MOLECULAR AND CELLULAR BIOLOGY, June 2007, p. 3936–3950
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 27, No. 11
Ubiquitin-Independent Proteasomal Degradation of Fra-1 Is Antagonized
by Erk1/2 Pathway-Mediated Phosphorylation of a Unique
Jihane Basbous,1Dany Chalbos,2,3Robert Hipskind,1Isabelle Jariel-Encontre,1and Marc Piechaczyk1*
Institut de Ge ´ne ´tique Mole ´culaire de Montpellier, CNRS, 1919 Route de Mende, Montpellier F-34293, France1; INSERM U540,
Montpellier F-34090, France2; and University Montpellier I, Montpellier F-34000, France3
Received 20 September 2006/Returned for modification 12 November 2006/Accepted 10 March 2007
Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos,
controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and
abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that
of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2
activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled
protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic,
pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how
it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer
contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated
by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological
induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos,
belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent
degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos
degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that
are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the
G0/G1-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression
of the other Fos family proteins.
The transcriptional regulator AP-1 is actually a large family
of dimeric protein complexes that bind to DNA motifs (AP-1/
TRE) found in a wide array of target genes (16). It is involved
in the regulation of many biological processes at the cellular
level, such as proliferation, differentiation, apoptosis, and re-
sponse to stresses, up to the whole organism, where it is in-
volved in organogenesis, immune responses, and control of
cognitive functions, among others (28, 29, 33, 43, 59, 67). AP-1
is also implicated in a variety of pathological situations, notably
tumorigenesis, and certain components can be oncogenes
and/or tumor suppressors depending on the cell context (20,
33, 44, 65, 68).
Within the variety of transcription factors that form AP-1,
the best known ones are the members of the Fos family,
namely, c-Fos, FosB, Fra-1, and Fra-2, and those of the Jun
family, namely, c-Jun, JunB, and JunD (16, 43). They all share
two adjacent, highly conserved domains: the basic DNA-bind-
ing domain and the leucine zipper (LZ), which mediates
dimerization. Together, these domains are known as the bZip
region. Fos proteins must heterodimerize with other AP-1
components to acquire transcriptional competence. In con-
trast, Jun proteins can also function as homodimers, even
though heterodimerization with partners like Fos proteins is
favored. Importantly, AP-1 can act as either a positive or neg-
ative regulator of transcription depending on its composition,
the target gene, the cell context, and the signals received from
the environment (16).
c-Fos, the prototype of the family, and Fra-1 are the most
studied Fos proteins. They are expressed constitutively in a
limited number of tissues (33). However, the c-fos and fra-1
genes are best characterized as immediate early genes, as they
are rapidly induced, i.e., within 15 min and 2 h, respectively, in
many cell types by a variety of extracellular stimuli. They also
show differences in protein expression. For example, upon
mitogen stimulation of quiescent cells, c-Fos accumulates tran-
siently, disappearing within a few hours, whereas Fra-1 appears
later and persists well beyond the G1phase of the cell cycle.
Importantly, variations in abundances are not limited to c-Fos
and Fra-1 in cells reentering the cell cycle but also concern
other AP-1 family proteins, which are responsible for contin-
uous and dynamic changes in AP-1 dimer composition (35, 36,
38). It is noteworthy that c-Fos participates in the transcrip-
tional activation of fra-1 (8, 68) together with other compo-
nents that have yet to be fully elucidated.
The expression of c-Fos and Fra-1 is altered in many tumors
(44, 68). c-Fos is oncogenic in several in vitro and in vivo
settings (20, 33), while Fra-1 is not transforming on its own (20,
33). However, it is associated with tumor progression, where it
can contribute to cell survival (66), proliferation (7), and in-
vasiveness (7). To avoid the deleterious effects of improper
expression, both the c-fos and fra-1 genes are regulated at
multiple transcriptional and posttranscriptional levels. In par-
* Corresponding author. Mailing address: Institut de Ge ´ne ´tique
Mole ´culaire de Montpellier, CNRS, 1919 Route de Mende, Montpel-
lier F-34293, France. Phone: (33) 4 67 61 36 71. Fax: (33) 4 67 04 02 31.
?Published ahead of print on 19 March 2007.
ticular, their transcription is activated by various mitogen-ac-
tivated protein kinase (MAPK) pathways, and their protein
products are targeted by the Erk1/2 (48, 68) and Erk5 (62)
cascades. Physiological regulation of the c-Fos protein by the
Erk1/2 pathway has been more extensively studied than that of
Fra-1. Both Erk1/2 and their effector kinases Rsk1 and Rsk2
phosphorylate c-Fos (14, 15, 26, 45, 46, 50, 51, 60), which alters
both its degradation rate (15, 23, 50, 51) and its transcrip-
tional activity (45, 46, 50). Similarly, phosphorylation cata-
lyzed by the Erk5 cascade stabilizes c-Fos and enhances its
ability to activate reporter genes (62). Importantly also, the
pattern of AP-1 proteins, including Fra-1, is perturbed in
Ras-, Raf-, and Mek1-transformed cells, where Erk1/2 plays
a key role in the expression and the phosphorylation of
several of them (18, 42, 64).
As for many key cell regulators, the tight control of Fos
protein degradation rates is essential to ensure the correct
timing and level of expression. Thus far, only the mechanisms
of c-Fos breakdown have been studied in detail. Most of the
studies have been performed in two different experimental
contexts where the protein is massively nuclear. One is consti-
tutive expression during asynchronous growth, and the other is
transient induction during the G0-to-G1-phase transition fol-
lowing mitogen stimulation of quiescent cells. In both situa-
tions, c-Fos is unstable, with a half-life in the hour range, and
the bulk of the protein is degraded by the proteasome (1, 9, 23,
55) independently of prior ubiquitylation (9), although a frac-
tion of c-Fos can undergo ubiquitylation in vivo in certain
circumstances (9). This is unusual, as most substrates require
polyubiquitylation to be addressed to and/or processed by the
proteasome (22). In line with this observation and the fact that
c-Fos is not detectably ubiquitylated in serum-stimulated cells
(9), Sasaki et al. recently reported the lack of ubiquitylation of
unstable, nuclear c-Fos induced by tetradecanoyl phorbol ac-
etate (56). However, they also elegantly showed that c-Fos,
when retained in the cytoplasm upon activation of the STAT3
pathway in the presence of the inactive Erk5 kinase pathway, is
subjected to ubiquitylation-dependent and proteasome-depen-
dent degradation (56). These data raise the possibility that
alternative pathways may contribute to c-Fos degradation in
different subcellular compartments. They may also explain why
c-Fos was originally found to be more unstable in the cyto-
plasm than in the nucleus (53).
Another peculiarity of c-Fos is that its breakdown is con-
trolled by differentially regulated autonomous destabilizers lo-
cated at its two extremities (9, 23). A C-terminal element is
functional in c-Fos during both asynchronous growth and the
G0-to-G1-phase transition, whereas an N-terminal destabilizer
is active only in G0/G1cells (9, 23). Moreover, the cytoplasmic
degradation of c-Fos described previously by Sasaki et al. de-
pends on a single destabilizer colocalizing with the N-terminal
one active in G0/G1cells (56). Further work will establish
whether the c-Fos N-terminal region contains a single or two
distinct destabilizing elements. Importantly, the activity of the
C-terminal destabilizer is reduced upon the phosphorylation of
two C-terminal serines by Erk1/2 and Rsk1/2 (9, 23). As a
consequence, this domain is less active in G0/G1cells, where
the Erk1/2 pathway is strongly activated, than in asynchro-
nously growing cells, where Erk1/2 is at best weakly active (48).
c-Fos turnover is, however, maintained in G0/G1phase by the
functional activation of the N-terminal destabilizer via an un-
defined mechanism (9, 23). Finally, the expression of Ras,
Mos, and Raf oncogenes, which activate the Erk1/2 pathway
(48), also inhibits c-Fos C-terminal destabilizer activity in pro-
liferating transformed cells.
There are a number of reports suggesting that Fra-1 is also
an unstable protein that is stabilized upon Erk1/2 MAPK path-
way activation. Thus, in the case of high Erk1/2 pathway activ-
ity resulting from either physiological stimulation by mitogens
(26, 49) or oncogenic activation of upstream signaling effectors
in thyroid (13), colon (66), and breast (7) cancer cells, Fra-1
accumulates to high levels and shows a characteristic diffuse
and retarded electrophoretic mobility due to phosphorylation
at multiple, unmapped sites. By contrast, Fra-1 shows reduced
phosphorylation and destabilization when Erk1/2 activity is
reduced, as is the case in control nontransformed thyroid cells
(13), in cells treated with a pharmacological inhibitor (66), or
upon mitogen withdrawal (26, 49). Finally, pharmacological
inhibition of the proteasome in colon tumor cells with a high
content of stabilized Fra-1 led to a further overaccumulation of
the latter protein. This suggested that the turnover of at least
a fraction of Fra-1 is controlled by proteasomal degradation
Several lines of evidence suggest that c-Fos and Fra-1 may
be degraded by, at least partially, similar mechanisms. In ad-
dition to the bZip domain, the two proteins show a second
region of high homology at the C terminus, where one of the
two destabilizers of c-Fos is located (9, 23). Several serines and
threonines phosphorylated by Erk1/2 and Rsk1/2 in c-Fos are
conserved in Fra-1 (see Fig. 1A and below) (45, 46, 49, 50, 69),
where only Thr231 of rat Fra-1 has been studied in depth (69).
Finally, the activation of the Erk1/2 and Erk5 pathways stabi-
lizes both c-Fos and Fra-1 (62). Nevertheless, the two proteins
show differences in their turnovers. For example, in Swiss 3T3
mouse embryo fibroblasts (MEFs) undergoing a G0-to-G1-
phase transition, Fra-1 has a half-life of more than 5 h, while
that of c-Fos is 45 min (26). To elucidate the molecular basis of
Fra-1 degradation and that of its physiological and pathologi-
cal Erk1/2 pathway-dependent stabilization, we have under-
taken here genetic and signaling studies of MEFs and several
cancer cell lines.
MATERIALS AND METHODS
Plasmids, cloning, and mutagenesis. Mutagenesis and clonings were carried
out using standard PCR-based techniques with a mutagenesis kit from Strat-
agene. All mutants were entirely sequenced. All details on expression plasmids
are available upon request. Table 1 summarizes the names of the Fra-1 variants
and chimeras, the mutations, and the name of the expression vectors. The
Myc2K/R–Fra-1K/R cDNA was synthetized by Genecust. The parental human
Fra-1 cDNA plasmid is a gift from H. Iba. The pIRES2-EGFP plasmid was
obtained from Clontech. The serum-inducible PM302 vector, which contains a
c-Fos insert, was described previously by Acquaviva et al. (1). The vectors for
mouse Mos (pcDNA3-HA-Moswt; vector CD294) and kinase-dead MosKD
(pcDNA3-HA-MosKD; vector CD295) (39) were kind gifts from S. Leibovitch.
The pECE-HA/p45mapkkDDvector (CD511) (11), a kind gift from G. Pages,
expresses a hemagglutinin-tagged version of the hamster MEK1 kinases in which
S218 and S222 have been mutated in D. All plasmids were purified using a
Cell culture and transfections. All cells were grown in Dulbecco’s modified
Eagle medium containing 10% fetal calf serum. HeLa, BALB/c 3T3, MCF7,
LS174T, and HCT116 cells are available from the American Type Culture Col-
lection. Transient transfections were carried out under standardized conditions
using the Jet PEI transfection reagent (Ozyme). Usually, 2 ?g of plasmid was
VOL. 27, 2007Fra-1 PROTEASOMAL DEGRADATION3937
used per 0.5 ? 106cells. Stable transfectants of BALB/c fibroblasts were ob-
tained after cotransfection of PM302-based Fra-1 expression plasmids and the
neomycin gene-expressing pCDNA3 vector (Clontech). Transfected cells were
selected in the presence of 1 mg/ml G418 and pooled for analysis. For serum
synchronization experiments, BALB/c 3T3 cells were serum starved for 36 h to
arrest them in G0phase and stimulated with fresh medium containing 20% fetal
calf serum. UO126 (Cell Signaling) was usually added at a concentration of 40
?M. To eliminate the possibility of UO126 exhaustion during the experiments,
efficient inhibition of the Erk1/2 pathway was always verified in immunoblotting
experiments with anti-phospho-Erk1/2 antibodies. MG132 (BiolMol) and epoxo-
mycin (Affiniti) were used at concentrations of 5 and 1 ?M, respectively.
Immunoblotting analysis and antibodies. Immunoblotting analyses were car-
ried out as previously described after the electrotransfer of sodium dodecyl
sulfate–10% polyacrylamide gel electrophoresis-fractionated proteins onto poly-
vinylidene difluoride membranes and using the Western Lightening chemilumi-
nescence kit from Perkin-Elmer Life Sciences (10). Luminograms of appropriate
exposures on Kodak X-OmatAR films are presented in the figures. When
necessary, bioluminescent signals were quantified using the camera-based
GeneGnome system from Syngene BioImaging and the GeneTool analysis program
provided by the supplier. Home-prepared 9E10 monoclonal antibody was used to
detect Myc-tagged proteins. All other antibodies were rabbit antisera. The anti-
enhanced green fluorescent protein (EGFP) antiserum was obtained from
Roche, and that against GAPDH (glyceraldehyde-3-phoshpate dehydrogenase)
was homemade. Erk1/2 was detected using the anti-p42/44 MAPK antibody and
phospho-Erk1/2 with the anti-phospho-p42/44 MAPK (Thr202/Tyr204) antibody
from Cell Signaling. Anti-Fra-1 antibodies (sc183) were from Santa Cruz Bio-
technology. The anti-phosphoserine 252 and 265 antibodies were obtained after
immunization of rabbits with the CSAHRKSpSSSSGD and SSDPLGpSPTLLAL
peptides coupled to keyhole limpet hemocyanin, respectively. They were pro-
duced and affinity chromatography purified by Eurogentec (Belgium). The pu-
rification procedure involved two successive steps using phosphorylated and
nonphosphorylated forms of the above-described peptides. Specific blocking by
phosphorylated peptides, but not by the nonphosphorylated ones, was verified
both in enzyme-linked immunosorbent assays using the phosphorylated peptides
as targets and by immunoblotting using extracts from HeLa cells cotransfected
with plasmids for wild-type Fra-1 and Mos. The horseradish peroxidase-conju-
gated anti-mouse and anti-rabbit immunoglobulin secondary antibodies were
obtained from Sigma.
Pulse-chase experiments. Twenty-four hours after transient transfection of
pIRES2-EGFP-based vectors for Fra-1, Fra-1-2S/A, and Fra-1-2S/D, 107cells
were radiolabeled in the presence of 150 ?Ci of a [35S]methionine-cysteine
mixture (NEG-772; Perkin-Elmer Life Sciences) per ml of culture medium for
1 h. Chases and cell lysis were conducted as described previously (9, 23). Immu-
noprecipitations were carried out with an anti-Fra-1 antibody (sc183; Santa Cruz
Biotechnology) using 20 ?l of magnetic protein G Dynabeads (Dynal). The
immunocomplexes were directly resuspended in the electrophoresis loading
buffer and fractionated through sodium dodecyl sulfate-containing 12% poly-
acrylamide gels. The quantification of signals on dried gels was performed using
the 445SI PhosphorImager 5 device from Molecular Dynamics and the Image-
Sequence alignments. Sequence alignments were performed using the Clustal
W (1.83) program available from EMBL-EBI (www.ebi.ac.uk/).
c-Fos and Fra-1 C-terminal domains. Our study of Fra-1
(271 amino acids) was prompted by its structural similarity with
c-Fos (380 amino acids) (Fig. 1A) and, in particular, the ho-
mology revealed by the alignment of their C-terminal domains
(Fig. 1B). In c-Fos, serines 374 and 362 are phosphorylated by
Erk1/2 and Rsk1/2, respectively. This facilitates the docking of
Erk1/2 to the upstream DEF (docking site for erk, FXFP)
domain FTYP and the subsequent phosphorylation of threo-
nines 325 and 331 (49, 50). Phosphorylation of S362 and S374
leads to c-Fos stabilization (15, 23, 50, 51), and that of T325
and T331 increases transactivation by c-Fos (45, 46, 50).
Serines and threonines are found at the equivalent positions in
Fra-1 (S265, S252, T223, and T217), as is the DEF domain.
Three additional threonine-proline motifs (T227, T230, and
FIG. 1. Comparison of Fra-1 and c-Fos and analysis of mutants.
(A) Structures of c-Fos and Fra-1. The bZIP domain is central. It is the
most conserved region between c-Fos and Fra-1. The second region of
high homology is the C-terminal domain of both proteins. DBD, DNA-
binding domain. (B) Comparison of c-Fos and Fra-1 C-terminal do-
mains. The C-terminal 80 amino acids of human c-Fos were compared
with the homologous region in human Fra-1 using the Clustal W
program. Identical amino acids are indicated with*, conserved ones
are indicated with :, and semiconserved ones are indicated with ●. The
Erk1/2 and Rsk1/2 target sites and the DEF domain of c-Fos are boxed
together with their conserved equivalents in Fra-1. The other putative
Erk1/2 target sites of Fra-1 are also boxed. (C) Structure of bicistronic
expression plasmids and principle of the immunoblotting assay.
pIRES2-EGFP is a CMV promoter-based eukaryotic expression vec-
tor. Wild-type (wt) and mutant Fra-1 proteins and EGFP chimeras are
cloned in a multicloning site (MCS) linker situated upstream of an
encephalomyocarditis virus internal ribosome entry site (IRES)-EGFP
expression cassette. Asynchronous cell cultures were transfected in
parallel with pIRES2-EGFP-based plasmids. Total cell extracts were
prepared 16 to 24 h later, which is a time that is sufficient to reach
equilibrium in protein accumulation. They were subsequently analyzed
by immunoblotting using specific antibodies against EGFP and the
protein to be analyzed (most often the 9E10 anti-Myc tag monoclonal
antibody). Note that for EGFP chimeras, a Myc6tag entails electro-
phoretic retardation (approximately 10 kDa) that allows easy discrim-
ination with EGFP used as an internal reference. Protein decay can, in
the first approximation, be considered to be exponential, which implies
that the relative steady-state levels of different proteins synthetized at
the same rate are (nearly) proportional to their half-lives. The com-
parison of protein-to-be-analyzed/EGFP ratios between samples,
which can be determined by densitometry scanning of luminograms or
direct chemiluminescence signal quantification with a camera-based
system, therefore gives the relative stabilities of compared proteins.
Importantly, using EGFP as an internal standard and comparing such
ratios intrinsically corrects for variations in protein synthesis rates,
whether those are due to slight differences in transfection efficiencies
or to variations in CMV promoter activity resulting from an alteration
of intracellular signaling (see Results).
3938 BASBOUS ET AL.MOL. CELL. BIOL.
T240) lie in the vicinity of the Fra-1 DEF motif and therefore
represent other potential sites for phosphorylation by Erks
Erk1/2 pathway-driven phosphorylations of serines 252 and
265, but not those of threonines 217, 223, 227, 230, and 240,
stabilize Fra-1 in asynchronous cells. We first addressed the
phosphorylation and stabilization of Fra-1 by the Erk1/2 path-
way in exponentially growing cells to determine if the two
events were linked directly or indirectly. A series of nonphos-
phorylatable and phosphomimetic mutants were generated in
human Fra-1 at positions corresponding to known Erk1/2 and
Rsk1/2 target sites in c-Fos (Fig. 1B and 2A and Table 1). The
stability of these mutants was evaluated in HeLa cells that
display low, albeit somewhat variable, Erk1/2 activity. We de-
veloped an assay system that is simpler than pulse-chase anal-
ysis to measure the relative stabilities of the numerous wild-
type, mutant, and chimeric proteins in our study (Fig. 1C). This
assay involves parallel transfections of asynchronous cells with
expression vectors derived from pIRES2-EGFP (Fig. 1C) that
give rise to both the proteins under study and EGFP from the
same bicistronic mRNA. The steady-state levels of the proteins
were analyzed using immunoblotting. EGFP, which is highly
stable in our system (23), serves as an internal standard for
normalization and allows the estimation relative protein sta-
bilities, if needed (see the legend of Fig. 1C). This approach
was validated (not shown) by verifying that differences in the
accumulation of c-Fos mutants relative to that of the wild-type
protein correlated with differences in half-lives measured by
pulse-chase in a previous work (23).
In Fra-1, individual phosphomimetic mutants of S252 (Fra-
1-S252D) and S265 (Fra-1-S265D) were both stabilized (Fig.
2B), with Fra-1-S265D appearing to be more stable than Fra-
1-S252D. Notably, the double S252D-S265D phosphomimetic
mutant (Fra-1-2S/D) showed a cumulative effect (Fig. 2B). In
contrast, the nonphosphorylatable Fra-1-S265A mutant and
the double S252A-S265A mutant (Fra-1-2S/A) were slightly
TABLE 1. Correspondences between names of Fra-1 mutants and chimeras, mutations, and protein structures and plasmid names
pIRES2-based expression vectors for
T217A, T223A, T227A, T230A
T217A, T223A, T227A, T230A, S252D, S265D
T217D, T223D, T227D, T230D
T217A, T223A, S252D, S265D
T240A, S252A, S265A
T240D, S252A, S265A
T240A, S252D, S265D
T240D, S252D, S265D
Lysineless mutant of Myc2-tagged Fra-1
Lysineless mutant of Myc2-tagged Fra-1; ?262-271
Lysineless mutant of Myc2-tagged Fra-1; S252A, S265A
Lysineless mutant of Myc2-tagged Fra-1; S252D, S265D
pIRES2-based expression vectors for
Myc6–EGFP–Fra-1 amino acids 231-271; S252A, S265A
Myc6–EGFP–Fra-1 amino acids 221-271
Myc6–EGFP–Fra-1 amino acids 231-271
Myc6–EGFP–Fra-1 amino acids 241-271
Myc6–EGFP–Fra-1 amino acids 251-271
Myc6–EGFP–Fra-1 amino acids 261-271
Myc6–EGFP–Fra-1 amino acids 231-271; S252D, S265D
PM302-based expression vectors for
VOL. 27, 2007Fra-1 PROTEASOMAL DEGRADATION3939
less stable than Fra-1, likely reflecting a low level of phosphor-
ylation-dependent stabilization of wild-type Fra-1 by weak
basal Erk1/2 pathway activity in our HeLa cells. Strikingly,
Fra-1-S252A was not destabilized, confirming the observation
with the S-to-D mutants that the phosphorylation of S252 has
a weaker effect than that of S265 on Fra-1 turnover (Fig. 2B).
Since phosphorylation occurs preferentially at sites that are
N terminal to DEF motifs after the docking of Erk1/2 (21, 50),
we analyzed mutants in threonines 217, 223, 227, and 230 alone
or in combination with mutants in S252 and S265 (Fig. 2C).
Mutation of T217 and T223 (Fra-1-2T/D), which are conserved
between Fra-1 and c-Fos, or of all four threonines (Fra-1-
4T/D) into alanines curiously led to a slight stabilization. This
increase (on average less than 50%) was modest relative to that
found with Fra-1-2S/D, either alone or together with the four
T-to-A mutants (Fra-1-4T/A-2S/D). Similarly, no increase in
stability was seen upon the mutation of the four threonines
into aspartic acids (Fra-1-4T/D). Taken together, these data
indicate that the phosphorylation of these threonines plays no
crucial role in the stabilization of Fra-1 turnover. Instead, it
may slightly attenuate the stabilizing effect of S252 and S265
phosphorylation. Given this minor effect, these threonines
were not investigated further.
We also studied the potential role of T240, located down-
stream of the DEF motif (Fig. 2D). Neither its mutation in D
nor its mutation in A affected Fra-1 accumulation, either alone
(Fra-1-T240D and Fra-1-T240A, respectively) or with S252
and S265 mutated to D (Fra-1-T240D-2S/D and Fra-1-T240A-
2S/D) or A (Fra-1-T240D-2S/A and Fra-1-T240A-2S/A). Im-
portantly, all stabilized constructs, like wild-type Fra-1, localized
predominantly within the nucleus in indirect immunofluores-
cence assays (not shown), indicating that stabilization was not
FIG. 2. Relative stabilities of C-terminal-domain serine and threonine mutants of Fra-1. pIRES2-EGFP-based expression plasmids of the
various Fra-1 mutants were transfected in asynchronously growing cells. Immunoblotting experiments were carried out 24 h later using total cell
extracts. Fra-1 proteins and EGFP were visualized using the 9E10 anti-Myc tag monoclonal antibody and an anti-EGFP antiserum, respectively.
(A) Structure of Fra-1 mutants. DBD, DNA-binding domain. (B) Analysis of serine 252 and serine 265 mutants in HeLa cells. (C) Analysis of
threonine 217, 223, 227, and 230 mutants in HeLa cells. (D) Analysis of threonine 240 mutants in HeLa cells. (E) Analysis of Fra-1 serine mutants
in BALB/c 3T3 fibroblasts. (F) Analysis of Fra-1 serine mutants in LS174T cells. (G) Analysis of Fra-1 serine mutants in MCF7 cells.
(H) Pulse-chase analysis of Fra-1, Fra-1-2S/A, and Fra-1-2S/D in HeLa cells. HeLa cells were transfected to express Fra-1, Fra-1-2S/A, and
Fra-1-2S/D, and pulse-chase analyses were carried out as described in Materials and Methods. All data presented are representative of at least
three independent experiments.
3940 BASBOUS ET AL.MOL. CELL. BIOL.
a direct consequence of gross intracellular protein redistribu-
tion. Moreover, expression of wild-type Fra-1, Fra-1-2S/A, and
Fra-1-2S/D in three other cell lines with low Erk1/2 pathway
activity, namely, BALB/c 3T3 mouse embryo fibroblasts,
LS174T human colon carcinoma cells, and MCF7 human
breast cancer cells, gave results similar to those described
above (Fig. 2E to G). This strongly suggests that the regulation
of Fra-1 stability by modification of serines 252 and 265 is not
cell type specific.
Pulse-chase experiments (Fig. 2H) were then used to esti-
mate the half-lives of wild-type Fra-1, Fra-1-2S/A, and Fra-1-
2S/D in asynchronous HeLa cells. Those of Fra-1 and Fra-1-
2SA were 80 ? 15 and 45 ? 15 min, respectively. This matched
the twofold difference usually seen in steady-state-level assays
such as the one presented in Fig. 2B and further validated that
approach. Consistently, with the 10- to 20-fold higher accumu-
lation of Fra-1-2S/D compared with Fra-1 in parallel transfec-
tion experiments, we were unable to determine the half-life of
Fra-1-2S/D, as only an approximately 30% decay could be
monitored in an 8-h chase.
Next, we demonstrated that the activation of the Erk1/2
pathway leads to the stabilization of Fra-1 via the phosphory-
lation of S252 and S265. The Erk1/2 and the Erk5 cascades are
blocked by U0126, a pharmacological inhibitor of the Erk1/2-
activating kinases Mek1 and Mek2 as well as of the Erk5
activator Mek5 (see references 34 and 48) (Fig. 3A). In con-
trast, the Erk1/2 cascade is activated selectively upon ectopic
expression of the Mek1/2 kinase Mos or of an activated form of
the Erk1/2-activating kinase Mek1 (MEK1DD). HeLa cells
were transfected with expression vectors for single or double
alanine mutants of S252 and S265 either alone or together with
one for Mos or a mutant Mos lacking kinase activity (MosKD).
The activation of Erk1/2 by Mos, but not by MosKD, was
shown by immunoblotting using antibodies specific to residues
phosphorylated by Mek1/2 (34, 48) (Fig. 3B). Mos led to in-
creased levels of EGFP, presumably by stimulating the pro-
moter activity of the pIRES2-EGFP-derived expression vectors.
More importantly, Mos induced much stronger expression of
wild-type Fra-1 than Fra-1-2S/A, indicating stabilization of the
former protein but not of the latter. Interestingly, the single S
mutants showed intermediate degrees of stabilization (Fig.
3B), with Fra-1-S252A being higher than Fra-1-S265A. This is
consistent with the increased accumulation of Fra-1-S265D
relative to that of Fra-1-S252D in Fig. 2B. Notably, all proteins,
including Fra-1-2S/A, showed retarded electrophoretic mobil-
ity in the presence of Mos. This is most likely due to the
phosphorylation of the DEF domain-proximal threonines
T217, T223, T227, and T230, as suggested previously by other
researchers (see above), since Fra-1-4T/D (Fig. 2C) and an-
other Fra-1-4T/D-2S/A (not shown) were also subjected to
electrophoretic retardation. These phosphorylations were not
further investigated because of the lack of an effect on Fra-1
stability in our setting. Identical results were obtained in
BALB/c MEFs and upon cotransfection of wild-type and mu-
tant Fra-1 with expression vectors for MEK1DD and Erk1 in
HeLa cells (not shown).
To facilitate further studies, we developed specific antisera
against Fra-1 peptides phosphorylated on S252 and S265. In
HeLa cells cotransfected with expression vectors for Mos and
either wild-type or serine-mutated Fra-1, these antisera specif-
ically detected the corresponding phosphorylated protein in
immunoblots (Fig. 4A). This indicates that these serines are
actual targets of the Erk1/2 pathway and that the phosphory-
lation of one serine was not dependent on that of the other.
FIG. 3. S252 and S265 phosphorylation-dependent stabilization of
Fra-1 upon Erk1/2 pathway activation. (A) Erk1/2 pathway. Erk1 and
Erk2 MAPKs (MAP kinase) (Erk1/2) are activated upon phosphory-
lation by the Mek1 and Mek2 MAPK kinases (Mek1/2). The UO126
drug can reversibly inhibit the latter as well as Mek5. Mek1/2 is itself
activated upon phosphorylation by kinases such as Raf and Mos. The
activation of Raf is controlled by Ras small GTPases, which also
control other pathways. Erk1/2 phosphorylates its substrates at S/T-P
motifs. It activates the Rsk1 and Rsk2 MAPK-activated kinases. Raf
and Mos specifically control the Erk1/2 pathway. (B) Effect of Mos on
the S252 and S265 mutants of Fra-1. HeLa cells were transfected to
express the indicated proteins (Fig. 2A) in the presence or in the
absence of vectors for the wild type (Mos) or a kinase-dead mutant
(MosKD) of Mos. Note that EGFP levels were higher in the presence
of Mos due to the stimulation of the CMV promoter. Taking this point
into account, chemiluminescent quantification indicated the absence
of Fra-1-2S/A stabilization. The level and the activation of Erk1/2 were
assayed using anti-Erk1/2 and anti-phospho-Erk1/2 antisera. The data
presented are representative of three independent experiments.
VOL. 27, 2007 Fra-1 PROTEASOMAL DEGRADATION3941
We used UO126 to block the activation of the Erk1/2 cas-
cade by Mos in HeLa cells coexpressing it and Fra-1. Fra-1
returned to its faster-migrating form in sodium dodecyl sulfate-
polyacrylamide gels within 2 h of inhibitor addition, and its
levels decreased by two- to threefold at most after 8 h of
inhibition (Fig. 4B). Thus, in the presence of UO126, Fra-1
had not recovered its original half-life of less than 1 h in its
unphosphorylated state (Fig. 2H). Immunoblots showed that
the dephosphorylation of S252 was fast in the presence of
UO126, while that of S265 was slower (Fig. 4B). This explained
why Fra-1 was not fully destabilized. This finding also raised
the possibility that basal levels of Erk1/2 activity may be nec-
essary for the efficient dephosphorylation of Fra-1-S265, since
UO126 treatment diminished Erk1/2 activity below that found
in nontreated HeLa cells (Fig. 4B). However, we cannot rule
out that the phosphatase specific for P-S265 may have been
rate limiting in this overexpression experiment. Notably, the
slowed electrophoretic mobility of both Fra-1-2S/A and Fra1-
2S/D induced by the coexpression of Mos is reversed by the
addition of UO126 (Fig. 4C). This indicates that the slowed
migration of Fra-1 essentially reflects its modification at mul-
tiple sites that are not S252 and S265.
Taken together, these data identify S252 and S265 as being
targets of the Erk1/2 pathway and implicate their phosphory-
lation, and not that of other putative Erk1/2 phosphorylation
sites, as the principal regulator of Fra-1 stability.
Phosphorylation of serines 252 and 265 in high-Erk1/2-ac-
tivity-displaying HCT116 cancer cells is responsible for stabi-
lization of Fra-1. HCT116 colon cancer cells display 10-fold-
higher Erk1/2 activity than LS174T, as estimated from
immunoblotting quantification of phospho-Erk1/2 in the two
cell types (Fig. 5A). This correlates with a high level of Fra-1
showing reduced electrophoretic mobility, reflecting hyper-
phosphorylation (66). We used several strategies to address
whether this high Fra-1 accumulation is contributed by S252
and S265 phosphorylation-dependent protein stabilization. In
FIG. 4. Erk1/2 pathway-induced phosphorylations of S252 and S265 and Fra-1 stabilization. (A) Erk1/2 pathway-induced phosphorylations of
Fra-1 S252 and S265. HeLa cells were transfected to express the indicated proteins (Fig. 2A) in the presence or in the absence of Mos. Total cell
extracts were probed with the various rabbit antisera designated for the detection of total Fra-1 (Fra-1), S252-phosphoylated Fra-1 (P-S252-Fra-1),
and S265-phosphorylated Fra-1 (P-S265-Fra-1) as well as total Erk1/2 and phosphorylated Erk1/2 (P-Erk1/2). The differences in Fra-1-2S/A
abundances in the presence and in the absence of Mos result largely from differences in the transcriptional activity of the expression vector as
shown in Fig. 3. (B) UO126 chase of HeLa cells coexpressing Fra-1 and Mos. The UO126 chase was started 16 h after cotransfection of
asynchronous HeLa cells transfected with Fra-1 and Mos expression vectors. Immunoblotting experiments were conducted with extracts from cells
taken at various time points. (C) UO126 chase in HeLa cells coexpressing Mos with either Fra-1-2S/A or Fra-1-2S/D. The experiment was carried
out as in B. ? corresponds to 8 h in the presence of UO126. The data presented are representative of three independent experiments.
FIG. 5. Stabilization of Fra-1 in HCT116 colon cancer cells.
(A) Relative Erk1/2 activities in HCT116 and LS174T cells. Cell ex-
tract immunoblots were probed with antibodies specific for the indi-
cated proteins. (B) Phosphorylation of endogenous Fra-1 on S252 and
S265. Extracts from nontreated cells and cells treated with UO126 for
16 h were analyzed by immunoblotting for visualization of the indi-
cated proteins. (C) Destabilization of overexpressed ectopic Fra-1 in
HCT116 cells upon Erk1/2 pathway inactivation. HCT116 cells were
transiently transfected in the presence or in the absence of UO126 for
16 h to inhibit the phosphorylation of Fra-1. Levels of ectopic total
Fra-1 were assayed with the 9E10 antibody, and S252-phosphorylated
Fra-1 and S265-phosphorylated Fra-1 were assayed with the relevant
anti-phosphoserine antiserum. Transfected Fra-1 was expressed well
over the level of endogenous Fra-1, which avoided interference with
endogenous Fra-1 when probing with the latter two antisera. The
efficiency of UO126 was demonstrated by assaying the levels of phos-
phorylated and nonphosphorylated Erk1/2. (D) Relative stabilities of
Fra-1, Fra-1-2S/A, and Fra-1-2S/D. Transfection experiments were
performed as described in the legend of Fig. 2.
3942 BASBOUS ET AL.MOL. CELL. BIOL.
a first step, we used immunoblotting to show that endogenous
Fra-1 is phosphorylated on S252 and S265 in HCT116 cells
(Fig. 5B). Next, the addition of UO126 to HCT116 cells led to
the dephosphorylation of S252 and S265, which correlated with
an 8- to 10-fold reduction in protein levels in a 16-h chase (Fig.
5B). Next, before resorting to the analysis of Fra-1 mutants, we
tested whether exogenous Fra-1 behaves similarly to the en-
dogenous protein. This was the case: when overexpressed by
the transfection of a bicistronic expression vector, the level of
exogenous wild-type Fra-1 decreased by approximately 10-fold
upon U0126 treatment, which correlated with faster electro-
phoretic mobility and the absence of phosphorylation of S252
and S265 (Fig. 5C). Finally, transfected Fra-1 accumulated to
a level comparable to that of Fra-1-2S/D in parallel transfec-
tion experiments, in contrast to the Fra-1-2S/A level, which was
much lower (Fig. 5D). Notably, the latter data contrasted with
those obtained using LS174T cells, where Erk1/2 activity was
low and where Fra-1 and Fra-1-2S/A showed only a modest
difference in their level of accumulation, whereas Fra-1-2S/D
accumulated to a high level (Fig. 2F).
Thus, while they do not exclude that other mechanisms also
contribute to the high accumulation of Fra-1 in HCT116 cells,
these data, when taken together, strongly suggest an important
role of the Erk1/2 pathway in Fra-1 stabilization via the phos-
phorylation of S252 and S265.
Stability of Fra-1 during the G0/G1-to-S-phase transition is
due to Erk pathway-dependent phosphorylation of serines 252
and 265. Fra-1 levels are low in most nondividing, nontrans-
and return to an intermediate level by 16 to 24 h poststimulation,
depending on the cell type. During the G0/G1-to-S-phase transi-
tion, Fra-1 is phosphorylated and relatively stable but becomes
destabilized if mitogens are removed (see above). Moreover,
mitogenic stimulation leads to the strong activation of the
Erk1/2 cascade within minutes, which then decreases to a mod-
erate level that persists for hours. Therefore, we investigated
whether the Erk1/2 pathway-dependent phosphorylation of
S252 and S265 regulates the stability of endogenous Fra-1 in
the G0/G1-to-S-phase transition.
First, we assessed S252 and S265 phosphorylation during
a G0/G1-to-S-phase transition in quiescent BALB/c MEFs
stimulated for growth by serum. The typical kinetics of en-
dogenous Fra-1 accumulation are presented in Fig. 6A. The
phosphorylation of S252 and S265 was detectable 2 h after
serum addition. Thereafter, the decrease in phospho-S265
levels paralleled those of total Fra-1 and of active Erk1/2, while
phospho-S252 decayed more rapidly. The addition of UO126 2
h after stimulation by serum led to (i) the rapid dephosphor-
ylation of Fra-1, as visualized by its faster electrophoretic mi-
gration at the 4- and 8-h points, (ii) the loss of phospho-S252
and phospho-S265 signals as early as 4 h poststimulation, and
(iii) the faster decay of Fra-1 starting at 8 h (Fig. 6B, left).
Although not proof per se, the above-described observations
are compatible with the idea of Erk-dependent stabilization of
Fra-1 through the phosphorylation of S252 and S265. To ad-
dress this point formally, we next resorted to an expression
system permitting the pulsed expression of mRNAs coding for
wild-type Fra-1, nonphosphorylatable Fra-1-2S/A, and phos-
phomimetic Fra-1-2S/D at the beginning of the G0-to-G1-
phase transition. The relevant open reading frames were
cloned in a vector faithfully reproducing the transient accumu-
lation of c-fos mRNA at the beginning of the G0-to-G1-phase
transition (1), i.e., with mRNA expression peaking by 45 to 60
min and being back to the basal level 90 to 120 min after
stimulation owing to (i) a minimal c-fos promoter carrying the
serum-responsive element responsible for mitogen-induced
transcriptional switching on and off and (ii) the natural 3? c-fos
untranslated region harboring the main mRNA destabilizer
(Fig. 6C). Thus, ectopic proteins are no longer synthetized to
significant levels after 90 to 120 min, which means that the
analysis of their steady-state levels by immunoblotting at later
time points reflects their stability. Stably transfected BALB/c
3T3 MEFs were arrested in G0phase by serum deprivation and
then stimulated by the readdition of serum in the presence or
in the absence of UO126 (Fig. 6D). Importantly, as c-Fos
promoter activation necessitates an active Erk1/2 pathway (25,
30), the drug was added 1 h after stimulation, i.e., a time
sufficient to reach maximal levels of ectopic proteins. Levels of
the latter, as well as Erk1/2 levels and activity, were monitored
over time by immunoblotting. The data presented in Fig. 6E
show that (i) in the absence of UO126, the wild-type Fra-1
level remained nearly constant, except for a slight decrease by
16 h that correlated with a reduction in Erk1/2 activity and
partial dephosphorylation visualized by faster electrophoretic
mobility, (ii) Fra-1 was dephosphorylated and destabilized in
the presence of UO126, (iii) Fra-1-2S/A expression was tran-
sient whether UO126 was present or not, (iv) the similar ki-
netics of expression of Fra-1, in the presence of UO126, and
Fra-1-2S/A, in the presence and in the absence of UO126,
indicated that the addition of UO126 had no incidence on our
transient expression system, at least under the conditions used,
and (v) Fra-1-2S/D was stable whatever the experimental con-
ditions. Altogether, the data indicate that Fra-1 is an intrin-
sically unstable protein. However, during the period extending
from G0to S phase, its degradation is antagonized by Erk1/2
pathway-dependent phosphorylation of S252 and/or S265. The
data also indicate a half-life for nonphosphorylated Fra-1 in
the 1-h range, which is consistent with the value obtained in
pulse-chase experiments with cells showing low Erk1/2 activity
Delineation of the Fra-1 destabilizer. We next delineated
the regions in Fra-1 responsible for its instability. The stability
of Fra-1 C-terminal truncation mutants was compared to that
of the wild-type protein in asynchronous HeLa cells. The re-
moval of 10 amino acids led to approximately a 12- to 15-fold
stabilization, as deduced from densitomer scanning analysis of
the luminogram shown in Fig. 7A. Further deletions did not
enhance this effect. We then tested whether the fusion of Fra-1
C-terminal fragments to the C terminus of EGFP would de-
stabilize this normally very stable protein. The addition of an
N-terminal Myc6tag (78 amino acids) allowed us to differen-
tiate between the EGFP–Fra-1 chimeras and EGFP that
served as the control for expression. A 2.2-fold destabilizing
effect was already detectable with the last 30 amino acids of
Fra-1, as deduced from densitomer scanning of the lumino-
gram shown Fig. 7B, and the fusion of the last 40 or 50 amino
acids further destabilized EGFP with similar efficiencies (12-
fold) (Fig. 7B). Taken together, the above-described data in-
dicate that a unique structural determinant contained within
VOL. 27, 2007 Fra-1 PROTEASOMAL DEGRADATION3943
the last 30 to 40 amino acids is sufficient to destabilize Fra-1 in
As c-Fos contains two destabilizers that are active in G0/G1
phase, we considered the possibility that S252 and S265 phos-
phorylation inhibits not only the C-terminal destabilizer in
serum-stimulated cells but also another one located towards
the N terminus. To test this, we compared the stability of Fra-1
mutants lacking the last 30 (Fig. 7C), 40 (not shown), and 50
(not shown) amino acids to that Fra-1-2S/A, which is unstable.
All three deletion mutants were stable for at least 16 h after
serum addition. As they contain neither the inhibiting phos-
phorylation sites nor the C-terminal destabilizer, this con-
firmed the lack of an N-terminal destabilizer whose action
could be antagonized by the phosphorylation of serines 252
and 265 during the G0/G1-S period.
Proteasomal degradation of Fra-1. We then asked to what
extent Fra-1 is degraded by the proteasome. Asynchronous
HeLa cells were first transfected to express wild-type Fra-1,
Fra-1-2S/A, and Fra-1-2S/D, respectively, and then treated
with the proteasome inhibitors MG132 or epoxomycin for 8 h
before immunoblotting analysis (Fig. 8A). Fra-1 and Fra-1-
2S/A levels increased more than 20- to 30-fold, which is con-
sistent with their half-lives in the 1- to 1.5-h range and indicates
that most, if not all, Fra-1 undergoes proteasomal degradation.
In contrast, there was no detectable change in Fra-1-2S/D
abundance, in keeping with our observation that the half-life of
phosphorylated Fra-1 exceeds 8 h (Fig. 2G). This also ruled out
a significant contribution by another major proteolytic system
to Fra-1 turnover under these experimental conditions. Con-
sistently, Fra-1 decay induced by UO126 treatment in HCT116
FIG. 6. Wild-type and mutant Fra-1 stability during a G0/G1-to-S-phase transition. (A) Phosphorylation of Fra-1 in serum-stimulated cells.
BALB/c 3T3 fibroblasts were brought in G0phase by serum deprivation for 36 h. They were then stimulated for growth by the readdition of culture
medium containing 20% serum. Immunoblotting experiments were conducted with extracts from cells stimulated for various periods of time with
the indicated antibodies. GAPDH (not shown) and Erk1/2 were used as an invariant internal standards. (B) Fate and phosphorylation of Fra-1
in serum-stimulated cells treated with UO126. In the left panel, cells were treated as described above (A), except that UO126 was added 2 h after
stimulation with serum. The right panel corresponds to control cells treated in parallel with no UO126 addition and allows the visualization of the
faster Fra-1 decay in the presence of UO126. (C) Structure of transient expression vectors. Fra-1, Fra-1-2S/A, and Fra-1-2S/D open reading frames
were cloned in the PM302 vector after the removal of its original c-Fos insert (1). They were stably transfected in BALB/c 3T3 fibroblasts. UTR,
untranslated region; SRE, serum-responsive element. (D) Design of the synchronization experiment. Deprivation of and stimulation by serum of
the various cells stably transfected with the plasmids described in C were performed as described in A. When required, UO126 was added 1 h
poststimulation, which gives sufficient time for Fra-1 accumulation. Ectopic mRNA levels peaked by 45 to 60 min and were back to the basal level
by 90 to 120 min after serum addition (1). (E) Immunoblotting assays. Immunoblotting experiments were carried out as described above (A) on
the cells stably transfected with the various PM302-based vectors. The data presented are representative of at least three independent experiments.
3944 BASBOUS ET AL.MOL. CELL. BIOL.
cells (Fig. 5B) was blocked by MG132 (Fig. 8B). We also
verified in two steps that the stabilization of phosphorylated
Fra-1 in the G0/G1-to-S-phase transition was due to the antag-
onism of proteasomal degradation and not of another proteo-
FIG. 7. Delineation of the Fra-1 destabilizer. (A) Analysis of C-ter-
minal-truncation Fra-1 mutants. Various C-terminal-truncation mutants
were cloned in the pIRES2-EGFP expression vectors, and their relative
accumulation in transiently transfected HeLa cells was assayed as de-
scribed in the legend of Fig. 2. DBD, DNA-binding domain. (B) Analysis
of EGFP–Fra-1 chimera in asynchronous HeLa cells. The various chime-
ras were cloned in the pIRES2-EGFP vector for transfection analysis in
HeLa cells as described above (A). Immunodetections of Fra-1 proteins
and EGFP were performed together with an appropriate combination of
anti-Myc tag and anti-EGFP antisera. MCS, multiple cloning site; IRES,
internal ribosome entry site. (C) Analysis of Fra-1 mutants during the
express the indicated proteins from PM302-based plasmids (Fig. 6C),
were serum synchronized and analyzed as described in the legend of
Fig. 6. Fra-1 was immunodetected with anti-Myc monoclonal antibod-
ies, and GAPDH was used as an internal invariant control. The data
presented are representative of at least three independent experi-
ments. UTR, untranslated region.
FIG. 8. Proteasomal degradation of Fra-1. (A) Proteasome-depen-
dent degradation of Fra-1 in asynchronous cells. HeLa cells were trans-
fected with pIRES2-EGFP expression vectors to express the indicated
proteins. Twenty-four hours later, they were treated with MG132 or
epoxomycin for 8 h. Immunoblotting experiments were carried out as
described in the legend of Fig. 2. (B) Inhibition of endogenous Fra-1
decay in HCT116 cells treated with UO126. HCT116 cells were treated
with UO126, as described in the legend of Fig. 5B, in the presence of
MG132 before immunoblotting analysis. (C) Inhibition of endogenous
Fra-1 decay by MG132 in serum-stimulated fibroblasts treated with
UO126. BALB/c 3T3 cells were serum stimulated as described in the
legend of Fig. 6B. UO126 and MG132 were added 2 h poststimulation.
(D) Proteasomal degradation of Fra-1-2S/A during the G0-to-G1-phase
transition. BALB/c cells stably transfected to express Fra-1-2S/A from a
PM302-based vector (Fig. 6C) were serum deprived for 36 h and then
stimulated by the addition of 20% serum. MG132 or epoxomycin was
added 1 h later. Immunoblotting assays were performed as described in
the legend of Fig. 6 by using GAPDH as an internal control. The data
presented are representative of at least three independent experiments.
(E) Stabilization of unstable Fra-1 mutants by MG132. HeLa cells were
transfected with pIRES2-derived expression vectors coding for the indi-
cated proteins for 24 h and treated for another 8 h in the presence of
MG132 before immunoblotting analysis. Normalization of luminograms
with EGFP in three independent experiments indicates comparable levels
of protein accumulation in the presence of MG132.
VOL. 27, 2007 Fra-1 PROTEASOMAL DEGRADATION3945
lytic pathway. First, serum-stimulated BALB/c 3T3 fibroblasts
were treated with both UO126 and MG132 2 h after the ad-
dition of serum. Endogenous Fra-1 did not decay during the
experiments, even though its faster electrophoretic migration
indicated that it was hypophosphorylated (Fig. 8C), which
contrasted with its disappearance in the absence of MG132
(compare with the 16-h time point in Fig. 6B, left). Second,
quiescent BALB/c 3T3 cells stably transfected with the serum-
inducible nonphosphorylatable Fra-1-2S/A mutant were serum
stimulated and treated with either MG132 or epoxomycin 1 h
later. Under these conditions, Fra-1-2S/A was stable for 16 h
(Fig. 8D), which contrasted with its disappearance in the ex-
periments presented in Fig. 6E. Finally, we verified that the
instability of the nonphosphorylatable Fra-1 mutants was re-
versed in the presence of MG132. Indeed, all these mutants
accumulated to a level comparable to that of the most stable
phosphomimetic mutants processed similarly in parallel exper-
iments (Fig. 8E). This confirmed that the bulk of Fra-1 under-
goes proteasomal degradation under the experimental condi-
Degradation of the bulk of Fra-1 does not depend on its
prior ubiquitylation. Ubiquitin is conjugated to proteins at the
ε-amino group of lysines and, in rare cases, the N terminus
(22). We previously showed that a nonubitiquitylable c-Fos
mutant with all lysines turned into arginines and with the N
terminus blocked by a Myc tag undergoes properly regulated
degradation by the proteasome, which thereby demonstrated
that prior ubiquitylation was not necessary for c-Fos destruc-
tion (9). We therefore tested whether a similar mutant of Fra-1
(Myc2K/R–Fra-1K/R) would undergo proteasomal proteolysis
and show regulated degradation like the wild-type protein. In
parallel transient transfection assays in HeLa cells, Fra-1 and
Myc2K/R–Fra-1K/R accumulated to the same level (Fig. 9A).
Moreover, the degradation of both proteins was dependent on
the proteasome, as shown by the stabilization by MG132 (Fig.
9A), and on the presence of a C-terminal destabilizer, since
mutants lacking the C-terminal 10 amino acids were stabilized
(Fra-1/1-261 and Myc2K/R–Fra-1K/R/1-261 in Fig. 7A and 9B,
respectively). Finally, the degradation of Fra-1 and Myc2K/R–
Fra-1K/R was similarly antagonized by Mos-driven activation
of the Erk1/2 pathway (Fig. 9C). Mutation of S252 and S265 to
A led to a slight destabilization, while mutating these two S’s to
D’s strongly stabilized both proteins (Fig. 2B and 9D). Taken
together, these data indicate that the ubiquitylation of Fra-1 is
not required for regulated proteasomal degradation.
The C-terminal 40 amino acids are sufficient for Erk1/2
pathway-dependent stabilization of Fra-1. To further evaluate
how S252 and S265 phosphorylation inhibits the Fra-1 desta-
bilizer, we determined the effect of Mos expression on the
stability of EGFP fused to the C-terminal 40 amino acids of
Fra-1 (Fig. 10A). In keeping with the above-mentioned data,
Mos entailed a higher accumulation of Myc6-EGFP due to the
transcriptional activation of the cytomegalovirus (CMV) pro-
moter of pIRES2-EGFP. However, the difference in accumu-
lations of Myc6-EGFP in the absence and in the presence of
Mos was smaller than that of Myc6-EGFP/231-271 transfected
under the same conditions (long-exposure luminogram), which
pointed to an increase of the stability of Myc6-EGFP/231-271
in the presence of Mos (Fig. 10B). This correlated with the
phosphorylation of the fusion protein, as detected by immu-
noblotting with the anti-S252- and anti-S265-phosphorylated
Fra-1 antisera (Fig. 10C). Although not excluding the influence
of other domains of the protein for more efficient phosphory-
lation, this indicated that the Fra-1 destabilizer contains suffi-
cient information for being recognized and antagonized by
Erk1/2 pathway kinases. The observation that Myc6-EGFP/
231-271 is less stable than Myc6-EGFP under these conditions
supports the idea that phosphorylation reduces the destabiliz-
ing activity without abolishing it. This was already suggested by
the fact that a small fraction of Fra-1-2S/D had disappeared at
the 8-h time point in the pulse-chase experiments presented in
Fig. 2G. Consistently, a mutant protein where S252 and S265
were mutated to D (Myc6-EGFP/231-271-2S/D) accumulated
to a level (i) higher than that of Myc6-EGFP/231-271 or of a
mutant where S252 and S265 were mutated to A (Myc6-EGFP/
231-271-2S/A) but (ii) lower than that of Myc6-EGFP in par-
allel transfection experiments (Fig. 10D).
We report here that under the conditions studied, the deg-
radation of the bulk of the intrinsically unstable Fra-1 protein
occurs in a proteasome-dependent but ubiquitin-independent
FIG. 9. Prior ubiquitylation is not necessary for proteasomal deg-
radation of Fra-1. (A) Proteasomal degradation of Myc2K/R–Fra-
1K/R. HeLa cells were transiently transfected to express Fra-1 and
Myc2K/R–Fra-1K/R from pIRES2-EGFP-based vectors. MG132 was
added 24 h later for a period of 8 h before immunoblotting analysis.
(B) Relative stabilities of Myc2K/R–Fra-1K/R and Myc2K/R–Fra-1K/
R/1-261. Experiments were carried out in HeLa cells with pIRES2-
EGFP-based vectors for the indicated proteins as described above (A).
(C) Stabilization of Myc2K/R–Fra-1K/R upon Mos expression. HeLa
cells were transfected with pIRES2-EGFP-based vectors for the indi-
cated proteins in the presence of the Mos expression vector. Immu-
noblotting experiments were conducted 16 h later. (D) Relative sta-
bilities of S252 and S265 mutants of MycK/R–Fra-1K/R. S252 and
S265 of Myc2K/R–Fra-1K/R were mutated either in A or in D, and the
resulting pIRES2-EGFP-based plasmids were transfected in parallel
with that for Myc2K/R–Fra-1K/R. Immunoblotting analysis of the var-
ious Fra-1 proteins was performed 16 h later.
3946 BASBOUS ET AL.MOL. CELL. BIOL.
manner and depends on a single destabilizer residing within
the C-terminal 30 to 40 amino acids. Fra-1 destruction is an-
tagonized upon the phosphorylation of two serines of the latter
domain that are targets of Erk1/2 pathway kinases. Consis-
tently, Fra-1 stabilization was observed under physiological
and oncogenic conditions of high Erk1/2 activity.
Fra-1 stabilization upon phosphorylation by kinases of the
Erk1/2 pathway. We have combined several approaches to
demonstrate that S252 and S265 are actual targets of the
Erk1/2 pathway. The approaches include the analysis of site-
specific mutants of Fra-1, cell signaling studies, and the use of
both a pharmacological inhibitor and phosphoserine-specific
antisera. The phosphorylation of one serine is not strictly de-
pendent on that of the other, but whether one event can mod-
ulate the other cannot be excluded at this stage. The decreased
electrophoretic mobility of Fra-1-2S/A upon the activation of
the Erk1/2 pathway is consistent with previous suggestions by
others that multiple sites are phosphorylated in Fra-1 (49, 69).
This also indicates that the latter phosphorylations do not
necessarily depend on prior S252 and/or S265 modifications.
However, we cannot rule out that they might have an effect, as
is the case with c-Fos (49, 50), under more moderate activation
levels of Erk1/2. Whatever the case, these additional phospho-
rylations do not affect Fra-1 turnover control, a situation sim-
ilar to that of c-Fos, where a mutation of T325 and T331
neither accelerates nor slows down the rate of degradation (45,
An important question is which kinases are responsible for
S252 and S265 phosphorylation. By analogy with c-Fos and
given our data, it is reasonable to assume that S265, which
resides in an SP motif, is a target for Erk1 and/or Erk2. Our
data do not rule out its phosphorylation on endogenous Fra-1
by Erk5 (62) in serum-induced BALB/c 3T3 cells, since this
pathway is also inhibited by UO126. In contrast, S252 is not
followed by a proline but instead is preceded by an arginine at
position ?3, a motif recognized by several kinases. The most
likely candidates are members of the Rsk and Msk families,
both of which are activated by Erk1/2 and phosphorylate nu-
merous transcriptional regulatory proteins (19, 24). Accord-
ingly, c-Fos is phosphorylated by Rsk in vitro and in vivo, a
modification that regulates its activity and turnover (14, 15,
50). Phosphorylation of neither Fra-1 by Rsk’s nor Fos family
proteins by Msks has been described. Future studies will de-
termine the contributions of these two kinase families in vivo.
The addition of UO126 to HeLa cells expressing phosphor-
ylated Fra-1 due to the presence of Mos entailed a dephos-
phorylation of S265 that was slower than that of S252 (Fig. 4B),
suggesting that the two phosphoserines are targeted by differ-
ent still-to-be-identified phosphatases. Although we do not
exclude the possibility that the phosphatase(s) specific for S265
may have been rate limiting under our conditions of Fra-1
overexpression, an interesting scenario is that basal Erk1/2
pathway activity may be required for the dephosphorylation of
S265. The possibility that a kinase responsible for the stabili-
zation of a protein is also necessary for the destabilization of
the same protein may appear contradictory. However, it is well
documented that both the nature and the intensity of the
processes under the control of Erk1/2 depend on not only the
timing but also the level of their activation (41, 48). Charac-
terizing the relative contributions of phosphorylation and de-
FIG. 10. Phosphorylation-driven antagonization of the Fra-1 desta-
bilizer. (A) Structure of EGFP–Fra-1 chimeras. DBD, DNA-binding
domain. (B) Mos-driven inhibition of Fra-1 destabilizer activity. Asyn-
chronous HeLa cells were transfected to express the indicated proteins
from a pIRES2-EGFP-based plasmid in the presence or in the absence
of the Mos expression vector. The immunoblotting analysis was carried
out 24 h later with anti-EGFP antibodies. A short-exposure lumino-
gram (SE) is presented in the left panel. Because of the signal satu-
ration for EGFP on this luminogram, transcriptional activation of
pIRES2-EGFP is barely appreciable but is similar to that shown in Fig.
3B. For better visualization of Myc6-EGFP/231-271, a longer-exposure
luminogram (LE) of the relevant part of the left panel is presented in
the right panel. (C) Phosphorylation of Myc6-EGFP/231-271 in the
presence of Mos. The protein extracts from cells transfected with a
pIRES2-EGFP-based vector for Myc6-EGFP/231-271 in the presence
or in the absence of Mos were probed with anti-phospho-S252 and
anti-phospho-S265 antisera. (D) Comparison of EGFP chimeras made
with wild-type and mutated Fra-1 destabilizer. Asynchronous HeLa
cells were transfected with pIRES2-based plasmids for the indicated
proteins, and immunoblotting analysis was carried out 24 h later. All
data presented are representative of at least three independent exper-
VOL. 27, 2007 Fra-1 PROTEASOMAL DEGRADATION3947
phosphorylation events will help elucidate how Fra-1 turnover
Several lines of evidence show that the principal mechanism
responsible for Fra-1 stabilization in HCT116 cells is Erk1/2
pathway-dependent phosphorylation of S252 and S265 (Fig. 5).
As high Fra-1 levels are observed in a variety of tumors also
displaying high MAPK activity (see above), it will be interest-
ing to determine whether the phosphorylation of S252 and
S265 is also a major stabilization factor in these situations.
Should this be the case, it is important to underline that other
mechanisms might also contribute to Fra-1 overaccumulation,
especially because the Erk1/2 pathway is already known to
positively regulate fra-1 gene transcription (68), including in
tumor cells (13, 66).
Proteasome-dependent, ubiquitylation-independent degra-
dation of Fra-1. Vial and Marshall previously reported that the
proteasome is responsible for the residual degradation of Fra-1
in HCT116 cells (66). Our work, even though it does not
exclude possible minor contributions by other proteolytic sys-
tems, establishes that Fra-1, when unstable, is essentially de-
graded by the proteasome (Fig. 8C). The vast majority of
substrates subjected to proteasomal hydrolysis are thought to
depend on prior ubiquitylation for being addressed and/or
recognized by the proteasome (22). We show that the degra-
dation, as well as the regulation of degradation, of the non-
ubiquitylatable Myc2K/R–Fra-1K/R mutant is undistinguish-
able from that of wild-type Fra-1. Thus, Fra-1 belongs to the
small group of proteins that can undergo ubiquitylation-inde-
pendent proteasomal destruction. The hamster E36-ts20 cell
line is thermosensitive for ubiquitylation activity due to a mu-
tation in the first enzyme (E1) of the ubiquitin cycle (37). It is
notable that Fra-1 is not stabilized upon a shift of these cells
cultured asynchronously at the nonpermissive temperature
(not shown). Even though caution is required for the interpre-
tation of data from this experiment due to the usually leaky
phenotype of E1-thermosensitive cell lines (54), these data are
consistent with our turnover analysis of Myc2K/R–Fra-1K/R.
Another point of consistency is our previous observation that
the activity of the C-terminal c-Fos destabilizer, closely related
to that of Fra-1, is independent of an active ubiquitin cycle (9).
It remains possible that a small fraction of Fra-1 underwent
ubiquitylation-dependent degradation in our experiments, as
approaches such as those that we have used for studying c-Fos
and Fra-1 measure the behavior of the bulk of a protein only.
It is also possible that Fra-1 may undergo ubiquitin-dependent
degradation under other conditions, as it is now clear that
some proteins can be addressed to the proteasome via several
mechanisms. This is well illustrated for the p53 oncosuppressor
(4) and c-Fos, whose cytoplasmic degradation can involve the
UBR1 E3 ubiquitin ligase (56).
Although the ubiquitylation of Fra-1 itself was not necessary
for proteasomal degradation in our experiments, one possibil-
ity to consider is the polyubiquitin chains present in trans on a
protein partner. Such an interactor cannot be an LZ dimeriza-
tion partner since (i) an LZ-deficient mutant of Fra-1 was as
unstable as the wild-type protein (not shown) and (ii) EGFP is
destabilized by a 40-amino-acid C-terminal fragment of Fra-1
that does not contain the LZ.
Very interestingly, Hoffman et al. showed previously that
transfected Fra-1 is also ubiquitylatable in an in vivo ubiquity-
lation assay, like that initially described by Treier et al. (63),
and that Fra-1 ubiquitylation is stimulated by the activation of
the Erk1/2 pathway, i.e., when the protein is stabilized (31).
This poses the question regarding the actual role of this mod-
ification, as ubiquitylation is also involved in the control of a
variety of protein functions independent of proteasomal pro-
teolysis (27, 57). The regulation of Fra-1 transcriptional activ-
ity is certainly to be considered a priority, as it is stimulatable
by the Erk1/2 pathway (69). Ubiquitylation is, however, not an
absolute prerequisite, as the lysineless Myc2K/R–Fra-1K/R
mutant was transcriptionally active in a transient luciferase
transcription assay performed using HeLa cells (not shown).
Fra-1 destabilizer and degradation of other Fos proteins.
We show here that Fra-1 breakdown depends on a unique
C-terminal destabilizer. This helps us to understand why c-Fos
expression is transient in mitogen-stimulated cells, whereas
that of Fra-1 is much longer lasting. The molecular explana-
tions are multiple. An obvious first explanation is the differ-
ential transcriptional regulations of the two genes. Thus, c-fos
mRNA induction in G0/G1phase is rapid and transient,
whereas that of Fra-1 occurs later and is longer before return-
ing to a basal level (2, 17, 68). A second mechanism is protein
stability control. Using pulsed ectopic mRNA expression for
wild-type and mutant Fra-1 proteins in serum-stimulated fibro-
blasts (Fig. 6), we show that the Erk1/2 pathway kinases are
sufficiently active for at least 16 h to phosphorylate S252 and
S265 and thereby prevent Fra-1 degradation during this period
of time. By contrast, c-Fos protein instability is preserved by
the activation of an N-terminal destabilizer even though the
activity of its C-terminal destabilizer is also reduced (23).
The Fra-1 destabilizer is contained within the last 30 to 40
amino acids at the C terminus. This region contains striking
similarities with the equivalent domains in the other Fos pro-
teins (Fig. 11). It is therefore plausible that its counterparts in
Fra-2 and FosB also show destabilizing activities, as these two
proteins are also unstable. Work is currently under way to
investigate this point and to determine whether (i) the desta-
bilizers of the four Fos proteins are equally efficient or not and
(ii) those of Fra-2 and FosB are also regulated by the Erk1/2
pathway-driven phosphorylation of the serine counterparts of
S252 and S265 of human Fra-1. Because the N-terminal re-
gions of c-Fos, Fra-2, and FosB share several homologous
segments that are not found in Fra-1, it will also be important
to address whether Fra-2 and FosB carry another N-terminally-
located destabilizer as c-Fos. In turn, the dissimilarity between
the N-terminal regions of c-Fos and Fra-1 raises the question
of whether UBR1-dependent, ubiquitin-dependent degrada-
tion of c-Fos (56) may also occur on Fra-1. Preliminary studies
using RNA interference to knock down UBR1 showed no
FIG. 11. Alignments of Fos protein C termini. The domains of the
four human Fos proteins corresponding to the C-terminal 40 amino
acids of Fra-1 were aligned using the Clustal W multiple sequence
alignment program.*:, and ● correspond to identical, conserved, and
semiconserved amino acids, respectively. The residues corresponding
to Fra-1-S252 and Fra-1-S265 are boxed.
3948BASBOUS ET AL.MOL. CELL. BIOL.
increase in Fra-1 levels in asynchronous HeLa cells (not
shown). However, to truly determine if Fra-1 behaves like
c-Fos with respect to UBR1-mediated degradation, we must
establish conditions where Fra-1 is quantitatively retained
within the cytoplasm.
The C-terminal half of c-Fos shows a disorganized structure
(12). Because (i) c-Fos and Fra-1 primary structures are closely
related in the C-terminal region and (ii) no particular motif or
structure could be identified in a bioinformatic analysis (not
shown), it is very likely that the Fra-1 C-terminal domain is
unstructured as well. It is therefore interesting that ubiquity-
lation-independent proteasomal degradation is thought to
preferentially concern poorly structured polypeptides possibly
exposing hydrophobic segments (32). Several possibilities
could be put forward to explain why the lack of structure would
favor efficient Fra-1 proteasomal degradation and how the
latter could be inhibited upon the phosphorylation of S252 and
S265. Adapter proteins for the recruitment and delivery of
ubiquitylated proteins to the proteasome have already been
described (22). A first possibility would therefore be the exis-
tence of comparable adaptors, maybe of the chaperone type,
recognizing loosely structured and/or hydrophobic peptide mo-
tifs. Another possibility would consist of the direct recognition
by the proteasome, as proposed for most proteins processed
independently of prior ubiquitylation. In fact, several types of
proteasomal complexes do coexist at any time in any eukary-
otic cell (52), which poses the question of which one(s) is
responsible for Fra-1 destruction. Thus, a large fraction of the
proteolytic core of the system, called the 20S proteasome, is
found free in vivo (61), whereas the rest associates with differ-
ent regulatory complexes (52) displaying different biochemical
properties. Although it has been considered to be a latent
protease for a long time, there is accumulating evidence that
the 20S proteasome contributes to cell protein hydrolysis (3, 5,
6, 32, 47, 52, 58). It is a four-ring cylinder-shaped multisubunit
complex with its proteolytic sites hidden in a central cavity
(52), and two orifices, one at each tip, are gated by flexible
interdigitated extensions of the subunits forming the outer two
rings. Various poorly structured protein substrates have now
been shown to be capable of perturbing the organization of
these extensions to gain access to the proteolytic chamber (32,
52). It is therefore possible that Fra-1 could do the same via its
unstructured C terminus. The best-known proteasomal regu-
lators are the 19S regulatory (or PA700) and 11S (or PA28?/?
and PA28?) activator complexes, which can attach alone, in
duplicate, or in combination at the extremities of the 20S
particle (52). The 19S complex recognizes ubiquitylated as well
as nonubiquitylated substrates (52). In contrast, PA28 activa-
tors, which influence the proteolytic activities of the 20S pro-
teasome, are not capable of recognizing ubiquitylated proteins,
although they may show some ubiquitylation-independent sub-
strate selectivity (40). These observations thus leave open the
possibility that the 19S and/or the PA28 regulators contribute
to Fra-1 degradation. Biochemical studies involving various
proteasomal complexes are under way to identify which pro-
teasomal complex(es) is responsible for Fra-1 hydrolysis as
well as whether recognition occurs directly via its C terminus or
indirectly through a peptidic adaptor.
M.P.’s laboratory is an “Equipe Labelise ´e” supported by the Ligue
Nationale contre le Cancer. This work has also been supported by
grants from the CNRS, the ARC, and the ACI program of the French
Ministry for Research. J.B. was supported by a fellowship for the Ligue
Nationale contre le Cancer.
We are grateful to G. Bossis for critical reading of the manuscript.
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