From the
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.
Gene products encoded by the early region 1A oncogene
(E1A)
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 X
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).
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.
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
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.
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).
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.
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
[
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.
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.
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.
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-
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.
We thank Barbara Tries and Daniela Schäfer for
excellent technical assistance and Ulla Schmücker for valuable
help with the automatic sequencing.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
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.
C 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).
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).
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).
),
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.
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.
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.
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.
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).
-
P]ATP for 20 min. The phosphorylation
reaction was terminated by extensive washing, and bound material was
eluted and analyzed on SDS-polyacrylamide gels.
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.
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).
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.