From the Laboratoire de Neurochimie-Anatomie, the
Laboratoire de Développement et Vieillissement du SNC,
the ** Laboratoire de Neurogénétique Moléculaire,
Institut des Neurosciences, CNRS-UMR 7624, Université Pierre
et Marie Curie, 75005 Paris, and § Institut de
Génétique Moléculaire, UMR 5535, IFR 24, CNRS,
34293 Montpellier Cedex 5, France
Received for publication, July 26, 2000, and in revised form, October 24, 2000
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ABSTRACT |
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The ternary complex factor Elk-1, a major nuclear
target of extracellular signal-regulated kinases, is a strong
transactivator of serum-responsive element (SRE) driven gene
expression. We report here that mature brain neurons and nerve growth
factor (NGF)-differentiated PC12 cells also express a second, smaller
isoform of Elk-1, short Elk-1 (sElk-1). sElk-1 arises from an internal
translation start site in the Elk-1 sequence, which generates a protein
lacking the first 54 amino acids of the DNA-binding domain. This
deletion severely compromises the ability of sElk-1 to form complexes
with serum response factor on the SRE in vitro and to
activate SRE reporter genes in the presence of activated Ras. Instead,
sElk, but not a mutant that cannot be phosphorylated, inhibits
transactivation driven by Elk-1. More pertinent to the
neuronal-specific expression of sElk-1, we show it plays an opposite
role to Elk-1 in potentiating NGF-driven PC12 neuronal differentiation.
Overexpression of sElk-1 but not Elk-1 increases neurite extension, an
effect critically linked to its phosphorylation. Interestingly, in the
presence of sElk-1, Elk-1 loses its strictly nuclear localization to
resemble the nuclear/cytoplasm pattern observed in the mature brain.
This is blocked by mutating a normally cryptic nuclear export signal in
Elk-1. These data provide new insights into molecular events underlying
neuronal differentiation of PC12 cells mediated by the NGF-ERK
signaling cascade.
Intracellular signaling mechanisms regulate many processes,
including cell proliferation, specification of cell fate, and differentiation. One universally used signaling pathway is the ligand-induced activation of receptor tyrosine kinases and their downstream signaling cascade, leading to the activation of the extracellular signal-regulated kinases
(ERKs),1 also known as
mitogen-activated protein kinases (MAPKs). ERKs propagate signals
through the phosphorylation of a wide range of proteins, including
other enzymes, proteins of the cytoskeleton, and transcription factors
(1). In particular, activated ERKs translocate to the nucleus, where
they can activate transcription factors and thereby regulate gene expression.
Elk-1, a major nuclear target of activated ERK proteins, is a member of
the ternary complex factor (TCF) family that also includes Sap1 and
NET/ERP/SAP2/Elk-3 (2-7). In cultured cell lines Elk-1 functions as a
nuclear transcriptional activator via its association with serum
response factor (SRF) in a ternary complex on the serum response
element (SRE) present in the promoter of many immediate early genes
(c-fos, egr1, egr2, pip92, and nur77) (8). Elk-1 has three major functional domains as follows: the N-terminal Ets-DNA binding domain that recognizes the 5'-CAGGA motif of
the SRE (4), a motif of 20 amino acids (the B domain) that mediates
protein-protein interaction with SRF (9-11), and the C-terminal part
of the protein, which contains consensus phosphorylation sites for ERK
but also the closely related MAPKs c-Jun N-terminal kinase/stress-activated protein kinase and p38 (12). These cascades are
activated by different extracellular signals, principally mitogens for
ERKs (13) and various stresses for c-Jun N-terminal kinase/stress-activated protein kinase and p38 (14, 15). Upon activation MAPKs can translocate to the nucleus (16) where they phosphorylate Elk-1 on Ser383 and Ser389, which
strongly potentiates SRE-dependent gene expression (6, 17-28).
We previously reported that Elk-1 was expressed, in post-mitotic
neurons, in both nuclear and cytoplasmic compartments (29). In the
process of investigating the functional significance of this unexpected
subcellular localization, we discovered a novel isoform of Elk-1,
sElk-1 for short Elk-1, the expression of which is linked to the
neuronal phenotype, determined in neuronal tissue as well as in
NGF-differentiated PC12 cells. This isoform arises from an internal
translation start site in the Elk-1 sequence and corresponds to a
truncated protein lacking the first 54 amino acids of the DNA binding
domain. We have investigated the molecular and functional properties of
sElk-1 relative to Elk-1, and we showed they have opposite roles in the
following: (i) SRE-driven gene expression and (ii) NGF-induced PC12
differentiation. Thus sElk-1 seems to be a key mediator of NGF/ERK
signaling cascade specifically in neuronal cells.
Immunohistochemistry--
The immunohistochemical procedure was
adapted from protocols described previously (29). Briefly,
free-floating sections were incubated (after rinse and saturation
steps) for 72 h at 4 °C with the primary antibodies. Rabbit
polyclonal antisera raised against Elk-1 (Santa Cruz Biotechnology),
Zif268 (Santa Cruz Biotechnology), and STAT3 (New
England Biolabs) were used at a dilution of 1:100. Then sections were
incubated 24 h at 4 °C with the secondary biotinylated antibody
(rabbit anti-IgG, 1:200), incubated for 90 min in
avidin-biotin-peroxidase complex solution (Vector Laboratories, final
dilution 1:50), and then placed in a solution containing 0.1%
3,3'-diaminobenzidine (50 mg/100 ml) and developed by
H2O2 addition (0.02%) for 60 min. After
dehydration, tissue sections were mounted onto gelatin-coated slides.
Tissue Preparation, Subcellular Fractionation, and Western
Blot--
Male Harlan Sprague-Dawley rats (weighing 200-250 g) were
killed by decapitation, and brains were quickly removed from the skull
and frozen at RNA Isolation and Analysis by Northern Blots--
Total RNA was
isolated from mouse and rat brains using TRIzol reagent (Life Science
Technologies) according to the supplier's recommendations. One step
enrichment for poly(A)+ RNA was performed in batch using
oligo(dT)-cellulose (Amersham Pharmacia Biotech). 10 µg of
poly(A)-enriched RNA was electrophoresed on formaldehyde-agarose gels
and blotted onto positively charged nylon as already described (20).
Filters were hybridized initially with a riboprobe corresponding to the
human ELK-1 coding region, stripped, and rehybridized
with a rat gapdh.
Plasmids--
Deletion of residues In Vitro Translation and Immunoprecipitation--
Coupled
in vitro transcription/translations were performed from
nontagged and HA-tagged Elk-1 constructs using the TNT-coupled reticulocyte lysate system (Promega) with T7 RNA polymerase and [35S]methionine at 20 µCi per reaction (ICN) according
to the manufacturer's instructions. For immunoprecipitation the
reaction was incubated overnight at 4 °C with pansorbin (Amersham
Pharmacia Biotech) and either an Elk-1 C-terminal antibody (Santa Cruz
Biotechnology) or a monoclonal antibody specific for the HA tag.
Cell Culture and Transfection--
PC12 cells were maintained in
RPMI 1640 culture medium (Eurobio) with 5% fetal calf serum, 10%
horse serum (Eurobio), 60 µg/ml penicillin G, and 100 µg/ml
streptavidin. For differentiation, the cells were plated at 5 × 103 cells/cm2 on a 4-well chamber slide
(SuperCell), precoated with 1 mg/ml polyethyleneimine in 0.03 M borate buffer, pH 8.3, and maintained in RPMI 1640 culture medium supplemented with 1% horse serum and 100 ng/ml NGF
2.5 S (Alomone Labs, Israel) for 2-5 days. Transient transfections of
PC12 cells were carried out by electroporation (0.200 kV, 950 microfarads; Bio-Rad). 2 × 106 cells, suspended in
400 µl of complete growth medium, were transfected with 10 µg of
the Elk-1 constructs. Transfected cells were scored for neuritic
formation after 48-96 h in the presence of 100 ng/ml NGF as described
previously (32, 33). Immunofluorescence experiments were performed
48 h after transfection. For cotransfection assays, PC12 cells
were transfected with both the GFP plasmid (pEGFP-N3, CLONTECH) and the various Elk-1 constructs in a
ratio of 1:5.
In Vitro Kinase Assays and Gel Retardation Analysis--
The
reactions contained 4 µl of in vitro translated protein in
50 mM Tris-HCl, pH 7.5, 10 mM
MgCl2, 1 mM EGTA, 2 mM
dithiothreitol, 100 µM ATP, 0.01% Brij 35, 10 mM Luciferase Assays--
NIH3T3 cells were grown in Dulbecco's
modified Eagle's medium containing 10% fetal calf serum (Life
Technologies, Inc.) and plated 16 h before transfection to ~30%
confluence. Medium was changed 1 h before transfection, and cells
were subsequently transfected with the CaPO4 method. Typically, 300 ng
of 3xSRE luciferase vector, 60 ng of cytomegalovirus
RasVal-12 expression vector, 50 ng of Elk-1 FL or
800 ng of Elk-1(AAG1) expression vectors were cotransfected with 5 ng
of SV40 Renilla luciferase expression vector and
complemented to 5 µg with nonspecific DNA. 8-10 h after
transfection, cells were placed in Dulbecco's modified Eagle's medium
without serum for 40 h, at which point cells were harvested, and
luciferase activities were determined according to the supplier's
protocol (Dual Luciferase, Promega).
Pull-down Assays--
35S-Labeled Elk-1 protein were
synthesized by coupled in vitro transcription/translation in
the presence of L-[35S]methionine (1000 Ci/mmol) using the TNT-coupled reticulocyte lysate system (Promega)
according to the supplier's recommendations. GST,
GST-CBP-(1100-1286), and GST-CBP-(1460-1891) (34, 35) were expressed
in bacteria, then purified by metal affinity chromatography, and then
directly bound to glutathione-Sepharose beads at 4 °C for 1 h.
The beads were then washed three times with a 100-fold excess of RJD
buffer (10 mM HEPES, pH 7.9, 5 mM
MgCl2, 50 mM NaCl, 17% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 0.05% Nonidet P-40) containing freshly added protease inhibitors (2.5 µg/ml aprotinin, leupeptin, pepstatin, 0.5 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride) and stored short term at
4 °C. 5 µl of in vitro translated Elk-1 was added to 10 µl of a 50% slurry of protein bearing beads in a total volume of 100 µl of RJD buffer and incubated with gentle agitation at
4 °C for 4 h. After three washes with 500 µl of
RJD buffer, the beads were collected, resuspended in 20 µl of 5× Laemmli buffer (0.025 M Tris-HCl, pH 6.8, 10%
SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue),
denatured for 5 min at 95 °C, and bound proteins analyzed by
SDS-polyacrylamide gel electrophoresis. Proteins were visualized by
Coomassie staining, followed by autoradiography of the dried gels.
Immunofluorescence--
PC12 cells were fixed with 4%
formaldehyde in phosphate-buffered saline (PBS) for 10 min,
permeabilized with 0.2% Triton X-100 in PBS for 5 min at room
temperature, and stained with a monoclonal antibody raised against
hemagglutinin (HA) (1:1000, Roche Molecular Biochemicals) for 1 h
at room temperature. Coverslips were rinsed in PBS and incubated for
1 h at room temperature with an anti-mouse Cy3-conjugated antibody
(1:500, Sigma).
Expression of an Antigenically Related Isoform of Elk-1 in Cells of
Neuronal Phenotype--
We previously noted, by immunocytochemical
analyses, the presence of Elk-1 in both nuclear and cytoplasmic
compartments in mature neurons ((29) illustrated in Fig.
1A). A similar subcellular distribution of Elk-1 was found using various antibodies directed against N-terminal and C-terminal parts of Elk-1 (29). This contrasts
with cultured cells, where Elk-1 is localized predominantly, if not
exclusively, to the nucleus (36) and its activation depends on ERK
nuclear translocation (32). This led us to characterize further Elk-1
expression in the central nervous system by Western blotting and
immunodetection with the C-terminal antibody (residues 407-426) used
for immunocytochemistry. Surprisingly, we observed in cerebral extracts
not only Elk-1 at 52 kDa but also a new form of Elk-1 migrating at 45 kDa (sElk-1, Fig. 1B). The two forms of Elk-1
were present in various cerebral structures, except the substantia
nigra pars reticulata.
To investigate the subcellular localization of the two isoforms of
Elk-1, we fractionated cortical extracts into the nucleus and
cytoplasm. As in our immunocytochemical analysis (see Fig. 1A), Zif268 is exclusively detected in the
nuclear fraction, whereas STAT3 is present only in the cytoplasmic
compartment (Fig. 1C). The same cortical extracts showed a
differential subcellular distribution for the two isoforms of Elk-1.
Although the 52-kDa protein is detectable in both cytoplasmic and
nuclear fractions, sElk-1 is exclusively present in the nuclear
fraction (Fig. 1D). In the latter fraction, Elk-1 and sElk-1
are expressed at similar levels. The strictly nuclear localization of
sElk-1 likely explains why it was absent in the substantia nigra pars
reticulata (see Fig. 1B), a structure where Elk-1 protein is
found in axon terminals but not cell bodies (29).
To characterize further the expression pattern of sElk-1
versus Elk-1, we analyzed Western blots from cortical
extracts as well as various peripheral tissues and cell lines. Although
the full-length protein was present in all tissues tested, sElk-1 was
exclusively observed in cerebral tissues (Fig.
2A). Notably, sElk-1 was not
detectable in either undifferentiated PC12 cells or in the cell lines
MMQ, COS7, and NIH3T3 (Fig. 2A). Taken together, these data
indicate that sElk-1 is specifically expressed in cerebral tissues.
Since Elk-1 proteins and mRNAs are restricted to neuronal but not
glial cells (29), sElk-1 seems to be linked to the neuronal phenotype.
Upon NGF treatment, PC12 cells acquire a phenotype that resembles
sympathetic neurons, with large neuritic processes (compare Fig. 2,
B and C). We reasoned that if sElk-1 expression
is characteristic of the neuronal phenotype, it should appear in
NGF-treated PC12 cells. Indeed, Western blot analysis shows that
extracts prepared from NGF-treated but not untreated PC12 cells contain
a version of sElk-1 that co-migrates with the version present in brain
extracts (Fig. 2D).
An Internal AUG Codon Is Used to Generate sElk-1--
The
neuronal specific expression of sElk-1 could arise through a number of
different mechanisms. One, the activation of neuronal specific gene
corresponding to sElk-1, is not supported by extensive data bank
queries or genomic hybridization results
(31).2
The second possibility was an alternative spliced version of Elk-1
mRNA capable of encoding sElk-1. However, RNase protection analysis
(6) and Northern blot analysis of total and poly(A)-enriched RNA from
brain tissues did not reveal an mRNA that would correspond to an
alternative spliced version capable of encoding this smaller form of
Elk-1 ((2), Fig. 3A).
Consistent with this we did not find, using extensive reverse
transcriptase-PCR analysis from brain tissues, any cDNA
corresponding to sElk-1 (data not shown). Thus, sElk-1 seems to be
generated via a post-transcriptional mechanism other than alternative
splicing. Specific proteolytic cleavage of Elk-1 in vivo
appeared unlikely due to the absence of any signature protease
recognition sites that would generate this protein (Proteol data bank;
Infobiogen). Furthermore, we reliably obtained the same level of sElk-1
in vivo in the presence or absence of proteases inhibitors
(data not shown).
Therefore, we investigated the possibility of an alternative
translation start site, since there is some precedent for this in
neuronal cells (37, 38). The Met residue located at position 55 could
serve as an internal translation initiation site (see Fig.
3B). In vitro transcription translation from an
Elk-1 WT expression vector generated two protein products
immunoprecipitated with the C-terminal Elk-1 antibody. One corresponded
to Elk-1 at 52 kDa, whereas the other migrated at the same molecular
weight as sElk-1 (45 kDa) (Fig. 3D, lane 1). To confirm that
these two proteins arose from initiation at one or the other AUG codon, we mutated these two ATGs to lysine-encoding AAGs termed Elk-1(AAG1) and -(AAG2), respectively (Fig. 3C). The Elk-1(AAG1) mutant
template failed to generate the full-length protein in vitro
(Fig. 3D, lane 2), and the Elk-1(AAG2) mutant did not yield
the 45-kDa protein (Fig. 3D, lane 3). We note that these two
templates give rise to higher levels of translated proteins than Elk-1
WT which encodes two protein products.
Different mechanisms can account for internal initiation of
translation. First, cap-dependent mechanisms including
leaky ribosome scanning, where ribosomes ignore potential start sites
presumably because the flanking sequence (the Kozak sequence) deviate
from the rules governing faithful initiation (39). Another
cap-dependent mechanism is discontinuous scanning or
ribosomal shunting (40). Last are cap-independent mechanisms directed
by an internal ribosomal entry site, where the ribosomal complex fails
to attach to the 5' cap. The first ATG codon in Elk-1 cDNA is
surrounded by an imperfect Kozak sequence
(TAGTGATGG); thus we mutated the sequence surrounding
the first ATG to a perfect Kozak consensus (Kozak1,
CCACCATGG) (Fig. 3C). In vitro
translation of this plasmid generates high levels of the two Elk-1
products (Fig. 3D, lane 4), suggesting that sElk-1 was not
generated by the leaky ribosome scanning model. In an effort to
understand further how sElk-1 was generated, we then noticed that the
sequence surrounding the second ATG (CCAACATGA) was in a
highly favorable Kozak context (7 nucleotides over 9) to drive
initiation of sElk-1. To address this, nucleotides surrounding the
second ATG were mutated to obtain a "poor" context (NoKozak2,
GGCAGATGA) (Fig. 3C). In this case, we completely
abrogated in vitro expression of sElk-1 (Fig. 3D, lane
5). Thus, the Kozak consensus played a key role in the mechanisms
of initiation at the second AUG codon (Met55).
The Subcellular Localization of Elk-1 Is Affected by sElk-1
Expression--
We generated expression vectors with an N-terminal HA
tag for Elk-1 WT, Elk-1(AAG2), or Elk-1(AAG1) and a GFP tag for
Elk-1(AAG2) constructs. As expected, HA-Elk-1 WT encodes both isoforms,
as shown by immunoprecipitation with Elk-1 antibody (Fig.
4A), but only the full-length
protein bears the HA epitope (Fig. 4B). The other expression
vectors encode exclusively HA-tagged (Fig. 4, A and
B) or GFP-tagged (data not shown) full-length Elk-1 or
sElk-1. Thus, these constructs allowed us to follow Elk-1 and sElk-1
(encoded by Elk-1(AAG2) and -(AAG1), respectively) by
immunofluorescence in transfected PC12 cells. When expressed alone,
both sElk-1 and Elk-1 were predominantly nuclear with the HA tag (Fig.
4C) or GFP tag (data not shown). However, coexpression of
both isoforms, using either HA-Elk-1 WT (Fig. 4C) or
cotransfection of both HA-Elk-1(AAG1) and GFP-Elk-1(AAG2) cDNAs
(Fig. 4D), led to relocalization of Elk-1 to the cytoplasm.
This phenomenon is linked to a leucine-rich sequence
(7LWQFLLQLLR16) in Elk-1 (see Fig.
3B) that resembles a nuclear export signal (NES), since
mutating this sequence caused Elk-1 to remain in the nucleus in the
presence of sElk-1 (Fig. 4C). These data suggest that the
expression of sElk-1 relocalizes Elk-1 intracellularly and help explain
the different subcellular localization of Elk-1 we observed in neuronal
versus other cell types.
Molecular Properties of sElk-1--
Deletion of the first 54 amino
acids in sElk-1 removes a major portion of the DNA binding domain (see
Fig. 3B) but leaves one
We then used transient transfection assays to evaluate the effect of
sElk-1 on SRE reporter gene activity. To potentiate DNA binding and
possible transregulation by sElk-1, we cotransfected constitutively
active RasVal-12 to generate activated ERK. The
transfections contained this expression vector, the SRE reporter
construct, and either Elk-1 WT or Elk-1(AAG1). SRE-driven gene
expression was increased by transfecting RasVal-12
alone, likely reflecting activation of endogenous TCF (Fig. 5B, 2nd lane). Whereas overexpression of Elk-1 WT potentiated SRE activity driven by RasVal-12, sElk-1 reduced this activity
(Fig. 5B, 3rd lane). Furthermore, the introduction of a
large excess of the sElk-1 vector significantly diminished the activity
of Elk-WT on SRE-driven gene expression, an effect reversed with the
Ser383/389 to Ala mutant (Fig. 5B, compare
5th lane with 6th
lane).
Given the low binding properties of sElk-1 on SRE (see Fig.
5A), we asked how sElk-1 inhibited Elk-1 properties on
SRE-driven gene expression. One possibility was that sElk-1 interacts
with the coactivator CBP, which is necessary for optimal SRE-driven gene expression by Elk-1 (35). Pull-down experiments performed with the
bromodomain of CBP clearly showed that both Elk-1 and sElk-1 interact
with this region in vitro (Fig.
5C).3 Thus,
altogether these data suggest that sElk-1 can act as a transcriptional
modulator not by competing with Elk-1 on SRE but for interaction with CBP.
Opposing Roles of sElk-1 and Elk-1 in NGF-induced PC12
Differentiation--
The pheochromocytoma cell line PC12 has served as
a model system to analyze intracellular mechanisms underlying neuronal
differentiation. Upon nerve growth factor (NGF) treatment, PC12 cells
stop proliferating and acquire a phenotype characteristic of
sympathetic neurons including extension of neurites (43). NGF-driven
PC12 differentiation critically depends on ERK signaling to Elk-1 (44).
This lead us to examine the respective roles of Elk-1 and sElk-1 in
NGF-driven PC12 differentiation. We transfected PC12 cells with
expression vectors coding for green fluorescent protein (GFP) and
either Elk-1 WT, Elk-1(AAG1) or Elk-1(AAG2). Neuronal differentiation was evaluated by scoring GFP-positive cells with neuritic extensions longer than the cell body 48 and 96 h after transfection as
described previously (32, 33). The experiments were performed in
the presence of NGF. After NGF addition, 30% of PC12 cells transfected with GFP alone were differentiated (data not shown). Overexpression of
sElk-1 significantly increased the number of differentiated cells (Fig.
6H) in the presence (Fig.
6B, Elk-1WT construct) or absence (Fig. 6D,
Elk-1(AAG1) construct) of Elk-1. Elk-1 alone (Fig. 6F,
Elk-1(AAG2) construct) failed to potentiate NGF-induced differentiation (Fig. 6H).
Since phosphorylation of Elk-1 and sElk-1 are critical for their
molecular properties, we tested the effect of overexpressing the same
proteins inactivated by mutating Ser383/389 to Ala.
Inactive sElk-1 (Fig. 6, C-E) no longer potentiated neurite extension (Fig. 6H), in direct contrast to Elk-1 (Fig.
6G), where the inactivating mutations led to a significant
increase in neuronal differentiation (Fig. 6H).
In conclusion, these data demonstrate that overexpression of sElk-1
alone at early stages of NGF treatment facilitates neuronal differentiation, an effect that is critically linked to its capacity to
be activated by ERKs. They also indicate that Elk-1 and sElk-1 play
opposite roles in neuronal differentiation.
We describe a novel, neuronal-specific isoform of Elk-1, sElk-1,
that originates from translation initiation at an alternative downstream start site in Elk-1 mRNA. Whereas viruses commonly produce several variants of a protein through internal initiation on
the same transcript, this mechanism is rarely documented in vertebrate
cells (45). Instead, alternative splicing is frequently used to
generate protein isoforms from a single gene. Nevertheless, internal
initiation is used in some instances to generate protein isoforms in
mammalian cells, as is the case for the transcription factors c/EBP
(46, 47), sCREM (37), and Egr3 (38). Notably the truncated versions
show markedly different properties as transcriptional regulators. We
propose a similar mechanism for sElk-1.
Elk-1 belongs to the TCF subfamily of transcription factors that are
characterized by the presence of a highly conserved Ets-DNA binding
domain in their N-terminal region (8). This adopts a
"winged-helix-turn-helix" structure due to the multiple
interactions of three An isoform of the TCF protein Net-b (49) is encoded by an alternatively
spliced mRNA and has a truncated C-terminal activation domain. This
splicing variant is insensitive to the Ras-MAPK pathway and functions
as a constitutive competitor for binding by other TCFs. This contrasts
with the mechanism we propose for sElk-1, which still contains its
phosphorylation sites, which are essential for its activity but is
strongly compromised in DNA binding. Thus, sElk-1 represents a novel
type of MAPK-dependent Ets protein that, while nuclear,
functions independently of DNA binding. It could act as a
transcriptional modulator directly, for example by competing with Elk-1
for interaction with the coactivator CBP, and/or indirectly, by causing
Elk-1 relocalization to the cytoplasm (see below).
Transfection of PC12 cells with the Elk-1 WT expression vector that
encodes both full-length and short Elk-1 enhanced NGF-driven differentiation, as measured by neurite extension. This effect can be
attributed to sElk-1, since its overexpression, but not that of
full-length Elk-1, potentiated differentiation to nearly the same
extent. Moreover, mutation of the major ERK phosphorylation sites at
Ser383/389 to Ala compromised potentiation by the sElk-1
vector. Thus, sElk-1 activation by the ERK signaling cascade
potentiates NGF-driven PC12 differentiation. In direct contrast,
overexpressing the Ser383/389 to Ala mutant of Elk-1
potentiated neuronal differentiation, albeit to lesser extent. This
inactive version can compromise transactivation by endogenous Elk-1
through competition for binding to specific promoter elements, such as
the SRE (50), thereby functioning, similarly to sElk-1, as a dominant
negative mutant of Elk-1. Altogether, these data show opposite roles of
Elk-1 and sElk-1 in NGF-driven differentiation of PC12 cells.
Although sustained activation of ERK by NGF gives rise to neuronal
differentiation in PC12 cells (43), in other cell systems signals that
cause sustained activation of ERK are implicated in cell proliferation
(51). One difference between NGF-treated PC12 cells and other cells
could be the presence of sElk-1. By inhibiting Elk-1 transactivating
properties at the SRE site, sElk-1 could block the activation of genes
implicated in cell proliferation. In support of this, c-Fos has been
argued to play a key role in cell cycle progression (52, 53). Although
NGF treatment of PC12 cells leads to a transient activation of
c-fos (54), microinjection of antibodies to c-Fos in PC12
cells significantly increases neuronal differentiation after exposure
to NGF (52). Indeed, early induction of c-fos could be
related to the mitogenic effect of NGF observed on the first days of
treatment (55, 56). Then, expression of sElk-1 could accelerate
neuronal differentiation by interfering with Elk-1 activation and
thereby c-fos expression, which would affect signaling to
genes that drive cell cycle progression.
Elk-1 is classically described as a nuclear target of activated
ERK. However, we show here that the scenario is different in cells of
neuronal phenotype, since we found it in both nuclear and cytoplasmic
compartments, including neuritic extensions. This redistribution was
related to sElk-1 expression. Extensive deletion studies have shown
that at least two regions of Elk-1 are required for nuclear
translocation (36). One is located in the N-terminal part of Elk-1
(amino acids 34-83) and contains a consensus sequence for nuclear
translocation (47GLRKNKTN54) that is deleted in
sElk-1. However, the other domain, spanning amino acids 137-157, would
appear to be sufficient for nuclear localization of sElk-1. The reason
why Elk-1 relocalizes upon the expression of sElk-1 is still unclear,
but it suggests that the two proteins compete for a limiting factor
that controls nuclear retention in neuronal cells. Furthermore, Elk-1
relocalization required the leucine-rich sequence
(7LWQFLLQLLR16), described previously as a
nuclear export sequence (NES) for the TCF Net (57), that normally is
silent in Elk-1. The nature of the nuclear retention factor and the
mechanism by which sElk-1 unmasks the export signal remain to be identified.
The presence of Elk-1 in cytoplasmic compartments could be
related to a specific function of ERK signaling in neuronal cells. Indeed, we have previously shown that phosphorylation of both ERK and
Elk-1 occurred in dendritic, cytoplasmic, and nuclear compartments upon
in vivo electric stimulation (29). The dendritic and nuclear
phosphorylation of Elk-1 was blocked by PD98059, an inhibitor of ERK
activity. In NGF-differentiated PC12 cells, strong activation of ERK by
phorbol ester leads to nuclear translocation of Elk-1 (data not shown).
This raises the interesting possibility that dendritic activation of
Elk-1 leads to its nuclear translocation, where it could serve as a
signal transducer and transcriptional activator mediating a later phase
of gene induction. Given the strong link between Elk-1 and ERK in
neuronal cells in vitro (28) as well as in vivo
(29), Elk-1 could conceivably translocate together with activated ERK
in the nucleus. Further experiments should clarify this intriguing hypothesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C. Sections of 500 µm thick were cut on an
ice-cold Cryomat. The different cerebral structures were dissected out
with a retinal scalpel and then stored until subsequent processing.
Subcellular fractionation of these cerebral structures was performed as
described previously (30). Brain extracts and cell lines were lysed in
a solubilization buffer as described previously (28). Lysates were
separated by 10% SDS-polyacrylamide gel electrophoresis before
electrotransfer. Blots were blocked with 5% non-fat dry milk and
incubated with rabbit polyclonal antisera raised against Elk-1 (1:500);
Zif268 (1:500), and STAT3 (1:1000). After rinsing the
blots were incubated with goat anti-rabbit horseradish
peroxidase-conjugated antibody (1:5000, Amersham Pharmacia Biotech)
before exposure to the ECL kit (Amersham Pharmacia Biotech).
473 to
70 of Elk-1
5'-untranslated region in the expression vector pSG5-Elk-1 (31) was
performed using the double-stranded DNA quick change mutagenesis
kit (Stratagene) with 5'-CACTATAGGGCGAATTCCCGGAGCTGCCACTGCC-3' as
primer. For site-directed mutagenesis the following oligonucleotides
served as primers: 1st ATG mutated,
5'-AGTGCTTCCCTAGTGAAGGACCCATCTGTGACG-3'; 2nd ATG mutated,
5'-AAGAACAAGACCAACAAGAATTACGACAAGCTT-3'; 1st ATG consensus Kozak,
5'-CGTGAGTGCTTCCCCCGCCATGGACCCATCTG-3'; 2nd ATG poor consensus Kozak,
5'-AAGAACAAGAAGCAGATGAATTACGACCAAGCTT-3'. For N-terminal HA-tagged
Elk-1 FL, an EcoRI/XbaI fragment of the full-length coding sequence of Elk-1 or Elk-1(AAG2) was generated by
PCR using 5'-CCGGAATTCGACCCATCTGTGACGCTG-3' and
5'-TGCTCTAGATCATGGCTTCTGGGG-3' and subcloned into an
EcoRI/XbaI fragment of the pcDNA3-HA.
HA-Elk-1(AAG1) was generated from HA-Elk-1 WT by deleting nucleotides
1-162 with 5'-GCCTCGGATCCAATTCAATTACGACAAGCTTAG-3' as primer. HA-Elk-1
(NES) was generated by PCR from HA-Elk-1 WT using
5'-GACCCATCTGTGACGGAACAAGGTAATGGCCAC-3' as primer. The N-terminal
GFP-tagged Elk-1 AAG2 was generated after PCR amplification of Elk-1
AAG2 and cloning into pcDNA3.1/NT-GFP-topo vector (Invitrogen).
Mutations of Ser383/Ser389 into
Ala383/Ala389 in Elk-1 cDNA fragment,
inserted either in pSG5 or in pcDNA3-HA expression vector, were
performed with primers 5'-AGCACTCTGGCTCCAATTGCACCCCGTGCTCCAGCC-3'. All the resulting mutagenized plasmids were sequenced and tested by in vitro coupled transcription/translation analysis.
-glycerophosphate, 5 mM 4-nitrophenyl phosphate, and 2.5 µg/ml each aprotinin, leupeptin, pepstatin, and
antipain. Where indicated the reaction contained 20 ng of purified and
activated recombinant Erk2 (Upstate Biotechnology Inc.). After a 30-min
incubation at 30 °C, the proteins were incubated under DNA binding
conditions with a 32P-labeled probe containing one copy of
the c-fos SRE as described elsewhere (3). In brief,
reactions (7.5 µl) contained 0.2 ng (4 fmol) of labeled probe, a
salt/protein/buffer mixture, 2.5 µg of poly(dI-dC)·(dI-dC),
recombinant core SRF-(90-245), and 2.5 µl of the appropriate control
or kinase reaction described above. After incubation for 30 min at room
temperature, complexes were resolved on a 5% polyacrylamide gel
containing 0.5× TBE at 1 mA/cm for 4 h. Complexes were visualized
by autoradiography at room temperature and by using intensifying
screens at
70 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
sElk-1, a brain-specific isoform of
Elk-1. A, immunocytochemical detection of Elk-1,
Zif268, and STAT3 in the cerebral cortex.
Zif268 is exclusively nuclear and STAT3 is
cytoplasmic, whereas Elk-1 is present in both nuclear and cytoplasmic
compartments of mature neurons (magnification × 630).
B, Western blot analysis of Elk-1 proteins in extracts
prepared from cerebral cortex (Cx), striatum
(Str), hippocampus (Hc), cerebellum
(Cb), and substantia nigra pars reticulata (SNr).
The Elk-1 C-terminal antibody reveals two bands, one at the apparent
molecular weight of Elk-1 protein and the other at 45 kDa,
corresponding to a small isoform of Elk-1 (sElk-1).
C and D, cerebral extracts were fractionated into
nucleus and cytoplasm and analyzed by Western blotting with
Zif268 and STAT3 antibodies (C) and an
Elk-1 C-terminal antiserum (D).
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Fig. 2.
sElk-1 expression is linked to the neuronal
phenotype. A, immunodetection of Elk-1 from
peripheral tissues and cell lines compared with cerebral cortex. Note
the absence of sElk-1 expression in non-neuronal extracts. Untreated
(B) or NGF-treated (C) PC12 cells were
photographed with an inverted microscope (magnification × 200).
D, comparative expression of Elk-1 in lysates from these
PC12 cells and cerebral cortex. Western blotting and immunodetection
were performed as described in the legend to Fig. 1.
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Fig. 3.
An internal AUG codon in Elk-1 mRNA
sequence is used as an initiation site to generate sElk-1
proteins. A, Northern blot detection of Elk-1 mRNA
expression from various mouse tissue total RNA (left panel)
and mouse brain poly(A) enriched RNA (right panel) extracts.
B, schematic representation of the three functional domains
of Elk-1: (i) the DNA binding domain (or Ets domain), (ii) the domain
that mediates interaction with the cofactor SRF, and (iii) the
C-terminal regulatory domain, which contains residues targeted by
MAPKs. The DNA binding domain of Elk-1 contains a methionine at
position 55 (Met55) and a leucine-rich sequence (residues
7-16) upstream from Met55. C, schematic diagrams
of the five expression vectors used to generate Elk-1 proteins in
vitro. Elk-1 WT contains the wild type sequence of Elk-1 cDNA
with the two in-frame ATGs (bold); Elk-1(AAG1) and Elk-1(AAG2) contain
the first or the second ATG converted to AAG, respectively. Elk-1
(Kozak1) contains the first ATG in a perfect Kozak consensus
context. Elk-1 (NoKozak2) contains the second ATG in a poor
Kozak consensus context. The dotted lines represent
nucleotides sequence of the 5'- or 3'-untranslated regions of Elk-1
cDNA. D, autoradiogram obtained after in
vitro coupled transcription-translation of Elk-1
constructs and immunoprecipitation (IP) with a C-terminal
Elk-1 antibody.
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Fig. 4.
sElk-1 expression drives Elk-1 within
the cytoplasm. A and B, in
vitro coupled transcription translation from expression vectors in
which a HA tag was fused to the N-terminal part of Elk-1(AAG2), Elk-1
WT, or Elk-1(AAG1). A, in vitro translated
proteins were immunoprecipitated (IP) with a C-terminal
Elk-1 antibody. B, immunoprecipitation with the HA antibody;
note that Elk-1 but not sElk-1 is immunoprecipitated in the case of
HA-Elk-1 WT. C, PC12 cells were transiently transfected with
10 µg of HA-Elk-1(AAG1), HA-Elk-1(AAG2), HA-Elk-1 WT, or HA-Elk-1 NES
and treated with NGF (100 ng/ml) for 24 h before fixation and
immunofluorescent detection of the HA tag (red). Nuclei were
visualized on the same field by Hoechst counterstaining
(blue). Nuclei of HA-immunoreactive cells (left
panels) are pointed by an arrowhead. For each
construct, 50-100 transfected cells with cytoplasmic and nuclear
staining (black bars) or nuclear staining (gray
bars) were counted from 3 independent experiments. D,
cotransfection of 10 µg of HA-tagged Elk-1(AAG1) (red)
with 2 µg of GFP-tagged Elk-1(AAG2) (green). The nucleus
of the transfected cell is pointed by an arrowhead in the
Hoechst-staining panel (blue). Note the cytoplasmic
localization of GFP-Elk-1(AAG2) in the presence of an excess of
HA-Elk-1(AAG1).
-helix in place, which in both
Elk-1 and the highly related TCF Sap-1a contacts the major groove of
DNA (41, 42). TCFs bind to the SRE through their interaction with the
SRF dimer, an interaction that might suffice to stabilize sElk-1
binding. To investigate this, we tested whether in vitro
translated sElk-1 formed ternary complexes together with SRF on the
c-fos SRE. In vitro translated Elk-1 WT extracts
generate the typical ternary complex (Elk-1·core
SRF2·SRE) (Fig.
5A). Similarly, the AAG2
mutant bound DNA, although more weakly than WT (data not shown).
We failed to detect this complex in extracts from in vitro
translated Elk-1(AAG1) showing that sElk-1 cannot readily form ternary
complexes under these conditions (Fig. 5A). Since
phosphorylation of TCF by ERK greatly enhances its ability to be
recruited into a ternary complex (17, 18), we tested whether sElk-1
binding activity was detectable after ERK activation. Incubation with
recombinant active ERK and ATP leads to the appearance of a slower
migrating induced complex (P-Elk-1·SRF2·SRE) generated
with in vitro translated Elk-1 WT (Fig. 5A), as
well as the appearance of a very weak complex with sElk-1 (Elk-1(AAG1)
mutant) (Fig. 5A). These data demonstrate that sElk-1
interacts poorly with SRF on the c-fos SRE upon ERK induction.
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Fig. 5.
Binding and transactivating properties of
sElk-1. A, band shift experiments were performed with
in vitro translated Elk-1 WT, Elk-1(AAG1), or without any
construct (URL), incubated in mock reactions ( ) or in the
presence of recombinant active Erk2 (+). Due to its much stronger
binding signal the Elk-1 WT reaction was diluted 15-fold prior
to the binding reactions to allow direct comparison to the mutant
protein. Binding reactions contained 100-base pair
32P-labeled restriction fragments spanning the
c-fos SRE and recombinant core SRF-(90-245), a deletion
mutant that allows identification of ternary complexes formed by
overexpressed Elk-1 WT and Elk-1(AAG1) independently of endogenous SRF.
The panel shows the region of the autoradiogram corresponding to the
complexes formed by Elk-1. B, luciferase assays from NIH3T3
cells transfected with either Elk-1 WT (50 ng) and/or Elk-1(AAG1) (1600 ng) or Elk-1 WT and Elk-1(AAG1) 2Ala, in the presence of a
3xSRE-TATAfos-luciferase reporter gene SRF and
RasVal-12. Statistical analysis from three independent
experiments were performed using a one-way analysis of variance between
groups. Post hoc comparisons were made using the Scheffe
test; **, p < 0.005 and
,
p < 0.005 when comparing to SRE/RasVal-12 or
SRE/RasVal-12/SRF/Elk-1 WT, respectively. C,
pull-down assays were performed using glutathione-agarose beads bearing
GST or the GST-CBP fusion and 35S-labeled Elk-1 and sElk-1
produced in vitro from the Elk-1 WT construct. The reactions
were separated on a 10% SDS-polyacrylamide gel electrophoresis
acrylamide, and the two Elk-1 isoforms were detected by
autoradiography. Note that Elk-1 and sElk-1 interact with the CBP
bromodomain.
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Fig. 6.
Overexpression of sElk-1 facilitates
PC12 neuronal differentiation. A-G, PC12 cells were
transiently transfected with 2 µg of GFP alone (A) or
in the presence of 10 µg of each Elk-1 construct: Elk-1WT
(B), Elk-1WT 2Ala (C), Elk-1(AAG1)
(D), Elk-1(AAG1) 2Ala (E), or Elk-1(AAG2)
(F), and Elk-1(AAG2) 2Ala (G) (magnification, × 400). H, after 48-96 h of treatment with NGF (100 ng/ml),
GFP-positive cells (green) were counted, and the number of
cells with neurites were scored. To be counted, the neurite had to be
longer than the cell body. Similar results were found after 48 and
96 h of transfection. Results are expressed as percent increase of
neuronal differentiation when compared with cells transfected with GFP
alone (n = 4 independent experiments at least for each
group of transfection). Statistical comparisons: ***, p < 0.001, unpaired Student's t test when comparing GFP
alone with GFP + another construct; , p < 0.05 when
comparing Elk-1 WT with Elk-1WT 2Ala;
, p < 0.05 when comparing Elk-1(AAG1) with Elk-1(AAG1)2Ala; and #,
p < 0.05 when comparing Elk-1(AAG2) with Elk-1(AAG2)
2Ala.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices with four anti-parallel
strands
(48). The
3-helix sits in the DNA major groove and interacts with
the core Ets recognition sequence GGA. The deletion in sElk-1 of the
first 54 amino acids in the DNA binding region removes two
helices and two
strands, which strongly impairs binding of sElk-1 to the
SRF·SRE binary complex. Nevertheless, according to the crystal structure of the the Elk-1 Ets domain bound to DNA, all of the amino
acids in the
3 helix that contact the GGA core sequence are still
present in sElk-1 (42), as are the majority of DNA backbone contacts.
These residues may suffice to generate an extremely weak ternary
complex with SRF on the SRE that we could only visualize upon sElk-1
phosphorylation. Thus, as with Elk-1, phosphorylation of sElk-1 induces
conformational changes that modify its binding properties.
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ACKNOWLEDGEMENTS |
---|
We thank G. Soulis for very helpful technical assistance; M. Garcia for the generous gift of PC12 cells and technical assistance; J. V. Barnier for gift of cell lines; and B. Della Gaspera for helpful discussions and advice. We also thank D. Stéhelin for the generous gift of mouse Elk-1 cDNA.
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FOOTNOTES |
---|
* This work was supported by the University Pierre et Marie Curie, the Ministère de l'Éducation et de la Recherche (to P. V.), the CNRS, by grants from the Association pour la Recherche Contre le Cancer (to J. C. and R. A. H.), the Fondation pour la Recherche Medicale (to R. A. H.), and the European Union BIOMED 2 Contract BMH4-CT-97-2215.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Both authors contributed equally to this work.
To whom correspondence should be addressed: Laboratoire de
Neurochimie-Anatomie, Institut des Neurosciences-Unité Mixte de Recherche 7624, CNRS-Université Pierre et Marie Curie, 75005 Paris, France. Tel.: 33-1-44-27-25-01; Fax: 33-1-44-27-26-69; E-mail: Jocelyne.Caboche@snv.jussieu.fr.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M006678200
2 R. A. Hipskind, unpublished results.
3 L. J. Nissen, J. C. Gelly, and R. A. Hipskind, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: ERKs, extracellular signal-regulated kinases; NGF, nerve growth factor; SRF, serum response factor; SRE, serum-responsive element; MAPK, mitogen-activated protein kinases; TCF, ternary complex factor; GFP, green fluorescent protein; HA, hemagglutinin; WT, wild type; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; GST, glutathione S-transferase; NES, nuclear export signal.
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