©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Repression of the c-Jun trans-Activation Function by the Adenovirus Type 12 E1A 52R Protein Correlates with the Inhibition of Phosphorylation of the c-Jun Activation Domain (*)

Dieter Brockmann (§) , Carsten Bury , Gabriele Kröner , H.-Christoph Kirch , Helmut Esche

From the (1) Institute of Molecular Biology (Cancer Research), University of Essen Medical School, Hufelandstra55, 45122 Essen, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The early region 1A 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic adenovirus subtype 12, represses the trans-activating function of the cellular transcription factor complex AP-1 consisting of c-Jun-c-Jun homodimers. In this report we demonstrate that the repression in vivo correlates with a direct physical interaction of the adenovirus protein with c-Jun in vitro. Interestingly, the 52R protein binds to the bZIP domain of c-Jun essential for dimerization and DNA binding but not to the c-Jun activation domain. This interaction does not prevent the promoter binding of c-Jun/AP-1. Moreover, the physical association between c-Jun and the TATA box-binding protein TBP is not disturbed by the 52R polypeptide. In fact, we show evidence that down-regulation of c-Jun activity by the adenoviral protein is due to the inhibition of phosphorylation of the c-Jun trans-activation domain. In vivo phosphorylation of the c-Jun activation domain is necessary for the interaction of c-Jun with specific cofactors such as CBP and therefore a prerequisite for the activation of target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating function of c-Jun by preventing its phosphorylation through a specific kinase necessary for the activation of the cellular transcription factor.


INTRODUCTION

Gene products encoded by the early region 1A oncogene (E1A)() of Ads are essential for the expression of all other viral genes (Berk et al., 1979; Jones and Shenk, 1979; Nevins, 1981). They also modulate the expression of specific cellular genes (for reviews, see Rochette-Egly et al. (1990) and Shenk and Flint (1991)). Moreover, early region 1A proteins are able to act as oncoproteins that cooperate with adenovirus E1B gene products to transform rodent cells in culture and, depending on the serotype ( e.g. Ad12), to induce tumors in immunocompetent animals (Huebner et al., 1962; Pope and Rowe, 1964; Mukai and Kobayashi, 1972). In oncogenic transformation protein functions of region E1A are necessary to immortalize primary cells (Houweling et al., 1980; Ruley, 1983; Zerler et al., 1986), whereas functions of region E1B are essential to obtain a fully transformed phenotype (Jochemsen et al., 1982; Byrd et al., 1988). The functions of region E1B can be substituted by specific cellular gene products, e.g. activated Ha-ras (Ruley, 1983; Byrd et al., 1988). The reasons for the difference in the oncogenicity of variant adenovirus serotypes are not yet clearly understood.

Region E1A of the in vivo oncogenic serotype Ad12 has a very complex expression pattern. Alternative splicing of a common precursor results in the generation of six mRNAs (13, 12, 11, 10, 9.5, and 9 S) which give rise to five different proteins (266R, 235R, 106R, 52R, and 53R) (Perricaudet et al., 1980; Sawada and Fujinaga, 1980; Brockmann et al., 1990). All proteins have identical amino-terminal ends but differ in their carboxyl termini due to the excision of different introns and/or frameshifts with the beginning of the respective second exon. Therefore, neither the 106R protein of the 11/10 S mRNAs nor the 52R protein of the 9.5 S mRNA, which are both unique for the highly oncogenic Ad12 (Brockmann et al., 1994), contain any of the conserved regions (CR1, CR2, CR3) which are thought, in combination with the very amino terminus, to be essential for the trans-regulatory functions of the two larger E1A proteins (Lillie et al., 1986; Schneider et al., 1987; Jelsma et al., 1989; Rochette-Egly et al., 1990; Stein et al., 1990; Wang et al., 1993).

Although the physiological roles of the 52R protein are still unclear, the study of its function(s) is of particular interest as Ad12 E1A virus mutants unable to express the 52R protein lose their transforming capacity. For example, the Ad12 host range mutant CS-1, which is adapted to efficient replication in Ad12 semipermissive simian Vero cells, is unable to transform primary rat embryo fibroblasts and to generate tumors in newborn hamsters. Due to the loss of the splice acceptor site, a functional 52R protein cannot be expressed from Ad12 CS-1 (Murphy et al., 1987; Opalka et al., 1992). Chimeric Ad5/Ad12 E1 genes are tumorigenic on condition that the Ad12 E1A amino terminus is included up to the leftmost border of CR3 (Jelinek et al., 1994). Jelinek and co-workers speculate that a so-called spacer segment, an Ad12 E1A-unique region of 60 nucleotides located between CR2 and CR3, may be involved in tumorigenicity. Interestingly, this spacer codes for the carboxyl-terminal end of the 52R protein (Brockmann et al., 1994). The importance of the spacer region is shown in an additional report of Telling and Williams (1994). These authors could show that hybrid Ad12 viruses, in which nucleotides 814-964 of the E1A gene (containing the spacer) were exchanged for the respective region of Ad5 E1A (containing no spacer), display wild-type transformation frequencies on baby rat and mouse kidney cells, but show a greatly reduced oncogenic capacity, as measured by tumor induction following virus inoculation in Hooded Lister rats. From these and other results the researchers drew the conclusion that the unique spacer region of Ad12 E1A is an oncogenic determinant of this virus. Since mutations in the spacer region also affect the 52R protein we started to characterize this Ad12 E1A-specific protein in detail.

First characterization of the 52R protein revealed that it contains a striking structure in its second exon: a basic region showing a high homology with one of the DNA binding subregions of the proteins of the Fos family (Brockmann et al., 1993), followed by a cysteine-rich motif of the type C XC XC XC (C = cysteine, X = other amino acid). We have shown that the 52R protein has a nonspecific DNA-binding activity for which both domains are essential (Brockmann et al., 1993). Moreover, using transient expression assays we could demonstrate that the 52R protein represses the trans-activating function of the cellular transcription factor AP-1 (Brockmann et al., 1994).

The AP-1 complex consists mainly of proteins encoded by the different genes of the jun and fos families (for reviews, see Curran and Franza (1988) and Angel and Karin (1991)). After forming homo- (Jun-Jun; c-Jun/AP-1) or heterodimers (Jun-Fos; c-Jun-c-Fos/AP-1) they bind to the TRE elements in the promoter regions of specific target genes ( e.g. the human collagenase gene) and induce their expression (Angel et al., 1987; Abate and Curran, 1990). For transcriptional activity c-Jun has to be stimulated by phosphorylation at two sites which are located in its trans-activation domain: Ser-63 and Ser-73 (Binetruy et al., 1991; Smeal et al., 1991, 1992). Two specific enzymes belonging to the MAP kinase group, termed JNK1 and JNK2, are responsible for this phosphorylation (Hibi et al., 1993).

Depending on its composition, the activity of the AP-1 complex can be differentially regulated by two E1A proteins. The protein product of the 12 S mRNA represses the trans-activating activity of c-Jun-c-Jun and c-Jun-c-Fos dimers (Offringa et al., 1990), whereas the Ad12 E1A 52R protein represses selectively the activity of c-Jun homodimers (Brockmann et al., 1994). Down-regulation mediated by the protein translated from the 12 S mRNA depends on CR1 and is due to the inhibition of the binding of the transcription factor to its target sequence (Offringa et al., 1990; Hagmeyer et al., 1993). The 52R protein of Ad12 E1A does not contain CR1, indicating that the repression mechanism has to be different. Consistent with this assumption, band shift as well as in vitro footprint analyses have shown that the promoter binding of c-Jun homodimers is not prevented by the 52R protein (Brockmann et al., 1994). Therefore we have analyzed whether the 52R protein blocks the trans-activating activity of c-Jun by interacting physically with the transcription factor complex.

In this report we demonstrate that the Ad12-unique E1A 52R protein binds to all three members of the Jun family of transcription factors (c-Jun, JunB, and JunD). The 52R protein associates with the bZIP regions of the Jun proteins containing the basic DNA-binding domain and the leucine zipper structure. Furthermore, our results show that the 52R protein prevents the phosphorylation of the c-Jun trans-activation domain in vitro, suggesting that the 52R protein regulates c-Jun activity by disturbing its interaction with a specific kinase(s).


MATERIALS AND METHODS

Plasmids

The isolation of the Ad12 E1A-specific 52R protein and the generation of the plasmid pGEX-52R expressing the 52R protein (GST-52R) as fusion protein consisting of the glutathione S-transferase from Schistosoma japonicum (NH terminus) as protein leader sequence and the 52R protein (COOH terminus) was described earlier (Brockmann et al., 1990, 1993).

The c- jun gene, which was obtained from A. J. van der Eb, Laboratory for Molecular Carcinogenesis, University of Leiden, Leiden, The Netherlands, was subcloned into the vector pGEX-2T (Pharmacia Biotech Inc.) to generate the plasmid pGEX-c- jun. The mutant pGEX-c- junTAD (amino acids 1-130), which expresses the c-Jun acidic trans-activation domain as GST fusion protein but which lacks the DNA-binding region and the leucine zipper, was generated by polymerase chain reaction using primers corresponding to nucleotides 1-27 (5`-end) and to nucleotides 371-391 (3`-end, complementary sequence) of the c- jun open reading frame. The c- jun subfragment was cloned via BamHI restriction sites, which were synthesized on the 5`-ends of both primers, into the BamHI cloning site of the vector pGEX-2T. The mutant pGEX-c- junbZIP (amino acids 233-331), which contains the basic region necessary for DNA-binding and the leucine zipper involved in dimerization but which lacks the acidic trans-activation domain of c-Jun, was obtained by isolating an AvaI (nucleotide 1108 of the c- jun gene) (Angel et al., 1988)/ BamHI fragment from pGEX-c- jun. After generating blunt ends the subfragment was cloned into the BamHI site of the vector pGem-3Z (Promega), which was filled in using dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I (Maniatis et al., 1982). From pGem-3Z-c- junbZIP a SmaI/ AccI fragment was isolated and, after generating blunt ends, cloned into the filled in EcoRI site of the vector pGEX-2T. pGEX-c- junLZ, which expresses the c-Jun leucine zipper as GST fusion protein, was cloned by polymerase chain reaction using primers corresponding to nucleotides 832-858 (5`-end) and to nucleotides 969-996 (3`-end, complementary sequence; amino acids 278-331) of the c- jun open reading frame. pGEX-c- junDB expressing the basic DNA-binding domain of c-Jun as GST fusion protein was cloned in the same way using primers corresponding to nucleotides 748-765 (5`-end) and to nucleotides 816-837 (3`-end; complementary sequence; amino acids 250-279). The 3`-end primer carries two stop codons at its end. The human junB and the mouse junD genes were obtained from J. Schütte, University of Essen Medical School, Essen, FRG. The plasmids pGEX-c-Jun1/223Ala and pGEX-c-Jun56/223 were a gift of M. Hibi and M. Karin, University of California, San Diego, CA.

The vector pGEX-(128/129) was a gift of M. A. Blanar, University of California, San Francisco, CA. This vector is a derivat of pGEX-2T and expresses a fusion protein consisting of the GST leader sequence, the recognition site for the anti-FLAG antibody (FLAG) and the recognition element for the catalytic domain of HMK which allows efficient phosphorylation of fusion proteins by HMK (Blanar and Rutter, 1992; Ron and Dressler, 1992). To obtain pGEX-(128/129)-52R the cDNA of the 52R protein was cloned in frame in the single EcoRI site following the coding region for the recognition element of the HMK. To obtain the fusion protein 6xHis-52R, which contains 6 histidine residues in front of the 52R protein, the 9.5 S cDNA was cloned into the BamHI site of the vector pQE-8 (Qiagen). Induction of the fusion protein expression and its purification via nickel-chelate affinity chromatography were performed as described by Qiagen.

pTM-hTFIID, coding for the human TATA box-binding protein TBP, was a gift of A. Berk, University of California, Los Angeles, CA.

The correct reading frame of the 52R fusion protein as well as of the Jun mutants was confirmed by sequencing using the automated laser fluorescent DNA sequencer of Pharmacia.

Cell Culture and Preparation of Whole Cell Extracts

HeLa cells and F9 embryonal carcinoma stem cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Jurkat cells were grown in RPMI 1640 supplemented with 10% fetal calf serum. Whole cell extract of TPA-treated Jurkat cells (50 ng/ml, 30 min) was prepared as described by Hibi et al. (1993).

CAT Assays

For transfection of F9 embryonal carcinoma stem cells, Petri dishes were precoated with 0.2% gelatin. 24 h after seeding the cells, cells were transfected (Graham and van der Eb, 1973; Parker and Stark, 1979) with 2 µg of the reporter plasmid, 2 µg of a mouse c- jun expression plasmid (pRc/RSV-m-c- jun), and up to 6 µg of the 52R expression plasmid (pRc/RSV-9.5S). 40 h after transfection, cells were harvested and lysed by freeze-thaw treatments. The protein concentration of the cellular extracts was measured using the Bradford (1976) method. Equal amounts of proteins were used to determine CAT activity (Gorman et al., 1982). CAT activity was quantified by liquid scintillation counting of the C spots on thin-layer chromatography plates.

In Vitro Transcription/Translation

In vitro transcription/translation was performed using the TNT-coupled reticulocyte lysate system of Promega and T7 or SP6 RNA polymerase, depending on the vector and orientation of the cloned insert.

Far Western Analyses

Crude extracts prepared from 1 ml of bacteria culture expressing the respective GST fusion protein were separated on 12.5% SDS-polyacrylamide gels. After gel electrophoresis proteins were electroblotted (Harlow and Lane, 1988) onto Hybond-C membranes (Amersham Corp.). Denaturing, renaturing, hybridization, and washing was performed as described by Ron and Dressler (1992).

As radioactively labeled probe, the 52R protein, which was cloned into the vector pGEX-(128/129), was used. For kinasing (Blanar and Rutter, 1992; Ron and Dressler, 1992) GST-(128/129) or GST-(128/129)-52R were purified as described earlier (Brockmann et al., 1993). Thereafter the glutathione-Sepharose-bound fusion proteins (10 µg) were incubated for 1 h at 37 °C in 60 µl of HMK buffer (20 mM Tris/HCl, pH 7.5, 1 mM dithiothreitol, 0.1 M NaCl, 12 mM MgCl), containing 6 µl of [-P]ATP (>185 TBq/mmol, Amersham) and 30 units of heart muscle kinase (Sigma). Free radioactivity and HMK were removed by washing three times with 1 ml of phosphate-buffered saline containing 5 mM NaF. Radioactively labeled fusion proteins were eluted two times with 150 µl of elution buffer (50 mM Tris/HCl, pH 8.0, 10 mM reduced glutathione, 1 mM dithiothreitol, 5 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml bovine serum albumin) by rocking for 30 min at room temperature. Usually 1 10 cpm of the labeled fusion proteins were used for 1 ml of hybridization solution.

Protein Binding Assay

Equal molar amounts of the different fusion proteins were used for the binding assays. The GST fusion proteins were coupled to glutathione-Sepharose 4B beads (Pharmacia) and incubated with up to 1 10 cpm of in vitro translated Jun or TBP in incubation buffer (10 mM Tris/HCl, pH 7.4, 5 mM MgCl, 50 mM NaCl, 0.5% Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin) containing 1.8 µg/µl bovine serum albumin. After 1 h at 4 °C the beads were washed five times with 500 µl of lysis buffer. For analysis of bound proteins, the beads were boiled in 1 Laemmli sample buffer and loaded onto 12.5% SDS-polyacrylamide gels (Laemmli, 1970). Labeled proteins were visualized by fluorography. For protein binding inhibition experiments, the protein binding assays were performed in the presence of a 10-fold molar excess of 6xHis-52R (compared to GST-c-Jun). Bovine serum albumin was added to the control reactions to keep the protein concentration constant in each assay.

Solid-phase Kinase Assay

The solid-phase kinase assays were performed as described by Hibi et al. (1993). For kinase inhibition experiments the fusion protein 6xHis-52R was added at 20-fold molar excess to the reaction solution (compared to GST-c-Jun). Bovine serum albumin was added to the control reactions to keep the protein concentration constant in each assay.


RESULTS

The Adenovirus Type 12 E1A 52R Protein Suppresses the trans-Activating Potential of the Cellular Transcription Factor Complex c-Jun-c-Jun

We have shown recently that the 52R protein down-regulates the trans-activation function of the cellular transcription factor c-Jun (Brockmann et al., 1994). To study the repression in more detail we performed transient expression assays in F9 mouse embryonal carcinoma cells lacking endogenous c-Jun activity (Chiu et al., 1988, 1989). Consequently, the expression of a reporter plasmid, whose expression is driven by the c-Jun binding site of the human collagenase promoter (Col-TRE/TK-CAT; Fig. 1A) is undetectable in these cells (Fig. 1 B). Co-transfection of 2 µg of the mouse c- jun expression plasmid pRc/RSV-m-c- jun results in a 12-fold activation of the TRE-mediated CAT-enzyme activity (Fig. 1 B). This trans-activation function of c-Jun is repressed by the 52R protein (Fig. 1 B) after co-transfection of a respective expression vector (pRc/RSV-9.5S; Fig. 1A). Down-regulation of c-Jun activity is dosage-dependent. In the highest concentration used (6 µg pRc/RSV-9.5S) the c-Jun-dependent CAT enzyme expression is repressed approximately 5-fold (Fig. 1 B). pRc/RSV lacking the 52R cDNA has no influence on CAT expression from the reporter plasmid Col-TRE/TK-CAT (Fig. 1 B).


Figure 1: The Ad12-specific 52R protein represses the trans-activating function of c-Jun. A, expression plasmids and reporter construct. In pRc/RSV-9.5S and pRc/RSV-m-c- jun, the expression of the Ad12 E1A 9.5S cDNA ( 9.5S) or the mouse c- jun cDNA ( m-c-jun) is driven by the Rous sarcoma virus long terminal repeat ( RSV/LTR). BGH, bovine growth hormone polyadenylation signal. In the reporter construct Col-TRE/TK-CAT, the CAT gene is driven by the thymidine kinase promoter of the herpes simplex virus ( TK, nucleotides -105 to +51) and one copy of the TRE element of the human collagenase gene ( Col-TRE, nucleotides -65 to -73). B, determination of CAT enzyme activity in cellular extracts obtained from F9 embryonal carcinoma stem cells. 2 µg of the Col-TRE/TK-CAT reporter construct were co-transfected with 2 µg of a mouse c- jun expression plasmid ( pRc/RSV-m-c-jun) and increasing amounts of a 52R expression plasmid ( pRc/RSV-9.5S) as indicated. pRc/RSV DNA was added to balance the total amount of the transfected DNA to 8 µg/dish. The results are an average of three independent experiments.



The 52R Protein Associates Physically with the bZIP Structure of c-Jun

Studying the repression mechanism we found that the 52R protein does not prevent the binding of c-Jun to its promoter target sequence (Brockmann et al., 1994). Therefore we speculated that the 52R protein might associate directly with c-Jun and disturb in this way the interaction between c-Jun and factors essential for transcriptional activation. To examine this possibility we performed far Western blot analyses. Protein lysates prepared from bacteria expressing the fusion protein GST-c-Jun (Fig. 2 B, lane 5) or the leader sequence GST (Fig. 2 B, lane 3) were resolved on a 12.5% SDS-polyacrylamide gel and blotted onto a nitrocellulose membrane. Bacterial lysates which do not contain the GST leader sequence or the GST-c-Jun fusion protein were used as controls (Fig. 2 B, lanes 2 and 4). Hybridization was performed with the P-labeled probe GST-(128/129)-52R. GST-(128/129) is a derivat of pGEX-2T and expresses a fusion protein consisting of the GST leader sequence and the phosphorylation site for HMK, which allows efficient phosphorylation of fusion proteins by HMK (Blanar and Rutter, 1992; Ron and Dressler, 1992). Control gels revealed that the affinity-purified probes were more than 95% pure (Fig. 2 D, lanes 4 and 7).


Figure 2: The 52R protein binds to the bZIP structure of c-Jun. A, schematic representation of the fusion proteins used for far Western blots. GST-c-Jun consists of the carboxyl terminus of the glutathione S-transferase as leader sequence ( GST) and the c-Jun protein ( c-Jun). In the c-Jun protein the acidic trans-activation domain ( TAD; hatched box), the basic DNA-binding region ( DB; vertically striped box) and the leucine zipper structure ( LZ; black box) are indicated. GST-c-JunTAD lacks the bZIP structure necessary for DNA-binding of c-Jun; GST-c-JunbZIP lacks the acidic trans-activation domain of c-Jun. GST-c-JunDB contains only the basic DNA binding region of c-Jun; GST-c-JunLZ contains only the c-Jun leucine zipper. The probe GST-(128/129)-52R consists of the GST leader sequence, the recognition site for the catalytic domain of heart muscle kinase, which allows efficient phosphorylation of the fusion protein by HMK, and the Ad12 E1A 52R protein (52R). B, bacterial lysates containing the induced (+) or noninduced (-) protein leader sequence glutathione S-transferase (GST; lanes 2 and 3), the fusion protein GST-c-Jun ( lanes 4 and 5), the fusion protein GST-c-JunTAD carrying the acidic trans-activation domain of c-Jun ( lanes 6 and 7), the fusion protein GST-c-JunbZIP carrying the basic DNA binding region and leucine zipper structure of c-Jun ( lanes 8 and 9), the fusion protein GST-c-JunLZ carrying the leucine zipper structure of c-Jun ( lanes 10 and 11) or the fusion protein GST-c-JunDB carrying the basic DNA-binding domain of c-Jun ( lanes 12 and 13) were separated on a 12.5% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane 1 represents the molecular mass marker, the sizes of the marker proteins are indicated on the left. The induced fusion proteins are marked by arrowheads. C, autoradiography of a far Western blot analysis of the gel described under B. The blot was hybridized with P-labeled GST-(128/129)-52R. The sizes of the molecular mass marker proteins are indicated on the left. The positions of the induced fusion proteins are marked by arrowheads. D, control of the purified hybridization probes GST-(128/129) ( lane 4; p) and GST-(128/129)-52R ( lane 7; p) on a 12.5% SDS-polyacrylamide gel after staining with Coomassie Blue. Lanes 2 and 3 represent bacterial lysates containing the uninduced (-) or induced (+) GST-(128/129) leader sequence, lanes 5 and 6 represent bacterial lysates containing the uninduced (-) or induced (+) fusion protein GST-(128/129)-52R; the induced fusion proteins are marked by arrowheads. Lane 1 represents the molecular mass marker, the sizes of the marker proteins are indicated on the left.



As shown in Fig. 2 C, GST-(128/129)-52R hybridizes to the GST-c-Jun fusion protein ( lane 5) but not to the GST leader sequence ( lane 3) or to lysate proteins obtained from uninduced bacteria ( lanes 2 and 4), indicating that the interaction is due to a specific association of the 52R protein with c-Jun. To exclude completely that the GST leader sequence is somehow involved in this interaction, we performed a far Western blot with radioactively labeled GST-(128/129) protein. No hybridization signals were obtained in these experiments (data not shown). These results clearly show that the 52R protein is able to bind directly and specifically to the cellular transcription factor c-Jun.

To identify the domain of c-Jun involved in the association with the 52R protein we hybridized a far Western blot carrying two c-Jun deletion mutants (GST-c-JunTAD, GST-c-JunbZIP; Fig. 2 A) with GST-(128/129)-52R. GST-c-JunTAD expresses the acidic trans-activation domain of c-Jun while GST-c-JunbZIP contains the basic DNA-binding domain and the leucine zipper structure of the transcription factor. As shown in Fig. 2 C, the 52R protein associates with the bZIP DNA binding domain of c-Jun ( lane 9) but not with its acidic trans-activation domain ( lane 7). Hybridization to GST-c-JunbZIP revealed two signals (Fig. 2 C, lane 9). The slower migrating protein represents the full-length GST-c-JunbZIP fusion protein, whereas the smaller protein might be either a degradation product of the Jun mutant or a protein generated by a preterminal stop due to the presence of a codon(s) infrequently utilized in the bacteria (Sambucetti et al., 1986; Sharp et al., 1988).

A further subdivision of the c-Jun bZIP domain into mutants containing either the leucine zipper (GST-c-JunLZ; Fig. 2 A) or the basic DNA binding domain (GST-c-JunDB; Fig. 2 A) revealed surprising results. Neither GST-c-JunLZ nor GST-c-JunDB on its own were able to interact with the 52R protein (Fig. 2 C, lanes 11 and 13). Several reasons might be responsible for this finding. First of all, dimerization of c-Jun might be a prerequisite for the physical association with the 52R protein. If this is true, no interaction can be observed using the mutant GST-c-JunDB which is unable to form dimers due to the lack of the leucine zipper structure. This explanation is supported by our finding that addition of bacterially expressed and affinity-purified GST-c-Jun to the hybridization solution of a far Western blot carrying the full-length GST-c-Jun fusion protein and the mutant GST-c-JunbZIP revealed more intensive hybridization signals as compared to that found in experiments without soluble GST-c-Jun (data not shown). In these assays addition of soluble GST-c-Jun results in a larger amount of c-Jun homodimers formed between the membrane-bound GST-c-Jun and the soluble c-Jun fusion protein as compared to the amount of c-Jun homodimers obtained through the renaturation of the far Western blot. On the other hand we cannot exclude that the small c-Jun domains used in these assays do not have the correct three-dimensional structure to bind to the 52R protein or that the 52R protein has two contact points in the bZIP domain: one is located in the DNA binding region, a second in the leucine zipper structure. Both of these binding sites might be necessary to form stable c-Jun-52R complexes.

The Ad12 E1A 52R Protein Binds to All Three Members of the Jun Family of Transcription Factors

The Jun family of cellular transcription factors consists of three members, c-Jun, JunB, and JunD, which are all highly conserved in their carboxyl termini (Angel et al., 1988; Hirai et al., 1989; Schütte et al., 1989). Therefore we have tested whether JunB and JunD might also be potential protein binding partners for the 52R protein.

A protein binding assay (Fig. 3 A) using in vitro translated JunB revealed that the 52R protein associates with JunB ( lane 5). In this experiment GST-c-Jun was used as a positive control as it forms efficiently heterodimers with JunB (Nakabeppu et al., 1988; Karin, 1991) (Fig. 3A, lane 6). No binding was observed using either the GST leader sequence (Fig. 3 A, lane 4) or the glutathione-Sepharose beads alone (Fig. 3 A, lane 3).


Figure 3: The 52R E1A protein associates with JunB and JunD. A, S-labeled, in vitro translated JunB was incubated either with glutathione-Sepharose beads ( GS-Beads, lane 3), the protein leader sequence glutathione S-transferase immobilized on GS beads ( GST, lane 4), the fusion protein GST-52R consisting of the GST leader sequence and the adenoviral 52R E1A protein immobilized on GS beads ( lane 5) or the fusion protein GST-c-Jun immobilized on GS beads ( lane 6). Bound material was analyzed on a 12.5% SDS-polyacrylamide gel and detected by fluorography. Lane 1 represents 20% of the input of the in vitro translated JunB per assay ( ivt JunB); lane 2 represents the molecular mass marker, the sizes of the marker proteins are indicated on the left. The full length JunB protein is marked on the right. B, bacterial lysates containing the induced (+) or uninduced (-) protein leader sequence glutathione S-transferase ( GST; lanes 2 and 3), the fusion protein GST-c-Jun ( lanes 4 and 5) or the fusion protein GST-(128/129)-JunDbZIP carrying the carboxyl-terminal end of the JunD protein ( GST-JunDbZIP; lanes 6 and 7) were separated on a 12.5% SDS-polyacrylamide gel and stained with Coomassie Blue. Lane 1 represents the molecular mass marker, the sizes of the marker proteins are indicated on the left. The induced fusion proteins are marked by arrowheads. C, autoradiography of a far Western blot analysis of the gel described for B. The blot was hybridized with P-labeled GST-(128/129)-52R. The sizes of the molecular mass marker proteins are indicated on the left. The positions of the induced fusion proteins are marked by arrowheads.



As seen in Fig. 3A, lane 1, in vitro translation of JunB gives rise to several polypeptides. The slower migrating band represents the full-length JunB protein, whereas the three other signals might be due to either degradation products of JunB or the usage of internal start codons in the in vitro translation reaction.

To study the interaction of the Ad12-unique 52R E1A protein with JunD and to confirm our data demonstrating that the association of the 52R polypeptide with the different Jun proteins is mediated through the Jun bZIP regions, we constructed a fusion protein consisting of the GST leader sequence and the bZIP DNA-binding domain of JunD (GST-JunDbZIP). A bacterial extract containing GST-JunDbZIP (Fig. 3 B, lane 7) was blotted onto a nitrocellulose membrane and incubated with radioactively labeled GST-(128/129)-52R. As shown in Fig. 3 C, GST-(128/129)-52R forms efficiently complexes with GST-JunDbZIP ( lane 7) and GST-c-Jun ( lane 5); no interaction was observed with the GST leader sequence (Fig. 3 C, lane 3) or with proteins of lysates obtained from uninduced bacteria ( lanes 2, 4, and 6). Taken together our results demonstrate that the 52R protein of Ad12 E1A binds to bZIP regions of all three members of the Jun family of cellular transcription factors.

The 52R Protein Does Not Inhibit the Binding of c-Jun to the TATA Box-binding Protein TBP

It is commonly believed that transcriptional activators such as c-Jun stimulate transcription through direct and/or indirect interactions between the activator protein complex and members of the basal transcriptional machinery. According to this model it was shown by Ransone et al. (1993) that the bZIP region of c-Jun binds to a 51-residue region (amino acids 221-271) of the carboxyl-terminal domain of TBP. The basic region as well as leucine zipper structure of c-Jun are necessary for this interaction. As the 52R protein binds also to the bZIP structure of c-Jun (Figs. 2 C and 3 C), it is reasonable to speculate that the adenovirus protein might block the c-Jun binding to TBP and repress in that way the activity of the cellular transcription factor. To test this assumption we performed protein binding assays in which immobilized GST-c-Jun fusion protein was incubated with in vitro translated S-labeled TBP. As described earlier by Ransone et al. (1993), the in vitro translated TBP binds to the GST-c-Jun fusion protein (Fig. 4 A, lane 7; unfortunately, the gel was cracked during drying). No unspecific binding was observed using the GST leader sequence coupled to the glutathione-Sepharose beads ( lanes 3 and 4). Moreover, the 52R protein also did not bind to the TATA box-binding protein ( lanes 5 and 6). Addition of a 10-fold molar excess of the 52R protein to the GST-c-Jun/TBP protein binding assay does not inhibit the interaction between the cellular transcription factor and TBP ( lane 8).


Figure 4: The 52R protein does not inhibit the physical interaction between c-Jun and TBP. A, S-labeled, in vitro translated human TATA box-binding protein TBP was incubated either with the protein leader sequence glutathione S-transferase immobilized on GS beads ( GST, lanes 3 and 4), the fusion protein GST-52R consisting of the GST leader sequence and the adenoviral 52R E1A protein immobilized on GS beads ( lanes 5 and 6) or the fusion protein GST-c-Jun immobilized on GS beads ( lanes 7 and 8) in the absence (-; lanes 3, 5, and 7) or presence (+; lanes 4, 6, and 8) of a 10-fold molar excess of the fusion protein 6xHis-52R. Bound material was analyzed on a 12.5% SDS-polyacrylamide gel and detected by fluorography (unfortunately the gel was cracked during drying). Lane 1 represents 20% of the input of the in vitro translated TBP protein per assay ( T); lane 2 represents the molecular mass marker ( M), the sizes of the marker proteins are indicated on the left. The position of the TBP protein is marked on the right. B, control of the protein amount used in the protein binding assays in A. Fusion proteins were separated on a 12.5% SDS-polyacrylamide gel and detected by staining with Coomassie Blue. Lanes 3 and 4 contain the GST leader sequence; lanes 5 and 6 contain the fusion protein GST-52R; lanes 7 and 8 contain the fusion protein GST-c-Jun. M ( lane 2), molecular mass marker; the sizes of the marker proteins are indicated on the left.



To control the amount of fusion proteins used in the protein binding assays the gel shown in Fig. 4A was Coomassie-stained before drying (Fig. 4 B). Fig. 4 B documents that nearly equal molar amounts of fusion proteins were used in the experiments described above. These results demonstrate that the 52R protein most probably does not interrupt the physical interaction between c-Jun and TBP. On the other hand, our data do not exclude that TBP and the 52R protein bind to distinct populations of the GST-c-Jun substrate in the in vitro binding assays. For example, it might be possible that TBP can bind monomeric as well as dimeric c-Jun, whereas the 52R protein requires c-Jun-c-Jun homodimers for binding. In this case we would detect monomeric c-Jun/TBP complexes, whereas the formation of a c-Jun-c-Jun/TBP complex, which would be functional in vivo, is inhibited by the 52R protein. Experiments are under way to analyze whether c-Jun, TBP, and the 52R protein are able to form trimeric complexes.

The 52R Protein Prevents Phosphorylation of the c-Jun trans-Activation Domain

The transcriptional activity of c-Jun is stimulated by phosphorylation at Ser-63 and Ser-73 within its NH-terminal activation domain (Binetruy et al., 1991; Smeal et al., 1991, 1992). Recently it was shown that two members of the MAP kinase group, termed JNK1 and JNK2, bind to this region (amino acids 33-79) and phosphorylate both residues (Hibi et al., 1993; Dérijard et al., 1994).

To test the hypothesis that the 52R protein might interfere with the activation pathway of c-Jun through JNKs, we performed solid-phase kinase assays as described by Hibi et al. (1993). Cellular extracts were prepared from TPA-treated Jurkat cells expressing high levels of JNKs and incubated with GST-c-Jun fusion proteins (immobilized on glutathione-Sepharose beads) in the presence or absence of a 20-fold molar excess of a 6xHis-52R fusion protein. After complex formation the reaction mixtures were incubated with [-P]ATP for 20 min. The phosphorylation reaction was terminated by extensive washing, and bound material was eluted and analyzed on SDS-polyacrylamide gels.

The GST-c-Jun fusion protein was only slightly phosphorylated using extracts from untreated Jurkat cells (Hibi et al., 1993) (Fig. 5 A, lane 3). This basal phosphorylation might be due to the presence of several different kinases in the extracts obtained from untreated Jurkat cells, e.g. extracellular signal-related kinases (Dérijard et al. (1994) and references therein), casein kinase II (Lin et al., 1992), and/or a small portion of active JNKs. Treatment of Jurkat cells with TPA activates the Jun-specific JNKs which phosphorylate c-Jun very efficiently (Hibi et al. 1993) (Fig. 5 A, lane 7). As expected, the GST leader sequence is not phosphorylated under these conditions (Fig. 5 A, lanes 1, 2, 5, and 6) demonstrating that the c-Jun part of the fusion protein is specifically phosphorylated in these assays. Most interestingly, addition of the 6xHis-52R fusion protein to the extract prepared from TPA-treated Jurkat cells results in the prevention of the phosphorylation of GST-c-Jun (Fig. 5 A, lane 8), whereas the basal phosphorylation is only very slightly affected (Fig. 5 A, lane 4). These results indicate that the 52R protein inhibits the phosphorylation of c-Jun by JNKs.


Figure 5: The 52R protein inhibits the phosphorylation of c-Jun. A, whole cellular extracts of TPA-untreated (-) or TPA-treated (+) Jurkat cells were incubated with the protein leader sequence glutathione S-transferase immobilized on GS beads ( GST, lanes 1, 2, 5, and 6) or the fusion protein GST-c-Jun consisting of the GST leader sequence and the c-Jun protein immobilized on GS beads ( lanes 3, 4, 7, and 8) in the absence ( lanes 1, 3, 5, and 7) or presence ( lanes 2, 4, 6, and 8) of a 20-fold molar excess of the fusion protein 6xHis-52R. After 3 h of incubation, followed by extensive washing, the solid-phase kinase assays were performed as described by Hibi et al. (1993). Bound phosphorylated material was analyzed on a 12.5% SDS-polyacrylamide gel and detected by autoradiography. The positions of the marker proteins are indicated on the left. The positions of the GST-c-Jun and GST proteins are indicated on the right. B, control of the fusion protein amount used in the solid-phase kinase assays as described in A. Fusion proteins were separated on a 12.5% SDS-polyacrylamide gel and detected by staining with Coomassie Blue. Lanes 1, 2, 5, and 6 contain the GST leader sequence; lanes 3, 4, 7, and 8 contain the fusion protein GST-c-Jun. The positions of the marker proteins are indicated on the left, the positions of the fusion proteins GST-c-Jun and GST are indicated on the right. C, phosphorylation of GST-c-Jun is due to a c-Jun amino-terminal protein kinase(s). Whole cellular extracts of TPA-untreated (-) or TPA-treated (+) Jurkat cells were incubated with the c-Jun mutants GST-c-Jun1/223Ala lacking the main phosphoacceptor sites (Ser-63 and Ser-73; lanes 1 and 2) and GST-c-Jun56/223 lacking amino acids 1-55 of c-Jun which are necessary for the physical interaction with JNKs ( lanes 3 and 4) as described in A. Bound phosphorylated material was analyzed on a 12.5% SDS-polyacrylamide gel and detected by autoradiography. The positions of the marker proteins are indicated on the left. The positions of the full-length GST-c-Jun mutants are marked by arrowheads. D, control of the fusion protein amount used in the solid-phase kinase assays as described in C. Fusion proteins were separated on a 12.5% SDS-polyacrylamide gel and detected by staining with Coomassie Blue. Lanes 1 and 2 contain the c-Jun mutant GST-c-Jun1/223Ala; lanes 3 and 4 contain the c-Jun mutant GST-c-Jun56/223. The positions of the marker proteins are indicated on the left, the positions of the full-length fusion proteins are marked by arrowheads. The faster migrating bands are degradation products (Hibi et al., 1993).



A second signal representing a protein with a molecular mass of 135 kDa can be seen in some lanes of the autoradiography presented in Fig. 5A. This band is not reproducible and most probably due to the phosphorylation of a protein which binds sometimes unspecifically to the glutathione-Sepharose beads or the GST leader sequence.

To confirm our assumption that GST-c-Jun is phosphorylated through JNKs in the experiments described above, we analyzed two c-Jun mutants in solid-phase kinase assays: 1) GST-c-Jun56/223, which lacks the binding site for the JNKs. This mutant cannot be phosphorylated by JNKs (Hibi et al., 1993; Dérijard et al., 1994) and 2) GST-c-Jun1/223Ala, which lacks the main phosphoacceptor sites (Ser-63, Ser-73). This mutant can be phosphorylated on Thr residues around position 90 (the T1 and T2 sites) (Hibi et al., 1993; Dérijard et al., 1994). As described by Hibi et al. (1993) and Derijard et al. (1994) GST-c-Jun1/223Ala is phosphorylated by the TPA-induced c-Jun-specific kinases (Fig. 5 C, lane 2). This mutant as well as the mutant GST-c-Jun56/223 gives rise to full-length fusion proteins as well as several degraded products if expressed in bacteria (Fig. 5 D, lanes 1-4). This is in accordance with data published by Hibi et al. (1993). Interestingly, not the full-length GST-c-Jun1/223Ala mutant but one of the faster migrating truncated fusion proteins is phosphorylated (compare Fig. 5, C, lane 2, with D, lane 2). The reason for this finding is not yet clear. GST-c-Jun56/223 is not phosphorylated at all (Fig. 5 C, lanes 3 and 4). Taken together our results strongly suggest that the 52R protein inhibits the phosphorylation of the c-Jun trans-activation domain by members of the JNK class of protein kinases.


DISCUSSION

In this study we show that the in vivo repression of the trans-activating activity of c-Jun by the Ad12 E1A 52R protein correlates with a physical interaction between the adenoviral protein and the cellular transcription factor in vitro. Unexpectedly, binding of the 52R protein does not occur to the TAD domain of c-Jun but to its bZIP structure necessary for dimerization and DNA binding. This association does neither prevent the promoter binding of the c-Jun homodimer nor the binding of c-Jun to the TATA box-binding protein TBP. In fact, the experiments presented here show that the 52R protein prevents the phosphorylation of the c-Jun activation domain by specific kinases, most probably by the c-Jun amino-terminal protein kinases, JNKs. From these data we draw the following model (Fig. 6). Depending on the TRE element, c-Jun is bound to the promoter region of a specific target gene (Hagmeyer et al., 1993) but does not induce its transcription due to the lack of the activation of its trans-activation domain. Activation occurs in response to a variety of extracellular stimuli through phosphorylation at Ser-63 and Ser-73 by JNK kinases. Expression of the 52R protein in these cells leads to its binding to c-Jun. This interaction results in the inhibition of the phosphorylation of the c-Jun TAD and is therefore responsible for the repression of the c-Jun trans-activation function. As the 52R protein binds to the bZIP structure of the cellular transcription factor, the repression mechanism is not due to a competition between the 52R protein and JNKs for the JNK-binding site in c-Jun, which spans amino acids 33-79 (Hibi et al., 1993). The physical interaction between c-Jun and the 52R protein is most probably responsible for a conformational change of the c-Jun protein which does not allow the binding of JNKs to c-Jun, a prerequisite for phosphorylation (Hibi et al., 1993), or the phosphorylation of the c-Jun TAD domain. On the other hand alternative mechanisms are still possible. It might be possible, for example, that the 52R protein binds to the JNKs, too, and prevents in this way their association with c-Jun or that the 52R protein somehow inhibits the phosphorylation reaction. Both mechanisms cannot be completely excluded. To confirm our model, phosphopeptide analyses are planned of c-Jun prepared from wild type 52R expressing cells versus cells expressing a mutant 52R protein that no longer represses c-Jun activity.


Figure 6: Model describing the mechanism by which the Ad12 E1A 52R protein might repress the trans-activating activity of c-Jun/AP-1. A c-Jun homodimer ( open symbols, acidic trans-activation domain; black box, basic DNA binding region; striped box, leucine zipper structure) is bound to its target region ( TRE) in the promoter of a specific gene but does not induce its expression due to the lack of the TAD activation. Activation of c-Jun occurs in response to a variety of extracellular stimuli through phosphorylation on Ser-63 and Ser-73 by JNK kinases. The amino-terminally phosphorylated c-Jun then interacts with the general transcription machinery and thus induces the expression of the target gene. Expression of the 52R protein in these cells leads to its binding to c-Jun. This interaction results in the inhibition of the phosphorylation of the c-Jun activation domain and is therefore responsible for the repression of the c-Jun trans-activation function.



A very interesting result of our and the studies of other groups is that the c-Jun bZIP domain is not simple a dimerization and DNA-binding motif but participates in several protein/protein interactions responsible for modulating c-Jun transcriptional activity. For example, this region mediates a physical association with TBP as shown by Ransone et al. (1993). The authors speculate that this interaction is in part responsible for recruiting TBP to form complexes to initiate RNA synthesis. Liu and Green (1994) have shown recently that the protein translated from the 13 S mRNA of E1A binds also to the c-Jun bZIP structure through its conserved region 3. This association leads to the trans-activation of a respective reporter construct. Furthermore, our results show that the bZIP structure can also function as a target for repressor molecules. All of these results demonstrate that the bZIP structure of c-Jun is a multifunctional domain which is involved in DNA binding (Gentz et al., 1989; Turner and Tjian, 1989), homo- and heterodimerization (Landschulz et al., 1988; Gentz et al., 1989; O'Shea et al., 1989), and transcriptional activation and repression (Ransone et al., 1993; Liu and Green, 1994) (this study).

Is there a correlation between the repression of the c-Jun transcriptional activity by the 52R protein and the oncogenicity of Ad12? Yamit-Hezi et al. (1994) have shown recently that c-Jun is involved in the regulation of MHC class I expression in D122 cells, a high metastatic cell line derived from 3LL carcinoma cells. MHC class I proteins present fragmented antigens on the cell surface to cytotoxic T lymphocytes which results in the elimination of, e.g. virus-infected cells. Therefore it is assumed that down-regulation of MHC class I gene expression is one factor involved in oncogenic transformation and tumor induction by Ad12 (Bernards et al., 1983). Due to these results one can speculate that the 52R protein is involved in oncogenic transformation by Ad12 through down-regulating MHC class I expression. However, in various cell types transcription of the MHC class I genes is controlled mainly by the class I regulatory element (Kimura et al., 1986; Baldwin and Sharp, 1987) which is among others the target sequence for the transcription factor NF-B (Isral et al., 1987; Baldwin and Sharp, 1988). Meijer et al. (1992) observed that the protein product of the Ad12 E1A 13 S mRNA reduces the DNA-binding activity of NF-B in baby rat kidney cell lines and represses in that way the MHC class I expression. The protein product of the 13 S mRNA of E1A of the non-oncogenic subtype Ad5 is unable to inhibit the DNA binding of NF-B. These results suggest that the oncogenicity of Ad12 might be due to a very efficient repression of MHC class I gene expression by two or more Ad12 E1A proteins in at least some cell systems. Currently we are cloning Ad12 E1A point mutants unable to express the 52R protein. These mutants will be tested for their oncogenic and tumorigenic potentials. In addition, co-transfection of c- jun and 52R expression plasmids with a MHC class I promoter construct will show whether the 52R can inhibit MHC class I gene expression induced by c-Jun.

An interesting feature of the Ad12 E1A gene is the expression of multiple proteins regulating gene expression activated by transcription factor complexes containing the c-Jun protein. c-Jun/AP-1 is repressed by the 52R protein and the 235R protein while c-Jun-c-Fos/AP-1 is repressed only by the 235R protein (Offringa et al., 1990; Brockmann et al., 1994). In contrast to the 52R protein, the 235R protein blocks the DNA binding of c-Jun homo- or heterodimers in a CR1-dependent mechanism (Offringa et al., 1990). Inhibition of promoter binding occurs without altering post-translational modifications in the DNA binding region of c-Jun (phosphorylation, redox regulation). Therefore it is speculated that this E1A protein influences the interaction of c-Jun with other cellular factors necessary for transcriptional activation (Hagmeyer et al., 1993). The advantage for Ad12 to express two proteins (the 235R and the 52R protein) with, in some cases, similar functions is not yet clear. Nevertheless it might be beneficial for the virus to modulate the activity of related transcription factor complexes specifically by two or more proteins during the productive infection of different cell types, cells at different stages of differentiation, and/or during the process of oncogenic transformation.


FOOTNOTES

*
This work was supported by the Deutsche Forschungsgemeinschaft through SFB 354/TP3 and through the Fonds der Chemischen Industrie. 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.

§
To whom correspondence should be addressed. Tel.: 49-201-7233687; Fax: 49-201-7235974.

The abbreviations used are: E1A, early region 1A oncogene; Ad, adenovirus; Ad5, adenovirus serotype 5; Ad12, adenovirus serotype 12; AP-1, cellular activator protein 1; bZIP, basic region/leucine zipper; CAT, chloramphenicol acetyltransferase; CR1, CR2, CR3, conserved regions 1-3; DB, basic DNA binding domain; E1B, early region 1B oncogene; GS, glutathione-Sepharose; GST, glutathione S-transferase; HMK, catalytic domain of heart muscle kinase; JNK, c-Jun amino-terminal protein kinase; LZ, leucine zipper; MAP, mitogen-activated protein kinase; MHC class I genes, major histocompatibility class I genes; TAD, trans-activation domain; TBP, TATA box-binding protein; TPA, 12- O-tetradecanoylphorbol-13-acetate; TRE, TPA-responsive element; R, amino acid residue.


ACKNOWLEDGEMENTS

We thank Barbara Tries and Daniela Schäfer for excellent technical assistance and Ulla Schmücker for valuable help with the automatic sequencing.


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