Vol. 68, No. 6

JOURNAL OF VIROLOGY, June 1994, p. 3527-3535

0022-538X/94/$04.00+0
Copyright © 1994, American Society for Microbiology

Analysis of AP-1 Function in Cellular Transformation Pathways
TAKEHISA SUZUKI,1 MASAO MURAKAMI,1 NOBUYUKI ONAI,' EIKO FUKUDA,'YOSHIKO HASHIMOTO,' MARTHA H. SONOBE,1 TAKASHI KAMEDA,
MASAO ICHINOSE,2 KAZUMASA MIKI,2 AND HIDEO IBAI*
Department of Tumor Virus Research, Institute of Medical Science, University of Tokyo,
Minato-ku, Tokyo 108, 1 and First Department of Intemnal Medicine, Faculty of
Medicine, University of Tokyo, Hongo Bunkyo-ku, Tokyo 113,2 Japan
Received 20 December 1993/Accepted 22 February 1994

JunD was also reported in these cells (14). In c-fos-overexpressing cells, cellular transformation seems to be mediated by
the heterodimeric complex of exogenous c-Fos and endogenous c-Jun (38). In the case of transforming junD mutants, it
was suggested that the heterodimeric complexes of endogenous Fra-2 and JunD mutants play a crucial role in cellulartransformation (18).
In our previous attempt to suppress specifically the transcriptional function of c-jun, we constructed a transdominantmutant designated supfos-1 (28). supfos-1 was originally described as a nontransforming v-fos mutant (40) which has an
insertion of four amino acids between the basic domain andthe leucine zipper structure (Fig. 1), and its gene product was
shown, both in vitro and in vivo, to form a heterodimer withc-Jun that lacks the specific AP-1 DNA binding (28). Thismutant, when introduced by retrovirus vectors, efficiently
suppresses c-jun transformation by sequestering the unstablec-Jun homodimer into the stable and nonfunctional supFoslJun heterodimer in the cells. Interestingly, nuclear extracts
from the supfos-1-expressing CEF reduced endogenous AP-1
DNA-binding activity (mainly contributed by Fra-2-Jun) to analmost nondetectable level by competing out endogenous
Fra-2 (27), suggesting that this mutant can also function as an
inhibitor of Fos family proteins. Several fos or jun mutants
have also been reported by other groups to function astransdominant suppressors by a mechanism similar to thatused by supfos-1 (38). Some of the transdominant mutants,
however, function without the leucine zipper structure, possibly by a squelching mechanism (38), while others retain both
dimer-forming and DNA-binding activities but lack the transactivation domain (6, 13, 20).
Most of the fos and jun family members belong to the

The c-fos and c-jun proto-oncogenes were originally identified as the cellular counterparts of the viral oncogenes carried
by Finkel-Biskis-Jinkins (FBJ) murine sarcoma virus (11) andavian sarcoma virus 17 (21), respectively. c-fos belongs to a
multigene family that includesfra-1 (9),fra-2 (23, 27), and fosB(42), and the fos gene family codes for nuclear proteins thatdimerize with the Jun family proteins, such as c-Jun (26), JunB(33), and JunD (16, 32), to form the transcription factor
complex AP-1. Dimerization occurs specifically through aleucine zipper structure: Jun family members can form lowaffinity homodimers and high-affinity heterodimers with the
Fos family, whereas Fos-related proteins do not form stablehomodimers (10, 25). Although these hetero- and homodimers
bind to similar DNA-binding sites (TGACTCA, AP-1-bindingsites) through the basic domains of both proteins, which arejuxtaposed by the dimerization, each dimer was shown to havea distinct transcriptional regulatory function, so that transcription can be positively and negatively modulated (37).
High-level expression of most members of the fos or jun
gene family has been reported to cause cellular transformationof chicken embryo fibroblasts (CEF) (17, 27, 36). JunD has notransforming activity, but it can acquire transforming potential
by spontaneous mutation (15, 18). These results indicate that
uncontrolled expression and qualitative change of any component of AP-1 can induce cellular transformation.
Logarithmically growing CEF express c-Jun and Fra-2 atrelatively high levels (36, 41), and basal-level expression of*
Corresponding author. Mailing address: Department of Tumor
Virus Research, Institute of Medical Science, University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108. Phone: 3-3443-8111, ext.
502. Fax: 3-3443-6604.

3527

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To understand the role of endogenous AP-1 activity in cellular transformation induced by oncogenes, we havemade use of a fos mutant (supfos-1) and a jun mutant (supjun-1), either of which can function as atransdominant inhibitor of AP-1-mediated transcriptional regulation. Chicken embryo fibroblasts (CEF)
infected with a series of transforming retroviruses were doubly infected with retrovirus carrying supfos-1 or
supjun-1, and suppression of cellular transformation was monitored in terms of reversion to normal cellularmorphology or acquisition of anchorage-dependent growth. Cellular transformation induced by several
exogenously expressed transforming genes of thefos orjun family was efficiently suppressed, as expected. CEF
transformed by v-src, v-yes, v-.fps, c-Ha-ras, and N-terminally truncated c-raf were also induced to revert to thenormal phenotype by these transdominant mutants, suggesting that functional transcription factor AP-1
activity is essential for the cellular transformation induced by these oncogenes. The suppression is not
attributable to nonspecific inhibition of cellular proliferation, because CEF transformed by v-ros or v-myc were
not induced to revert to the normal phenotype. We next analyzed changes in all known components of chicken
AP-1 induced by v-src, c-Ha-ras, or activated c-raf transformation. The levels of both Fra-2 and c-Jun
expression were elevated two- to fourfold, and hyperphosphorylation of Fra-2 was also observed. We further
showed that Fra-2-c-Jun heterodimer is mainly responsible for the elevated AP-1 DNA-binding activity in thesetransformed cells, and we propose that this heterodimer play a crucial role in the transformation induced bythese oncogenes.

3528

J. VIROL.

SUZUKI ET AL.
1 6 0
P L E R

supFosll

supJunl

15

1

I
1

3 3 1 a a

24 7

I

v
L---a7/

FIG. 1. Protein structures of supFos-1 and supJun-1. supFos-1 is a
derivative of v-Fos (FBJ), which has an insertion composed of fouramino acids (aa) between the basic domain (striped box) and theleucine zipper motif (black box). supJun-1 is an N-terminally truncatedmutant of c-Jun (human) and starts from an internal methionine 247.

category of immediate-early genes and are promptly induced

c-Jun

supJun-1c-Jun+competitor---

supJun-

+

.,

gee

FIG. 2. supJun-1 binds to AP-1-binding sites as a homodimer orheterodimer with c-Jun. Each protein was synthesized separately, andmixtures were incubated for 30 min at 37°C. DNA-binding activity wasdetermined by gel shift analysis using 0.2 ng of a 32P-labeled oligonucleotide containing the AP-1-binding site (FSE2) and 4 Kg of reticulocyte lysate mixture in the presence (+) or absence (-) of 20 ng of
unlabeled oligonucleotide as a specific competitor. Arrowheads indicate positions of specific AP-1 DNA-binding activity.

MATERIALS AND METHODS
Plasmid construction. The 0.8-kb SmaI fragment containing
the entire cDNA of human c-H-ras was isolated from pSPTHras (from the Japanese Cancer Research Resources Board)(30), ligated to BamHI linkers, and inserted into the unique
BglII site of pDS3 (17) to generate pHras. pND-raf was
generated from the human c-raf-1 cDNA, p627 (from JCRB)(5), by PCR using a 5' primer that covers the artificial
methionine codon preceded by an artificial Kozak consensus
sequence and a BamHI site (5'-AGGGATCCACCATGGAAAGAGAGCGGCACCAGT-3') and a 3' primer that contains
the stop codon (5'-CCGGATCCCTACTAGAAGACAGGCA
GCCTCGG-3'). The PCR product was digested with BamHI
and inserted into the unique BglII site of pDS3. pND-raf
encodes the same amino acid sequence as the 20A clone
constructed by Stanton et al. (35), which lacks the N-terminal
313 amino acids of human c-raf. The 0.5-kb NaeI-EcoT4I
fragment of pHJ (gift from R. Tjian) (4) was filled in, ligated to
BglII linkers, and inserted into the BglII site of pDS3 togenerate psupjun-1, which produces an N-terminally truncated
Jun from the internal methionine 247. The truncated Jun
retains the entire basic domain and the intact leucine zipper
structure, as shown in Fig. 1. The 0.83-kb EcoRI-BspMI
fragment of pSPfra-1 (rat) (9) was filled in and ligated withblunt-ended BglII-cut pDS3 to generate pFlR1, which carries
the entire rat fra-] gene.
For RNA probe synthesis, four DNA templates were constructed.
The 450-bp BstXl-EcoRI fragment of pJun (gift from P. Vogt) (21)encoding the C-terminal half of the v-jun gene was cloned into
pBlueskm to generate pjunRPA, which was linearized at the PvuII
site and used as a template. The 260-bp PstI-SacI fragment ofchicken v-fos (12) was inserted into pSPT19 to generate pfosRPA,
which was linearized at the NdeI site and used as a template. The
250-bp BstEI-NcoI fragment of chicken fra-2 (26) was filled in andligated to pSP64, which was doubly digested with EcoRI and BamHI
and blunt ended, to generate pfra2RPA. The pfra2RPA was linearized at the Pvull site and used as a template. The chicken junDconstruct was generated by PCR, using the published sequence (14).
The 245-bp SadH-TaqI fragment of the PCR product was inserted
between the AccI and SacII sites of pBlueskm to generate thepjunDRPA. pjunDRPA was linearized at the intemal DdeI site andused as a template. For Northem (RNA) blotting of the chicken
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA, thec-DNA that encodes the entire GAPDH was generated by thereverse transcription RT-PCR technique, using the published sequence (29).
Cells and viruses. CEF were prepared, grown, and infectedwith viruses as reported previously (28). For metabolic labeling, CEF were grown in 60-min-diameter dishes and labeledwith 500 ,uCi of [35S]methionine for 60 min. For the productionof recombinant viruses such as supjun-1 (carrying N-terminallytruncated jun), ND-raf (carrying N-terminally deleted raf), andH-ras (carrying normal c-Ha-ras) viruses, psupjun-1 and pHras
were completely digested with Sall and ligated to the Sall

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by several external stimuli as well as transient expression ofsuch oncogenes as src, ras, and raf, which are believed to belocated upstream of fos and jun family members in the signaltransduction pathway (2). It is noteworthy that cellular transformation induced by constitutive expression of these oncogenes seems to require functional endogenous c-Fos or c-Jun.
This was suggested by the reversion of transformed cells to thenormal phenotype following introduction of antisense RNA(19) for the fos gene, anti-Fos antibody (31), or a transdominant mutant of fos or jun. Because AP-1 activation by several
oncogenes has been studied in a number of different cell lines
by using either stable or transient transfections, it is not easy todevelop a general model from these observations. It seems
necessary to perform extensive screening of oncogenes which
require endogenous AP-1 for their transforming activity anddetailed analysis of the induced changes in all of the AP-1
components, using the same cell system.
In the first part of this report, we introduce a new transdominant suppressor, supjun-1, and show that both this mutant

and supfos-J can function as general inhibitors of AP-1. By
introduction of the dominant negative mutants into CEF
transformed by a series of oncogenes that do not belong to thefos orjun family, we were able to categorize the oncogenes into
two groups; one group requires functional AP-1 activity fortransforming activity, while the other does not. We havefurther analyzed all known components of the AP-1 complex in
CEF transformed by oncogenes that belong to the formergroup in an attempt to understand the molecular mechanisms
by which these oncogenes affect endogenous AP-1 activity.

VOL. 68, 1994

CELLULAR TRANSFORMATION AND ENDOGENOUS AP-1

tem (Amersham).

RNA preparation and RNase protection analysis. Total
RNA was extracted and purified from infected CEF by the acid
guanidium-thiocyanate-phenol chloroform method (22). RNAprobes for RNase protection analysis and RNA molecularweight markers were synthesized from the linearized template
DNA (0.5 ,ug) by T7 or SP6 RNA polymerase (30 U) in 40 mM
Tris Cl (pH 8.0)-8 mM MgCl2-2 mM spermidine-50 mM

NaCI-10 mM dithiothreitol-1 mM ATP, GTP, and UTP-10,uM CTP-50 ,uCi of [y-32P]CTP (3,000 Ci/mmol)-20 U of
RNase inhibitor at 37°C for 30 min. RNA probes were purified

by polyacrylamide gel electrophoresis and hybridized to 10,ug
of total cellular RNA in 80% formamide-40 mM piperazineN,N-bis(2-ethanesulfonic acid) (PIPES)-400 mM NaCl at 45°C
for 12 h. After digestion with 40,ug of RNase A per ml and 2
jig of RNase T, per ml at 42°C, samples were analyzed in 6 M
urea-polyacrylamide gels and detected by autoradiography.
RESULTS

Another highly effective transdominant mutant, supJun-1.
To obtain effective transdominant mutants other than supfos-I(Fig. 1), we tested some nontransforming mutants offos orjun
described here or previously (40) for the ability to suppress
c-jun transformation, using the method previously describedfor supfos-J (28). To introduce c-jun and the candidate mutant
into a single cell, we used replication-competent retrovirus
vectors as described previously (28). Two sets of vectors thatdiffered in subgroup specificity (A or B) were created for eachgene. CEF are resistant to infection by retroviruses of the same
subgroup, but they can be superinfected by retroviruses of adifferent subgroup. Thus, it was possible to obtain large
populations of doubly infected CEF expressing both c-Jun andthe candidate mutant at similar levels without any selection
procedure.
One mutant, designated supjun-1, which lacks the entire
transactivation domain (Fig. 1) was shown to have an equivalent or slightly enhanced suppressing activity compared withsupfos-1; CEF infected with supjun-J (A) (subgroup A) is
resistant to morphological transformation after superinfection
with JH-1 (B) (carrying human c-Jun). This suppression wasnever observed when supjun-1 (A) was substituted with
DS3(A) (control vector). At the same time, JH-1(A)-transformed CEF reverted to the normal phenotype upon superinfection with supJun-1 (B). The suppression was clearly quantitated by analyzing colony-forming activity in soft agar (Table
1). Similar results were obtained when JH-1 was substituted forT2 and T3 viruses (Table 1), which encode transforming junDmutants.
Although supJun-1 protein lacks all regions that have beenreported to function as transactivation domains (3), it shouldhave specific DNA-binding activity, because it retains theentire basic region and the intact leucine zipper structure. Thiswas confirmed by gel shift analysis (Fig. 2) of the mixture of
supJun-1 and wild-type Jun which were produced in vitro;
three specific band shifts were generated by the supJun-1
homodimer, the supJunl-c-Jun heterodimer, and the c-Jun
homodimer. This result is in a clear contrast to supFos-1, which
was shown to have no DNA-binding activity (28), and we
decided to compare the two mutants in a further analysis.
Either supfos-1 or supjun-1 can function as a general
inhibitor of AP-1 activity. We next tested whether supfos-I or
supjun-1 can suppress not only c-jun transformation but alsotransformation induced by oncogenes that belong to the fos
family. We previously reported the full transforming activity of
FM4 and F2C1 viruses, which carry c-fos (mouse) (17) andfra-2 (chicken) (27), respectively. The fra-1-carrying virus
constructed here (FiR1) exhibited similar transforming activity, as judged from the titer of transforming virus in the virus
stock or its efficiency of colony formation in soft agar (data notshown). As is the case with c-jun, the transformation inducedby c-fos, fra-1, and fra-2 was efficiently suppressed by subsequent infection with supfos-J or supjun-1 when judged bycolony-forming activity (Table 1) and cellular morphology

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digest of pREP to form the structure of the replicationcompetent provirus (subgroup A); pND-raf was partially digested with Sall, and a 1.3-kb fragment that covers the raf
sequence was isolated and used for ligation with the Sall digestof pREP. Ligated DNAs (2 jig) were transfected into CEF as
described previously (17), and replication-competent virus
stocks were collected from the culture 5 or 6 days after
transfection. Viruses were never propagated before use tominimize genetic changes of the virus genome. For productionof the subgroup B virus, pREP was substituted with pREP-B.
Similar viruses containing the human c-jun gene (JH-1 [36]),
the mouse c-fos gene (FM4 [17]), the chickenfra-2 gene (F2C1[27]), the v-src gene of Rous sarcoma virus (N4 [17]), atransdominant negative mutant of v-fos (supfos-J [28]), and no
oncogene (DS3 [17]) have been described previously. The
natural avian retroviruses Rous sarcoma virus Prague C,
Fujinami sarcoma virus (Rous-associated virus-1 [RAV-11),
Y73-associated virus (YAV), UR2 (RAV-1), and MC29
(RAV-1 or RAV-2) were also used to introduce the v-src, v-fps,
v-yes, v-ros, and v-myc genes, respectively.
Colony formation. CEF that were sequentially infected with
two species of viruses were trypsinized 4 days after the second
infection. Approximately 3,000 doubly infected CEF weremixed with 3 x 105 freshly prepared CEF feeder cells andseeded in suspension in soft agar (0.4%) on top of a bed ofhard agar (0.8%) as discussed previously (28). Colonies, whichwere formed after 2 weeks of incubation at 38.5°C, werecounted (average of three plates) at 3 weeks after seeding.
Gel shift analysis. Gel shift analysis using proteins synthesized in rabbit reticulocyte and using a 32P-labeled 62-bp
double-stranded DNA probe containing the FSE2 AP-1 DNAbinding site was described previously (37).
The nuclear extracts were prepared from infected CEF as
described previously (28). Gel shift analysis of the nuclear
extract was done as described previously (28, 41), using a
32P-labeled 65-bp HindIll-Aval fragment of pcoll, which has
an insertion of an oligonucleotide containing the AP-1 DNAbinding site of the human collagenase gene at the BamHI siteof pUC119. In some experiments, the antiserum was added tothe mixture 15 min before addition of the labeled probe. The
samples were analyzed by electrophoresis on a nondenaturing
5% polyacrylamide gel at 4°C, and shifted bands were detectedby autoradiography.
Immunoprecipitation and Western blot (immunoblot) analysis. 35S-labeled cell lysates were prepared under denaturingconditions (boiling in 2.0% sodium dodecyl sulfate [SDS]) andimmunoprecipitated as described previously (17, 40). Polyclonal antisera raised against Fos peptide 1 (40) and Fra-2
peptide 2 (27) have been described previously.
The total cell extracts prepared under denaturing conditions(80 ,ug of each) were resolved by electrophoresis on an
SDS-10% polyacrylamide gel, and the proteins were transferred to a polyvinylidene difluoride membrane (Millipore
Immobilon P). Immunoblots were treated with 5% dried milk
and then incubated with affinity-purified anti-Jun antiserum.
The filter was incubated with anti-rabbit immunoglobulin G
conjugated to horseradish peroxidase, and the protein bands
were visualized by the ECL Western Blotting Detection Sys-

3529

3530

J. VIROL.

SUZUKI ET AL.
TABLE 1. Suppression of colony formation by supfos-J and supjun-I viruses

Ratio"

c-fos
fra-]

fra-2
c-jun
junD mutant
junD mutant
v-src
v-src
v-yes
v-fps
c-H-rasN-terminally deleted raf
v-myc
v-ros

Oncogene-supfos-I'

supfos- I -oncogened

Oncogene-supjun-Isupjun--/oncogene

ND
ND
ND
ND
0.65
ND
ND
ND
0.36
0.13
0.40
0.31
0.90
1.07

0.28
0.35
0.33
0.25
0.56
0.31
0.25
0.27
ND
ND
0.28
0.24
1.1
ND

0.25
ND
ND
0.10
ND
ND
0.32
0.32
0.23
0.18
0.18
0.14
0.98
0.86

0.18
ND

0.13
0.05
0.25
0.30
0.17
0.20
ND
ND
0.09
0.06
0.96
ND

aPrC, Rous sarcoma virus Prague C; FSV, Fujinami sarcoma virus.
" The number of colonies formed by sup]un-I(A)/FM4(B)-infected CEF as shown in Fig. 4D was divided by that formed by DS3(A)/FM4(B)-infected CEF (Fig. 4A).
The ratio shown is the average of three independent experiments. ND, not determined.
"The experimental protocol in which transforming virus was infected and supfosl was infected subsequently.
d The experimental protocol in which supfosl was infected first and transforming virus was infected subsequently.

(data not shown). The overall results were reproducible evenwhen the order of introduction of the transdominant mutant
and fos family oncogene was changed (Table 1). For example,
when DS3(A)-infected CEF were subsequently infected with ac-fos virus [FM4(B)], cells were clearly transformed, assuminga polygonal cellular morphology with a high saturation density(Fig. 3B). When CEF were initially infected with supjun-1,

however, subsequent infection with c-fos virus did not induce
cellular transformation (Fig. 3G), and the cells exhibited amorphology similar to that of CEF infected with DS3(A)alone. These results further suggest that either of thesetransdominant mutants can function as a general inhibitor ofboth the Fos and Jun family proteins (AP-1).
Screening of other oncogenes whose transforming activity

FIG. 3. Cellular morphology of doubly infected CEF. CEF were first infected with DS3(A) (A to E) or supjun-J(A) (F to J) 4 days later
superinfected with FM4(B) (B and G), H-ras(B) (C and H), ND-raf(B) (D and I), or N4(B) (encoding v-src) (E and J) and kept under minimal
essential medium containing soft agar for 5 days before being photographed. The bar corresponds to 100 ,um.

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FM4
FlRl
F2C1
JH-1
T2
T3
N4
PrC
Y73
FSV
H-ras
ND-rafMC29
UR2

supjun-I

supfos-I

Oncogene

Virus'

VOL. 68, 1994

CELLULAR TRANSFORMATION AND ENDOGENOUS AP-1

3531

N1

superinfection, they were trypsinized, seeded into soft agar (60-mm-diameter plates), and propagated for 3 weeks at 38.5°C before being
photographed.

was suppressed by the transdominant mutants. Using these
inhibitors of AP-1, we next screened CEF transformed by aseries of naturally isolated or recombinant transforming retro-viruses to test whether endogenous AP-1 function is essential
for their transformation. Among the recombinant viruses,
H-ras and ND-raf were constructed in this study and encode
the normal human Harvey c-ras gene or N-terminal truncatedhuman c-raf, respectively. Both viruses transformed CEF moderately, as judged from the morphological changes (Fig. 3C
and D) and titer of focus formation [around 105 focus-forming
units/ml for H-ras(A) or ND-raf(A)]. These results are in good
agreement with those observed in murine cell lines (8, 35).
As shown in Table 1 and Fig. 4, supfos-1 and supjun-1 canefficiently suppress colony formation in soft agar caused by
v-src, v-yes, v-fps, c-Ha-ras, and ND-raf, though in all of thesecases, suppression by supjun-J was stronger than suppression
by supfos-1. On the other hand, colonies formed by v-myc or
v-ros were never suppressed by superinfection with either ofthese transdominant mutants. The reduction of anchorageindependent growth of the doubly infected CEF is in good
agreement with the reversion to the normal cellular morphology in monolayer cultures, some examples of which are shownin Fig. 3 (compare Fig. 3C to E with Fig. 3H to J, respectively).
In the v-src-introduced culture, however, partially refractile
cells were still detectable (Fig. 3J), and their population
increased gradually when the cultures were maintained for
longer periods of time. It is also noteworthy that CEF transformed by v-myc or v-ros did not revert to the normal
morphology upon superinfection with these two transdominantmutants (data not shown).
Analysis of the AP-1 components in CEF transformed byseveral oncogenes. As discussed above, we categorized sevenoncogenes into two groups by using transdominant mutants;
five (v-src, v-yes, v-fps, ND-raf, and c-H-ras) require endogenous AP-1 activity for their transforming activity, while two(v-ros and v-myc) do not. Since either of the two transdominantmutants used in this study can function as a general inhibitor of
AP-1, we next analyzed all known chicken AP-1 components(c-fos, fra-2, c-jun, and junD) in cells transformed by several
oncogenes to examine the molecular mechanisms by which
AP-1 is involved in cellular transformation.
From logarithmically growing CEF, we prepared total cell
extracts and analyzed the absolute amounts of endogenous Jun
and JunD by immunoblotting with an anti-Jun antiserum thatis cross-reactive to JunD (Fig. 5A). Compared with DS3infected or uninfected CEF, CEF transformed by v-src, v-fps,
and v-ros expressed three- to fourfold more c-Jun, while about
twofold-greater expression was observed in v-yes-, c-Ha-ras-,and ND-raf-transformed CEF. It should be pointed out thatthe c-jun-transformed CEF expressed much higher levels ofexogenous human c-Jun (about five times the endogenous
c-Jun level). On the other hand, v-myc-transformed CEF
expressed a similar level of c-Jun, comparable to DS3-infectedor uninfected CEF. The JunD bands were too weak comparedwith c-Jun bands to allow comparison of the expression levels
among these cells quantitatively.
For analysis of endogenous c-Fos and Fra-2, logarithmicallygrowing CEF were metabolically labeled with [35S]methionine.
From cellular lysates prepared under denaturing conditions,proteins were immunoprecipitated with the anti-Fra-2 antiserum, which specifically precipitates Fra-2 (Fig. SB), and theanti-Fos antiserum, which precipitates all Fos family proteins.
None of the logarithmically growing CEF showed any detectable c-Fos expression (data not shown), but basal expression of
Fra-2 was detectable in all cells. In CEF transformed by v-src,
ND-raf, and c-H-ras, Fra-2 formed a broad band of 41 to 46
kDa, compared with a sharper, thin band of 41 to 42 kDa
detected in DS3-infected CEF. After bacterial alkaline phosphatase treatment of the immunoprecipitates, the protein
bands of all cells migrated as sharp 41-kDa bands, indicatingthat the slowly migrating population of the protein represents
hyperphosphorylated forms (Fig. 5B) as we have previously
observed in Fra-2 immediately after growth stimulation (39).
Phosphorylation of Fra-2 was most intensive in v-src-transformed cell and was also clear in ND-raf- or c-Ha-ras-transformed CEF. By comparing the dephosphorylated form of

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FIG. 4. supjun-1-infected CEF are resistant to transformation by several oncogenes. CEF were first infected with DS3(A) (A to C) or

supjun-J(A) (D to F) and 4 days later superinfected with FM4(B) (A and D), H-ras(B) (B and E), or N4(B) (C and F). At 4 days after

J. VIROL.

SUZUKI ET AL.

3532

(4

A
44
z

-

Q

7

I-

1- - ,v~tn

Xz z n

X

Z

C

h4

Q

x

ZZ

-

4W.-

ame 4m 4I

FIG. 6. Endogenous AP-l DNA-binding activity in nuclear extracts
from infected CEF. The AP-l-binding activity present in extracts
prepared from CEF infected with DS3, H-ras, ND-raf, or N4 (carrying

_~
>

.1

-

x,,

.-

f,

a
Z

46k>

p* phosphorylated

Fra-2

~ *

nonphosphorylated Ira-2

40ki-

C

1

2

3

4

5

v-Src

6

-

46kor

40kp

v-src) was determined by gel shift assays using a 32P-labeled DNA
probe containing the AP-1-binding site of the collagenase gene. Someof the nuclear extracts were treated with anti-Jun or anti-Fra-2 peptide
1 antiserum as well as preimmune serum.

Fr-a-2zso *- C-JUln

FIG. 5. Expression of Jun (A and C) aand Fra-2 (B and C) in
infected CEF. (A) Total cell lysate (80 V Lg of protein each) was
resolved on an SDS-l0% polyacrylamide gel, and proteins weredetected with anti-Jun antiserum, using thee ECL Western Blotting
Detection System. FSV, Fujinami sarcoma viirus. The open and closed
triangles indicate the positions of endogenous chicken c-Jun and JunD,
respectively. The arrowhead indicates the pos ition of human c-Jun. (B)CEF were labeled with [35S]methionine forprepared under denaturing conditions and treated with anti-Fra-2
peptide 2. Immunoprecipitates containing thie same amount of radioactivity were incubated without or with bacte rial alkaline phosphatase(BAP), resolved on an SDS-10% SDS polya crylamide gel, and visualized by fluorography after exposure of X-ray films for 32 days (-BAP)
or 12 days (+BAP). (C) supjun-J(A)-infect ed (lanes 2, 4, and 6) orDS3(A)-infected (lanes 1, 3, and 5) CEF were superinfected with
N4(B) as shown in Fig. 3J or E. After labeling
I h, Fra-2, c-Jun, and v-Src were immunopr(
peptide 2 (lanes I and 2), anti-Jun (lanes 3 an 4), and anti-v-Src (lanes
polyacryl5 and 6) antisera, respectively, resolved on anamide gel, and visualized by fluorography.

gcwitha[dwS]methaoninefor
Id
SDS-10%-S

Fra-2 protein, we can roughly estimate that the expression
levels in CEF transformed by v-src, H-ras, and ND-raf areabout three-, two-, and twofold greater than those in DS3infected CEF, respectively. Although v-myc-transformed cells
expressed low levels of nonphosphorylated or hypophosphorylated forms of Fra-2 as in DS3-infected CEF, v-ros-transformed CEF produced elevated levels of highly phosphorylated Fra-2 as in v-src-transformed CEF (data not shown). In
the CEF transformed by exogenously expressed fra-2, the Fra-2
expression level was increased about sevenfold (Fig. 5B).
When supjun-l-infected CEF were superinfected with N4(Fig. 3J), the cells were metabolically labeled with [35S]methionine and Fra-2 and Jun expression was analyzed by immunoprecipitation. The results indicated that expression levels of
Fra-2 and Jun as well as the extent of Fra-2 hyperphosphorylation were not affected by the expression of supJun-1 (Fig. 5C;
compare lane 1 and 2 or lanes 3 and 4), indicating that
supJun-1 suppresses the transformation not by affecting thecomponents of endogenous AP-1. We have also observed thatsupjun-1 does not affect expression of the exogenous v-src gene(Fig. 5C; compare lane 5 and 6).
We

next

determined the AP-1 DNA-binding activity in

gel shift
ND-raf-, and c-Ha-ras-transformed CEF,
analysis of nuclear extracts (Fig. 6). When a DNA probe

v-src-,

using

containing the AP-1 site from the human collagenase gene wasused, DS3-infected CEF generated a thin band of gel shiftcomplex, while greatly enhanced bands with the same mobility
were observed when CEF expressing v-src, ND-raf, or c-Ha-ras
were used. All of the shifted bands disappeared upon additionof either the anti-Jun or the anti-Fra-2 antiserum but wereinsensitive to a preimmune serum. The apparently increasedisAP-1-binding activity in the presence of preimmune
serum

possibly attributable to a carrier effect of increased protein
concentration. This result indicates that the Fra-2-c-Jun complex is mainly responsible for the band shifts and also for theenhancement of DNA-binding activity in these transformed
cells.

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VOL. 68? 1994

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FIG. 7. Expression levels of mRNAs that encode AP-1 components
in infected CEF. Levels offos,fra-2, c-jun, and junD transcripts in CEF
infected with DS3, H-ras, ND-raf, and N4 (carrying v-src) viruses wereanalyzed by RNase protection assay. Open triangles indicate positionsof untreated probes; closed triangles correspond to positions of probes
protected by the transcripts. Samples were analyzed in a 3.5% (fos,jun,and fra-2 transcripts) or 5% (for junD) polyacrylamide gel.

Elevated levels of Fra-2 and Jun proteins result from
regulation at the transcriptional level. CEF transformed by
v-src, ND-raf, and c-Ha-ras expressed elevated levels of Fra-2
and c-Jun proteins. We next tested the expression levels of
c-fos, fra-2, c-jun, and junD mRNAs by RNase protection
assay. As shown in Fig. 7, c-fos mRNA was undetectable in allof the logarithmically growing cells, while growth-stimulated
CEF expressed it clearly. The fra-2 and c-jun mRNA levels
were elevated two- to fourfold in v-src, ND-raf-, or c-Ha-rastransformed cells. In RNase protection assays, intactness andquantity of the RNA samples were tested by ethidium bromide
staining of the gel or by Northern blot analysis using a probe
for a housekeeping gene encoding GAPDH. This result suggest that the enhancement of Fra-2 and c-Jun expression inthese cells is mainly regulated at the transcriptional level.
Although junD mRNA expression in v-src-transformed cells
was slightly higher, the junD mRNA levels were similar among
other cells.
DISCUSSION

Although supFos-1 was initially designed as a specific suppressor of Jun family proteins by forming a nonfunctional andstable complex of supFos-1 and Jun (28), we have shown herethat it can also efficiently suppress the transformation inducedby fos family proteins. Since heterodimer formation withendogenous c-Jun is thought to be essential for the transformation by exogenously expressed Fos family proteins (38),supfos-1 would function as a competitive inhibitor of exogenous Fos in this case. Like supfos-l, supjun-J suppressed either

fos or jun transformation, and it is noteworthy that supjun-]
was a more potent suppressor of either fos or jun transformation (Table 1). This stronger suppression might be attributable
to the direct blocking of transcription by the AP-1 DNAbinding activity of supJun-1. On the other hand, non-DNAbinding heterodimers formed by supFos-1 are passively removed from the transcriptional system. supJun-1 forms
homodimers and heterodimers with c-Jun, as shown in vitro(Fig. 1), and stable heterodimeric complexes with Fra-2 weredetected in CEF (our unpublished result). We would expect
only a very limited transactivation activity in all of these homoor heterodimers, because the in vitro transcriptional analysis
indicated that a similar truncated Jun showed only marginaltranscriptional activity even in combination with full-length
Fos (1). supJun-1 shares some properties with A9 (20) andTam-67 (6), which were previously reported as transdominantmutants of Jun that lack transactivation domains. Both of themutants were shown to have inhibitory effects on AP-1mediated transcription in transient expression systems. Ourmodel of the inhibition mechanism favors the quenchingmechanism proposed by Brown et al. (6).
We found that cellular transformation induced by suchoncogenes as src, frs, yes, ras, and raf was suppressed by both of
the transdominant mutants. Therefore, we decided to analyze
the amounts of all known chicken AP-1 components (c-Fos,
Fra-2, c-Jun, and JunD) in transformed cells induced by eachof 13 oncogenes. The expression level of either Fra-2 or c-Jun
in these transformed CEF was about two to four times that of
the untransformed CEF, but we think that the elevation ofeither protein alone is not sufficient to cause the cellulartransformation; the amounts of exogenously expressed Fra-2
and c-Jun that induce cellular transformation are about sevenand five times, respectively, that of the endogenous protein in
infected CEF. Elevation of both proteins, however, would
contribute synergistically to the enhancement of AP-l DNAbinding activity (Fig. 6) and possibly also to the induction ofcellular transformation. Although we did not detect elevatedexpression from a reporter plasmid containing a single AP-1binding site in these transformed CEF, three- to fivefold
elevation was detected when a reporter with three tandem
repeats of an AP-1 site was used instead (our unpublishedresults).
Cellular transformation induced by v-ros or v-myc was notaffected by either of the transdominant suppressors, suggestingthat endogenous AP-1 function is not essential for theirtransforming activity. It is interesting that the nuclear oncogene v-myc and the gene encoding the transmembrane tyrosine
kinase v-Ros show clear differences in their induction levels ofAP-1 components. v-myc induced no change in the endogenous AP-1 components, indicating that the v-myc transformation pathway is independent of AP-1. v-ros-transformed cells
have elevated levels of c-Jun, although these enhancements are
not essential for cellular transformation. Since v-ros encodes atransmembrane tyrosine kinase and is a homolog of thesevenless gene, it might be expected to influence the ras and raf
signalling pathways. Therefore, we think that there is anotherpathway that alone is sufficient for the ros transformation.
Although in this study we did not address the phosphorylation status of the c-Jun protein in vivo, several reports haveindicated that phosphorylation of amino-terminal serines 63
and 73 of c-Jun accompanies the transformation of murine
cells induced by v-src, activated c-Ha-ras, and activated raf-1,
and it was also shown that phosphorylation is important for rat
fibroblast transformation induced by high-level expressions ofboth c-jun and activated c-Ha-ras (2, 3, 34). Two lines ofevidence, however, indicate that these findings are not directly

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c-jun

3533

3534

SUZUKI ET AL.

ACKNOWLEDGMENTS
We gratefully acknowledge fruitful discussions with A. Nomoto. We
thank T. Curran and P. Vogt for supplying pSP-fra-1 and pJun,
respectively. We thank T. Yoshida and H. Okuno for the construction
and characterization of FlRl virus. We also thank JCRB for supplying
pSPT-Hras and p627. We thank Y. Suzuki, E. Suzuki, and N. Masuda
for assistance in preparation of the manuscript.
This work was supported by a research grant from the Princess
Takamatsu Cancer Research Fund and by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science and Culture ofJapan. M. H. Sonobe is supported by a Fundacao de Amparo a
Pesquisa do Estado de Sao Paulo postdoctoral fellowship.

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applicable to the chicken cell system. First, the N-terminal sites
are apparently constitutively phosphorylated even in resting
CEF and are only modestly affected by mitogenic stimulation.
Second, a c-Jun mutant in which the N-terminal phosphorylation site is changed from a serine to an alanine residue retains
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specific DNA-binding activity. The 40-kDa Fra-2 protein synthesized in reticulocyte lysates also retains efficient dimerforming activity with c-Jun and binding activity to AP-1 sites.
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to be established.
We would like to point out that these two transdominantmutants have the particular advantage that they are downstream in the signal transduction pathways (2), and both
exhibit the blocking of cellular transformation caused by a wide
range of oncogenes. Since supFos-1 and supJun-1 have only amodest effect on normal growth, we are now introducing thesegenes into primary cells to look for a differentiation processthat involves endogenous AP-1 activation.

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