Recruitment of the Retinoblastoma Protein to c-Jun Enhances
Transcription Activity Mediated through the AP-1 Binding Site*
Junko
Nishitani
§,
Toru
Nishinaka
§,
Chi-Hong
Cheng§,
Walter
Rong
,
Kazunari K.
Yokoyama
, and
Robert
Chiu
§¶**
From the
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
Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba,
Ibaraki 305, Japan
 |
ABSTRACT |
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 |
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.
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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
-galactosidase expression in cells co-transfected with
RSV-
gal plasmid.
Plasmids and Fusion Proteins--
E1A
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,
exon 21),
and GST-RB (379-928,
exon 22) were described previously (36).
GST-RB (251-928,
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,
379-572 and
622-714), two PCR
fragments were initially synthesized using plasmid pGST-RB (251-928,
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 pJ3 ) 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 p hRbc) and pJ3 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
pJ3 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 p3J (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.
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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
( -c-Jun) (lane 5), or anti-RB monoclonal
antibody ( -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
( -RB) (lane 1), anti-c-Jun antiserum
( -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.
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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.
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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.
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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,
622-714,
exon 21 (
703-737), and
exon 22 (
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 (
379-573 and
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.
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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 ( ) in Western blot probes
with anti-Gal4 antibody. C, schematic diagram illustrating
RB-c-Jun interaction in the two-hybrid assay in vivo.
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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, E1A
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 pJ3 (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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