, H., Sugita, T., Tanabe, O., Kinoshita, S., Nishio, Y., Nakajima, T., Hirano, T., and Kishimoto, T.(1990) EMBO J.9, 1897-1906 [Abstract]
  5. Poli, V., Mancini, F. P., and Cortese, R.(1990) Cell63, 643-653 [Medline] [Order article via Infotrieve]
  6. Chang, C.-J., Chen, T.-T., Lei, H.-Y., Chen, D.-S., and Lee, S. C. (1990) Mol. Cell. Biol.10, 6642-6653 [Medline] [Order article via Infotrieve]
  7. Cao, Z., Umek, R. M., and McKnight, S. L.(1991) Genes & Dev.5, 1538-1552
  8. Williams, S. C., Cantwell, C. A., and Johnson, P. F.(1991) Genes & Dev.5, 1553-1567
  9. Descombes, P., and Schibler, U.(1991) Cell67, 569-579 [Medline] [Order article via Infotrieve]
  10. Maire, P., Wuarin, J., and Schibler, U.(1989) Science244, 343-346 [Medline] [Order article via Infotrieve]
  11. Landschulz, W. H., Johnson, P. F., Adashi, E. Y., Graves, B. J., and McKnight, S. L.(1988) Genes & Dev.2, 786-800
  12. Mueller, C. R., Maire, P., and Schibler, U.(1990) Cell61, 279-291 [Medline] [Order article via Infotrieve]
  13. Mukaida, N., Shiroo, M., and Matsushima, K. J.(1989) J. Immunol.143, 1366-1371 [Abstract/Free Full Text]
  14. Nishizawa, M., Tsuchiya, M., Watanabe-Fukunaga, R., and Nagata, S. (1990) J. Biol. Chem.265, 5897-5902 [Abstract/Free Full Text]
  15. Tanaka, T., Akira, S., Yoshida, K., Umemoto, M., Yoneda, Y., Shirafuji, N., Fujiwara, H., Suematsu, S., Yoshida, N., and Kishimoto, T.(1995) Cell80, 353-361 [Medline] [Order article via Infotrieve]
  16. Wegenka, U. M., Buschmann, J., Lütticken, C., Heinrich, P. C., and Horn, F.(1993) Mol. Cell. Biol.13, 276-288 [Abstract]
  17. Lütticken, C., Wegenka, U. M., Yuan, J., Buschmann, J., Schindler, C., Ziemiecki, A., Harpur, A. G., Wilks, A. F., Yasukawa, K., Taga, T., Kishimoto, T., Barbieri, G., Pelle Grinni, S., Sendtner, M., Heinrich, P. C., and Horn, F.(1994) Science263, 89-92 [Medline] [Order article via Infotrieve]
  18. Akira, S., Nishio, Y., Inoue, M., Wang, X.-J., Wei, S., Matsusaka, T., Yoshida, K., Sudo, T., Naruto, M., and Kishimoto, T.(1994) Cell77, 63-71 [Medline] [Order article via Infotrieve]
  19. Hunter, T., and Karin, M.(1992) Cell70, 375-387 [Medline] [Order article via Infotrieve]
  20. Metz, R., and Ziff, E.(1991) Genes & Dev.5, 1754-1766
  21. Trautwein, C., Caelles, C., van der Geer, P., Hunter, T., Karin, M., and Chojkier, M.(1993) Nature364, 544-547 [CrossRef][Medline] [Order article via Infotrieve]
  22. Wegner, M., Cao, Z., and Rosenfeld, M. G.(1992) Science256, 370-373 [Medline] [Order article via Infotrieve]
  23. Buck, M., Turler, H., and Chojkier, M.(1994) EMBO J.13, 851-860 [Abstract]
  24. Trautwein, C., van der Geer, P., Karin, M., Hunter, T., and Chojkier, M.(1994) J. Clin. Invest.93, 2554-2561 [Medline] [Order article via Infotrieve]
  25. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  26. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  27. Ferreri, K., Gill, G., and Montminy, M.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 1210-1213 [Abstract]
  28. Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 192-196 [Abstract]
  29. Roberts, S. G. E., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R.(1993) Nature363, 741-744 [CrossRef][Medline] [Order article via Infotrieve]
  30. Lin, Y-S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R.(1991) Nature353, 569-571 [CrossRef][Medline] [Order article via Infotrieve]
  31. Lin, Y.-S., and Green, M. R.(1991) Cell64, 971-981 [Medline] [Order article via Infotrieve]
  32. Kyte, J., and Doolittle, R. F.(1982) J. Mol. Biol.157, 105-132 [Medline] [Order article via Infotrieve]
  33. Deléage, G., Tinland, B., and Roux, B.(1987) Anal. Biochem.163, 292-297 [Medline] [Order article via Infotrieve]
  34. Frain, M., Swart, G., Monaci, P., Nicosia, A., Stämpfli, S., Frank, R., and Cortese, R.(1989) Cell59, 145-157 [Medline] [Order article via Infotrieve]
  35. Kuo, J. C., Conley, P. B., Chen, L., Sladek, F. M., Darnell, J. E., and Crabtree, G. R.(1992) Nature355, 457-461 [CrossRef][Medline] [Order article via Infotrieve]
  36. Goodrich, J. A., Hoey, T., Thut, C. J., Admon, A., and Tjian, R.(1993) Cell75, 519-530 [Medline] [Order article via Infotrieve]
  37. Cress, W. D., and Triezenberg, S. J.(1991) Science251, 87-90 [Medline] [Order article via Infotrieve]
  38. Gonzalez, G. A., and Montminy, M. R.(1989) Cell59, 675-680 [Medline] [Order article via Infotrieve]
  39. Gonzalez, G. A., Menzel, P., Leonard, J., Fischer, W. H., and Montminy, M. R.(1991) Mol. Cell. Biol.11, 1306-1312 [Medline] [Order article via Infotrieve]
  40. Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M.(1994) Nature370, 226-229 [CrossRef][Medline] [Order article via Infotrieve]
  41. Stein, B., Cogswell, P. C., and Baldwin, A. S.(1993) Mol. Cell. Biol.13, 3964-3974 [Abstract]

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