Recruitment of the Retinoblastoma Protein to c-Jun Enhances Transcription Activity Mediated through the AP-1 Binding Site*

Junko NishitaniDagger §, Toru NishinakaDagger §, Chi-Hong Cheng§, Walter RongDagger , Kazunari K. Yokoyamaparallel , and Robert ChiuDagger §**

From the Dagger  Dental Research Institute/Oral Biology and Medicine, School of Dentistry, § Surgical Oncology, School of Medicine, and the  Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095-1668 and parallel  Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan

    ABSTRACT
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Abstract
Introduction
References

The retinoblastoma susceptibility gene product (RB) is a transcriptional modulator. One of the targets for this modulator effect is the AP-1 binding site within the c-jun and collagenase promoters. The physical interactions between RB and c-Jun were demonstrated by co-immunoprecipitation of these two proteins using anti-c-Jun or anti-RB antisera, glutathione S-transferase affinity matrix binding assays in vitro, and electrophoretic mobility shift assays. The C-terminal site of the leucine zipper of c-Jun mediated the interaction with RB. Although the B-pocket domain of RB alone bound to c-Jun, a second c-Jun binding site in the RB was also suggested. Mammalian two-hybrid-based assay provided corroborative evidence that transactivation of gene expression by RB required the C-terminal region of c-Jun. We conclude that RB enhances transcription activity mediated through the AP-1 binding site. Adenovirus E1A or human papillomavirus E7 inhibits RB-mediated transcription activity. These data reveal that the interactions between these two distinct classes of oncoproteins RB and c-Jun may be involved in controlling cell growth and differentiation mediated by transcriptional regulation.

    INTRODUCTION
Top
Abstract
Introduction
References

The retinoblastoma susceptibility gene product, p105Rb (RB),1 is generally believed to be an important regulator in the control of cell proliferation or differentiation (1-3). Although the biochemical mechanisms of RB function remain unclear, one possibility is that it exerts transcriptional regulation (4). Indeed, several transcription factors have been identified as targets of modulation by RB, which may be directly involved in modification of chromatin structure and results in regulation of a set of genes (5-10) required for controlling cell growth.

Transcription regulation is a control mechanism that is critical for fundamental biological processes, such as cell growth and differentiation. Proteins involved in transcriptional control either bind specific DNA sequences or act as co-activators or adapters forming a complex with the transcription factor. These transcription factors and co-activators are often targeted by viral oncoproteins during oncogenic transformation of cells. For instance, the ability of adenovirus E1A to transform cells is closely associated with its ability to interact with RB. The ability of E1A to transactivate E2F-mediated transcription maps to the conserved regions 1 and 2 (CR1 and CR2) of E1A, both of which are involved in RB binding (11). It has also been reported that E1A represses AP-1 activity (12) and that the ability of E1A to down-regulate AP-1 activity is dependent on CR1. Similarly, the transforming and immortalizing activities of human papillomavirus (HPV) E7 have been mainly attributed to the ability of E7 that binds to RB and related proteins such as p107 and p130 (13, 14). The binding regions of E7 are homologous to the adenovirus E1A and simian virus 40 large antigen RB binding domains (13, 15, 16). These regions of homology include CR1 and CR2, which are present in both the 12 S and 13 S forms of E1A (17).

Many studies have shown that the HPVs associated with malignant lesions readily cause transformation in vitro. Although the E7 gene alone was sufficient for the transformation of established rodent cell lines and to exhibit anchorage-independent growth (18), cooperation with an activated ras oncogene was required for the immortalization and transformation of primary rat cells (19). Mutations at positions 24 and 26 of E7 inhibited binding of RB and transformation of NIH 3T3 cells and rat embryonic fibroblasts (19, 20).

The nuclear proto-oncogene c-jun was originally defined as a cellular homologue of a transforming gene of an avian sarcoma virus 17 (21-23). The products of the c-jun gene form homodimeric or heterodimeric complexes with the products of the fos or fra gene family to regulate transcription of target genes by binding to a specific DNA sequence in the promoter region (24-27). In addition to the transcription regulation, c-jun also has a functional role in cellular proliferation, differentiation, and transformation (reviewed in Ref. 28).

The RB protein localizes in the nucleus and represses the expression of the c-fos gene (29), the gene product of which, Fos, is one component of the heterodimeric transcription factor AP-1 (24, 25). Therefore, it is possible that RB can be functionally linked to c-jun and thus play a role in transcriptional control. Constitutive activation of the nuclear proto-oncogene c-jun leads to increased transcription of a set of genes that generate malignant phenotypes in some cells (30), whereas inactivation of the Rb gene causes many human cancers (31, 32). In addition, these two classes of genes have also been demonstrated to involve in cell differentiation (3, 33).

In the present study, we demonstrate that transient expression of human RB significantly stimulated the expression of the human c-jun and collagenase promoter in murine fibroblast, 3T6, and NIH 3T3 cells. Deletion and mutagenesis analysis reveals that RB transactivates gene expression mediated through the AP-1 binding site. In addition, we demonstrate a direct physical interaction between c-Jun and RB by using co-immunoprecipitation, GST-affinity matrix binding assays, and electrophoretic mobility shift assays (EMSAs). The interaction domains are mapped to the C-terminal region of c-Jun and the B-pocket of RB. Transactivation of c-jun expression by RB is inhibited by the expression of adenovirus E1A or HPV E7, suggesting that the RB-c-Jun complex may involved in controlling cell growth and differentiation.

    EXPERIMENTAL PROCEDURES

Cells and Transient Transfection Assays-- Cells were cultured as follows: NIH 3T3 cells in Dulbecco's modified Eagle's medium (DMEM) with 10% calf serum, 3T6 mouse fibroblasts and CV-1 cells (green monkey kidney cell line) in DMEM with 10% fetal bovine serum, Molt4 human acute lymphoblastic leukemia cells in RPMI 1640 medium plus 10% fetal bovine serum, and F9 cells in F-12/DMEM with 10% fetal bovine serum. Cells were grown in a humidified incubator at 37 °C in an atmosphere of 6% CO2. All cell lines were transfected by the Ca3(PO4)2 DNA coprecipitation method. Cells were harvested 48 h after transfection, and CAT activities were measured by percentage of acetylation. For standardization, equal amounts of protein were used in each reaction. Transfection efficiency was normalized according to the level of beta -galactosidase expression in cells co-transfected with RSV-beta gal plasmid.

Plasmids and Fusion Proteins-- E1ADelta 51-116 (deletion mutant in CR1 transforming domain) and E1Am928 (missense mutation in CR2 region) have been described previously (34). c-Jun (1-311), c-Jun (1-244), and c-Jun (1-221) proteins were in vitro translated using pGEM4Zc-jun as a template digested with BspHI, BstXI, or PstI, respectively. c-Jun331m14, c-Jun331m9, and c-Jun331m15 were in vitro translated from pGem4Zm14, pGem4Zm9, and pGem4Zm15 (35), respectively. c-Jun (1-281) protein was in vitro translated from pGEM-c-jun281, which was prepared by cloning a PCR-amplified fragment (1-281) into a pGEM4Z vector at the HindIII and BamHI sites. Primers 5'-CCCAAGCTTATGACTGCAAAGATGGAAACGACCTTC-3' and 5'-CGCGGATCCTCACTCCAGCCGGGCGATTCTCTCCAG-3' were used for PCR amplification with pGEM4Zc-jun (1-331) as a template. GST-RB (379-928), GST-RB (379-792), GST-RB (379-928, Delta exon 21), and GST-RB (379-928, Delta exon 22) were described previously (36). GST-RB (251-928, Delta 622-714) was prepared by cloning a PCR-amplified fragment from an RB mutant (obtained from E. Harlow) into pGEX 4T-1 vector at the BamHI and XhoI sites. GST-RB (638-792) was prepared by cloning a PCR-amplified fragment with 5'-CGCGGATCCACCCAGAAGCCATTGAAATCTACC-3' and 5'-CCGCTCGAGTCAAAA-CTTGTAAGGGCTTCGAGGAATG-3' as primers and GST-RB (379-928) as a template. Similarly, GST-RB (379-572) was cloned into pGEX 4T-1 vector at the BamHI and XhoI sites. For constructing GST-RB (251-928, Delta 379-572 and Delta 622-714), two PCR fragments were initially synthesized using plasmid pGST-RB (251-928, Delta 622-714) as the template. The primers 5'-CGCGGATCCCGAACACCCAGGCGAGGTCAGAACAGG-3' and 5'-CCCAAGCTTCATAACAGTCCTAACTGGAGTGTGTGG-3' amplified RB amino acids 251-380; primers 5'-CCCAAGCTTATTAAACAATCAAAGGACCGAGAAGGACC-3' and 5'-CCGCTCGAG-TCATTTCTCTTCCTTGTTTGAGGTATC-3' amplified amino acids 572-928, with amino acids 622-714 deleted. The amplified fragments were ligated in-frame at the HindIII site before cloning into the BamHI-XhoI site in pGEX4T-1. A 1.9-kilobase-pair fragment (c-Jun 1-331) was PCR-amplified from pGEM4Zc-jun using primers 5'-CCGGAATTCATGACTGCAAAGATGG-AAACGACC-3' and 5'-CGCGGATCCTCAAAATGTTGCAACTGCTGCGTTAG-3'. The PCR-amplified fragment was fused to GAL4 (1-147) and then cloned into the HindIII site of the mammalian expression vector, pRc/CMV (Invitrogen). A c-Jun frameshift-mutant was generated by PCR amplification using RSVmc-jun as a template. The amplified fragment containing C-terminal amino acid residues 194-331 was fused to GAL4 (1-147) for a negative control fusion plasmid, GALDB-Cmc-Jun. Expression vector containing GAL4 (1-147) fused to N-terminal c-Jun (1-223) was obtained from M. Karin. RB (301-928) was PCR-amplified from phRB, using primers 5'-CGGGATCCCGAATTCTCTTGGACTTGTAAC-3' and 5'-CGGGATCCAAGC-TTGCAAGGTCCTGAGATCCTC-3'. The PCR product was cloned into pGAD424 (CLONTECH) at the BamHI site to create a translational fusion with GAL4 (768-881) before inserting into the HindIII site of pRc/CMV.

Co-immunoprecipitation Assays-- Cellular proteins were biosynthetically labeled for 3 h by the addition of L-[35S]methionine (500 µCi/ml, Amersham Pharmacia Biotech) to subconfluent cell layers in methionine-free DMEM (CV-1) or RPMI 1640 medium (Molt 4) with 2% dialyzed fetal bovine serum. Labeled cells were lysed in 1.0 ml of ice-cold radioimmune precipitation buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% Nonidet P-40, 0.2% SDS, 0.2% deoxycholate, and 1 mM phenylmethylsulfonyl fluoride) with 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 µg/ml aprotinin. Lysates were incubated overnight with specific monoclonal or polyclonal antibodies. The antigen-antibody complexes were incubated with a mixture of protein A- and protein G-Sepharose at 4 °C for 2 h, washed five times with 1 ml of lysis buffer, followed by boiling in SDS-buffer to dissociate the complexes. Dissociated complexes were diluted with cold radioimmune precipitation buffer, re-immunoprecipitated with specific antiserum, and analyzed by SDS-PAGE. Gels were dried and exposed to x-ray film at -70 °C.

In Vitro Binding Assays-- GST-RB fusion proteins were immobilized on glutathione-Sepharose 4B beads equilibrated with NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, and 1 mM dithiothreitol). In vitro translated, [35S]methionine-labeled c-Jun and its various mutant proteins were prepared using TNT T7 Quick Coupled Transcription/Translation kit (Promega). The translated proteins were incubated with GST-RB (379-792) or GST-2T-Sepharose beads in NETN buffer containing 0.5 µg BSA for 1 h. The beads were washed three times with NETN buffer, boiled in SDS-PAGE loading buffer for 3 min, and then resolved by 12.5% SDS-PAGE. Gels were fixed, dried, and exposed to x-ray film.

Western Blot Analysis-- Immunocomplexes precipitated by anti-RB antibody (PMG3-245, PharMingen; IF-8 and C-15, Santa Cruz Biotechnology) or preimmune serum were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with anti-c-Jun antibody, followed by horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G. Immunoreactive bands were detected by ECL developing reagents (Amersham Pharmacia Biotech).

EMSAs-- Complimentary oligonucleotides containing TGACATCA sequences of human c-Jun/AP-1 binding site were annealed and labeled as described previously (37). EMSAs were performed in a binding buffer (12 mM Hepes, pH 7.9, 5 mM MgCl2, 4 mM Tris-HCl, pH 7.9, 60 mM KCl, 0.6 mM Na2EDTA, pH 8.0, 0.6 mM dithiothreitol, 12% glycerol) consisting of 2 µg of poly(dI-dC)-(dI-dC) (Amersham Pharmacia Biotech) and 6 µg of acetylated bovine serum albumin in a total volume of 20 µl. GST-RB fusion protein was expressed using the Baculovirus expression vector system and purified by a GSH-Sepharose 4B matrix. c-Jun was bacterially expressed using the pET expression system. When required, antibodies were preincubated with purified GST-RB fusion protein for 1 h at 4 °C before the addition of c-Jun lysate. Incubation was continued for 15 min at room temperature, at which time 20,000 cpm of labeled AP-1 binding probe was added, and the reaction was continued for another 15 min at room temperature. For competition experiments, a 50-fold molar excess of double-stranded AP-1 oligonucleotide was preincubated with c-Jun for 15 min at room temperature before the addition of the probe. The bound complexes were resolved on a 4% nondenaturing polyacrylamide gels with 2.5% glycerol under low ionic conditions (0.25X TBE). Gels were then fixed, dried, and exposed to x-ray film with an intensifying screen at -80 °C.

    RESULTS

RB Transactivates Expression of the c-jun and Collagenase Genes through the AP-1 Binding Site-- RB represses the expression of the c-fos gene (29), the gene product of which, Fos, is one component of the heterodimeric transcription factor AP-1 (24, 25). To test the hypothesis that RB may regulate the expression of c-jun through the AP-1 binding site, we demonstrated that RB stimulated c-jun expression by approximately 10-15-fold in the NIH 3T3 cells and that this stimulation required an intact AP-1 binding site (Fig. 1A). Similar results were also observed in the murine fibroblast cell line 3T6 (Fig. 1A). Analogous experiments with the collagenase promoter, containing or lacking an AP-1 site, also demonstrated that RB induced transcription is mediated through the AP-1 site (Fig. 1A). Stable transfectants carrying collagenase promoter -73/+63 or -60/+63 fused to chloramphenicol acetyltransferase reporter gene also demonstrated that RB enhances gene expression required AP-1 site (Fig. 1B). Furthermore, to examine whether RB actually regulates the endogenous c-jun gene expression, we performed Northern blot analysis to compare c-jun expression in SAOS2 cells ectopically expressed Rb gene with that in wild-type SAOS2 cells. As shown in Fig. 1C, expression of Rb in SAOS2 cells enhances the endogenous c-jun expression about 1.8-fold after standarization with glyceraldehyde-3-phosphate dehydrogenase as an internal control.


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Fig. 1.   Function of RB in transcriptional regulation. A, the AP-1 binding site is a target for activation of gene expression by RB. 2.5 µg of CAT reporter construct was cotransfected with either 7.5 µg of RB (phRB) or control plasmid (pUC18 or pJ3Omega ) into NIH 3T3 cells or 3T6 cells by the calcium phosphate coprecipitation method. -132jun·CAT and -73col·CAT contain an AP-1 binding site, -132mjun·CAT and -60col·CAT lack the functional site. Both phRB (previously named pOmega hRbc) and pJ3Omega have been described previously (27). B, RB enhances transcription in stable transfectants carrying -73col·CAT gene containing AP-1 binding site. 3T6 cells were transfected with -73col·CAT or -60col·CAT and pSV2neo. After selection by incubation with 0.5 mg/ml G418 for 3 weeks, the selected cells were transfected with 2.0 µg of phRB expression plasmid or pJ3Omega as a negative control. Cell lysates were prepared and analyzed for CAT activity. C, RB enhances endogenous c-jun expression. SAOS2 cells, which lack functional RB, were transfected with p3JOmega (lane 1) or phRB (lane 2) and pSV2neo expression plamids. After selection by incubation with 0.8 mg/ml G418 for 4 days and with 0.5 mg/ml G418 for 10 days, total RNA were prepared from the selected cells for Northern analysis of c-jun expression. Glyceraldehyde-3-phosphate dehydrogenase probe was used as an internal control.

Physical Interactions between the RB and c-Jun Proteins-- To investigate whether AP-1-mediated transactivation by RB requires a direct physical association between the RB and c-Jun proteins, we immunoprecipitated TPA-stimulated [35S]methionine-labeled Molt 4 cell lysates with either anti-c-Jun polyclonal antibody or anti-RB monoclonal antibody (PMG3-245, PharMingen). Under nondenaturing conditions, RB coprecipitated with c-Jun in immunoprecipitates prepared with anti-c-Jun antibody (Fig. 2A, lane 2). One-third of the immunocomplex above was then solubilized and re-immunoprecipitated with rabbit preimmune sera, anti-c-Jun, or anti-RB antibody. Immunoprecipitation with anti-c-Jun antibody revealed a 39-kDa c-Jun protein; RB monoclonal antibody immunoprecipitated RB protein and its degraded products (Fig. 2A, lanes 5 and 6). Similar results were also obtained from CV-1 cells, although the amount of coprecipitated RB with c-Jun was less than that prepared from Molt 4 cells (Fig. 2B, lane 1). This observation could be due to differences in the availability of RB in the cells (38).


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Fig. 2.   Detection of physical association between c-Jun and RB proteins. A, immunoprecipitation of c-Jun or RB proteins from TPA-stimulated [35S]methionine-labeled Molt 4 cells. Labeled cell lysates were precipitated with preimmune serum (lane 1), anti-c-Jun polyclonal antiserum (lane 2), or anti-RB monoclonal antibody (Rb-PMG3-245, PharMingen) (lane 3). Immunoprecipitated complexes using anti-c-Jun antiserum were reimmunoprecipitated with a specific antiserum: rabbit preimmune serum (PI) (lane 4), anti-c-Jun antiserum (alpha -c-Jun) (lane 5), or anti-RB monoclonal antibody (alpha -RB) (lane 6). The positions of the RB and c-Jun protein bands are indicated by arrows. B, coimmunoprecipitation of RB with c-Jun in [35S]methionine-labeled CV-1 cells. Labeled cell lysates were precipitated with anti-c-Jun antibody and then re-immunoprecipitated with anti-RB monoclonal antibody (alpha -RB) (lane 1), anti-c-Jun antiserum (alpha -c-Jun) (lane 2), or rabbit preimmune serum (PI) as a control (lane 3). The positions of the RB and c-Jun protein bands are indicated by arrows. C, Western blot analysis of c-Jun in the immunocomplex precipitated with anti-RB antibody. Molt 4 cell lysates containing ectopic expression of c-Jun were precipitated with anti-RB antibody (PMG3-245, IF-8 or C-15). Preimmune serum was used as a negative control. c-Jun was then analyzed by Western blot with anti-c-Jun antibody.

In contrast, the anti-RB monoclonal antibody did not coprecipitate c-Jun with RB (Fig. 2A, lane 3). To confirm the interaction between RB and c-Jun in Molt 4 cells, c-jun expression plasmid was transfected into Molt 4 cells to enhance the capacity of interaction. We detected c-Jun with anti-c-Jun antibody by Western blot in these cell lysates originally precipitated by anti-RB antibody (Fig. 2C, lanes 1 and 2) but not by antibody raised against the C-terminal RB amino acid residues 914-928 (Fig. 2C, lane 3). These results clearly demonstrated that c-Jun physically associates with RB in vivo.

RB Enhances Binding Activity of c-Jun at AP-1 Binding Site-- To investigate whether RB-c-Jun complex binds to the AP-1 site, we performed EMSAs using labeled human c-jun TRE oligonucleotide containing the AP-1 binding sequence, TGACATCA. Bacterial expressed recombinant c-Jun and baculovirus-expressed GST-RB were used for EMSA. In the presence of bacterially expressed c-Jun lysates, a single binding complex with TRE probe was observed (Fig. 3, lane 3). A supershift in mobility (Fig. 3, lane 1) and a dramatically decreased binding activity (Fig. 3, lane 2) were observed in the presence of anti-c-Jun antibody and in an addition of 50-fold molar excess of cold double-stranded TRE competitor, respectively. These data suggest that c-Jun-DNA binding is specific. Purified GST-RB fusion protein quantitatively enhanced c-Jun-binding activity (Fig. 3, compare lane 3 to lanes 4-6). This enhanced binding activity can be blocked by preincubation with anti-RB antibody (Fig. 3, lane 7). The interaction with anti-RB antibody is specific because an unrelated antibody against serum-responsive factor had no effect in the presence of c-Jun or in conjunction with GST-RB (Fig. 3, lanes 8 and 9). These data further demonstrated that RB is involved in the c-Jun DNA-binding activity.


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Fig. 3.   EMSAs of AP-1 complex in the presence of RB and c-Jun. Purified bacterially expressed c-Jun and baculovirus expressed GST-RB were preincubated before the addition of 32P-labeled double-stranded oligonucleotide containing a single AP-1 binding site. Preincubation of GST-RB with anti-RB antibody was performed before adding 50 ng of c-Jun. The AP-1 complexes were analyzed on a 4% nondenaturing polyacrylamide gel with 2.5% glycerol under low ionic strength conditions.

Identification of a RB-interacting Domain of c-Jun-- A direct physical association between RB and c-Jun was further investigated by incubating [35S]methionine-labeled c-Jun with GST-RB-Sepharose beads and analyzing the bound protein by SDS-PAGE (Fig. 4A, lane 1). To localize the region of c-Jun important for direct interaction with RB, [35S]methionine-labeled mutant derivatives with sequential deletion from the C-terminal portion of c-Jun were analyzed by GST affinity matrix-based assays for their abilities to interact with RB. Deletion mutant c-Jun311 (1-311) still interacted with GST-RB (Fig. 4A, lane 4), although the binding declined steadily with further truncation of c-Jun to amino acid 281 (Fig. 4A, lane 7). Further deletions beyond amino acid 281 totally abolished binding (Fig. 4A, lanes 10 and 13), suggesting that the C-terminal region of c-Jun is important for interaction with RB. Binding studies with leucine zipper mutants, c-Jun331m9 (mutation at amino acid residues 286 and 294) and c-Jun331m14 (mutation at amino acid residues 286, 287, and 294) (35), that contain mutations in the second and third leucines required for c-Jun homodimerization still retained binding to RB (Fig. 4A, lanes 16 and 19). However, the mutant c-Jun331m15 (mutation at amino acid residues 287 and 308) failed to bind to RB (Fig. 4A, lane 22). Thus, the region of c-Jun required to interact with RB appears to be the C-terminal portion of c-Jun containing part of the leucine zipper motif (Fig. 4B).


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Fig. 4.   Direct interaction between RB and c-Jun in vitro. A, identification of an RB-interacting domain within c-Jun. In vitro translated, [35S]methionine-labeled c-Jun and its various mutant proteins were analyzed by their binding capability with GST-RB Sepharose. B, schematic representation of in vitro translated c-Jun proteins are presented.

Identification of a c-Jun-interacting Domain of RB-- To localize the domain(s) of RB required for interaction with c-Jun, GST-RB (379-928) and its various mutants were analyzed for binding with in vitro translated c-Jun (1-331). As shown in Fig. 5A (see schematic representation in Fig. 5B), in vitro translated c-Jun bound to the discrete B-pocket domain but not the A-pocket domain of RB (Fig. 5A, lanes 11 and 12). c-Jun also bound RB with internal deletions in the B-pocket domain, Delta 622-714, Delta exon 21 (Delta 703-737), and Delta exon 22 (Delta 738-775) (Fig. 5A, lanes 3-5), suggesting that c-Jun can bind to another site within amino acids 379-928 of RB. To determine whether a highly homologous A-pocket region can complement the partially deleted B-pocket mutants, we constructed a double deletion mutant, GST-RB 251-928 (Delta 379-573 and Delta 622-714), with deletions of the entire A-pocket and portions of the B-pocket for binding assays. We observed that c-Jun bound the double deletion mutant, but at a significantly reduced level (Fig. 5A, lanes 9 and 10 compared with lanes 2 and 3). This result suggested that although the A-pocket domain failed to bind c-Jun as a discrete element, its presence could complement the partially deleted B-pocket domain. A similar observation was reported with p300, which requires two separate regions to interact with c-Jun (39).


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Fig. 5.   Mapping the region of RB required for interacting with c-Jun. A, in vitro translated, [35S]methionine-labeled c-Jun was incubated with GST-RB (379-928) and its various deletion mutants on an affinity matrix. The bound c-Jun was resolved by 12.5% SDS-PAGE and detected by autoradiography. B, schematic representation of GST-RB (379-928) and its various deletion mutants.

Two-hybrid Assays of c-Jun-RB Interaction in Vivo-- Finally, we examined the RB-c-Jun interaction in NIH 3T3 cells by the two-hybrid-based assay (see schematic diagram in Fig. 6C). We observed a dose-dependent transactivation by RB in the presence of full-length c-Jun, but not with N-terminal c-Jun (Nc-Jun) or the frameshift-mutated C-terminal c-Jun (Cmc-Jun) (see schematic representation of constructs in Fig. 6A), suggesting that RB requires the C-terminal region of c-Jun to enhance transcriptional activity (Fig. 6B). However, we also observed a decrease in CAT activity with increased RB concentration beyond 4 µg in our experiment with a 5X Gal4 binding site reporter, pG5E4·CAT (Fig. 6B). Nevertheless, the levels of Gal4-RB proteins expressed in the transfected cells showed a dose-dependent linear increase in this range, using Western blot probed with anti-Gal4 antibody (Fig. 6B). The data presented in Fig. 6B are consistent with the in vitro binding data, which implicated the C-terminal region of c-Jun in mediating interaction with the RB. Interaction between these two proteins may be responsible for the observed stimulation of gene expression by RB.


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Fig. 6.   In vivo interaction of c-Jun-RB using the two-hybrid assay. A, constructs used for the transient expression assays. B, a graphic depiction of a representative CAT assay illustrating dose-dependent transactivation by RB. For the in vivo expression assays, 2.5 µg of pG5E4·CAT reporter and 2.5 µg of either GAL4DB-c-Jun, GAL4DB-Nc-Jun (N-terminal), or GAL4DB-Cmc-Jun (C-terminal) expression plasmids were coprecipitated with various concentrations of GAL4AD-RB before adding to 5 × 105 NIH 3T3 cells. After 16 h, the Ca3(PO4)2 precipitates were removed, and the cells were harvested 48 h later. A relative CAT activity of 1.0 was arbitrarily chosen for 4, 8, or 1% CAT conversion for GAL4-c-Jun, GAL4-Nc-Jun, or GAL4-Cmc-Jun, respectively. Gal4-RB proteins expressed in the transfected cells showed dose-dependent linear increase (open circle ) in Western blot probes with anti-Gal4 antibody. C, schematic diagram illustrating RB-c-Jun interaction in the two-hybrid assay in vivo.

Activation of c-Jun-mediated Transcription by RB Is Relieved by the Expression of Adenovirus E1A and Papilloma Virus E7-- E1A-RB complex has been implicated in controlling cell growth (16). To test whether E1A affected the ability of RB to stimulate transcription through the AP-1 site, cotransfection of E1A and RB expression plasmids with -73col·CAT was performed. The wild-type E1A, but not the transforming domain CR1 deletion mutant, E1ADelta 51-116, abrogated RB-stimulated gene expression (Fig. 7A). This result agrees with the previous report that E1A inhibits gene expression mediated by the AP-1 binding site (12). Similarly, RB-mediated regulation of the collagenase promoter was also inhibited by the expression of another RB-binding protein, HPV E7, but not by its mutants E7m24 or E7m91 (20) (Fig. 7B). Mutations in E7m24 and E7m91 are located in the regions important for the transforming function of the E7 protein (20). These results suggested that the formation of E1A-RB or E7-RB complex can reduce the transcriptional activity of c-Jun protein and thereby disrupt the activation of c-Jun-regulated genes normally subjected to positive regulation by RB.


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Fig. 7.   Adenovirus E1A and human papillomavirus E7 inhibit RB-mediated transactivation. A, adenovirus E1A inhibits RB-mediated transactivation. Five µg of -73Col·CAT reporter constructs were cotransfected with wild-type c-Jun or its frameshift-mutant, mc-Jun (0.1 µg each), phRB (5.0 µg), wild-type E1A (5.0 µg), or its mutants, and various combinations as indicated. B, HPV E7 inhibits RB-mediated transactivation. Five µg of reporter construct (-73Col·CAT or -60Col·CAT) was cotransfected into NIH 3T3 or a stable transfectant carrying HPV E7, E7m91, or E7m24 with pJ3Omega (5.0 µg) or phRB (5.0 µg) expression plasmid. Results are expressed as the relative CAT activity ± S.D.


    DISCUSSION

When we initiated this study, our first data showed that the transient expression of RB transactivates -1.1 kilobase/+740 c-Jun·CAT activity (data not shown). To map the RB-responsive element, we used several c-jun promoter deletion mutants, including -132 c-jun and -79 c-jun, fused to CAT reporter gene. We consistently observed that RB transactivates c-jun·CAT activity and suggested that transactivation was mediated through the AP-1 site. In addition, we further tested AP-1-mediated transactivation by RB using -73col·CAT containing a single AP-1 site and -60col·CAT lacking AP-1 site as a reporter. The results consistently showed that RB transactivation is dependent upon the presence of an AP-1 site. Furthermore, we also demonstrated that RB actually enhances the endogenous c-jun gene expression in SAOS2 cells.

The discrepancy between our data and a previous report by Robbins et al. (29) that could be due to the use of different constructs (5X versus 1X AP-1 site) and different promoters. Nevertheless, both AP-1 site-mediated transactivation and transrepression have been reported previously (reviewed in Ref. 26). Differential modulation could depend upon the promoter or the cell type. To further confirm our data, we used two-hybrid-based assay with 5X GAL4 binding site as the reporter to simulate the 5X AP-1 reporter as used by Robbins et al. (29). We observed dose-dependent transactivation by RB as shown in Fig. 6B. These results suggested that the concentration of RB might dictate transcriptional repression or activation. At higher concentrations, RB possibly recruits other RB binding cellular factors equivalent to E1A or E7 proteins, resulting in transrepression instead of transactivation. Similar results were observed with JunB repression of gene expression mediated through a single AP-1 binding site. This repression was converted to transactivation when 3X AP-1 site was used as a reporter (40).

We have demonstrated that RB enhanced c-Jun DNA-binding activity in vitro by EMSAs (Fig. 3). In addition, we have demonstrated the C-terminal region of c-Jun containing part of the leucine zipper was involved in the interaction with RB. These data suggest that the hydrophilic part but not the hydrophobic part of the amphipathic helix of c-Jun is involved in the interaction with RB. Although RB also interacts with c-Fos (41), we did not observe either enhanced or reduced binding activity between RB and c-Jun when recombinant c-Fos was included in the reaction mixtures (data not shown). RB could interact with different members of the AP-1 family, resulting in a different effect on AP-1 activity or its specificity. We also systematically analyzed different regions of RB that may be involved in the interaction of RB with c-Jun. The results revealed that both the A- and B-pockets in RB can complement each other, although the B-pocket alone can interact with c-Jun in vitro. A similar observation has also been reported with p300, which requires two separate regions to interact with c-Jun (39). It has also been demonstrated that neither the A- nor the B-pocket of RB has any E2F repressor activity (42). However, A- and B-pockets coexpressed on separate proteins regained transcriptional repressor activity (42), suggesting that these two pocket domains interact to form a functional repressor.

Previous reports suggested that RB transactivates gene expression through the SP-1 site (37, 43, 44). The mechanism for this transactivation remains unclear, although the accessory factor(s) that required for this transactivation has been suggested (37). Our present studies showed that RB-mediated transactivation also maps to the AP-1 site by the mechanism of direct interaction between RB and c-Jun proteins. We here provide direct evidence demonstrating the role of RB as a coactivator of c-Jun in vivo. Similarly, p300, another E1A-binding protein, also functions as a coactivator of CREB and c-Jun (39, 45).

Is RB-c-Jun complex essentially required for cell growth control? Our data do not directly address this question. However, it has been reported that adenovirus E1A represses transcription of the collagenase gene via the phorbol ester-responsive element (12). The mechanism of repression has not yet been identified. Here, we offer a possible mechanism that E1A inhibits AP-1-mediated transcription by targeting c-Jun coactivator RB (Fig. 7A). Furthermore, this RB-mediated transactivation is inhibited by the expression of adenovirus E1A but not by the CR1 transforming domain deletion mutant, implying that AP-1-mediated transactivation by RB is important for cell growth control, as suggested by Nead et al. (41). RB-/- cells would explore the physiological role of RB-c-Jun complex in cell growth control. Induction of c-Jun, c-Fos, and JunB has been reported during keratinocyte differentiation (46), suggesting that c-Jun/AP-1-mediated transactivation by RB is important for cell differentiation. Therefore, inhibition of keratinocyte differentiation by E7 protein may be mediated by the repression of c-Jun via targeting its coactivator RB (Fig. 7B). These results agree with report by Nead et al. (41) that RB is a potential modulator of c-jun expression during keratinocyte differentiation. Therefore, the functional interplay between RB and c-Jun might well represent an important mechanism for controlling transcription, cell growth and differentiation.

    ACKNOWLEDGEMENTS

We thank Michael Karin for a generous gift of anti-c-Jun antisera and leucine zipper mutants of c-Jun, Robert Weinberg for RB expression plasmid, D. M. Livingston and Ed Harlow for several GST-RB constructs and their deletion mutants, K. Vousden for E7 and its mutated expression plasmids, Z. Gu for the NIH 3T3 stable transfectants carrying E7 and its mutants, and H. Zhou and Y. K. Cui for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA 66746 (to R.C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dental Research Institute, School of Dentistry, University of California, Box 951668, Los Angeles, CA 90095-1668. Tel.: 310-825-0535; Fax: 310-825-0921; E-mail: rchiu{at}surgery.medsch.ucla.edu.

    ABBREVIATIONS

The abbreviations used are: RB, retinoblastoma susceptibility gene product; HPV, human papillomavirus; EMSA, electrophoretic mobility shift assay; DMEM, Dulbecco's modified Eagle's medium; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CR, conserved region; CAT, chloramphenicol acetyltransferase.

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