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INTRODUCTION |
The c-fos proto-oncogene is a prototypic member of the
set of "immediate early" genes whose transcription is rapidly
activated in the absence of protein synthesis by mitogenic signals (1). Within the promoter of the c-fos gene is a
26-bp1 regulatory sequence,
the serum response element (SRE), that mediates the rapid and transient
induction of the c-fos gene by serum and growth factors (2).
The SRE binds a ubiquitous transcription factor, the serum response
factor (SRF), which is essential but not sufficient for serum and
growth factor activation of SRE (3-5). SRF is an obligatory member of
a multiprotein complex that occupies the SRE and serves as a
convergence point for multiple signal transduction pathways. Thus,
growth factors that activate the Ras-Raf-extracellular signal-regulated
kinase cascade and phorbol esters target a family of transcription
factors containing an N-terminal Ets domain that form a ternary complex
with SRF and SRE DNA (6). c-fos promoter mutants that cannot
bind the ternary complex factor (TCF) remain responsive to serum
induction through a second TCF-independent pathway that still requires
SRF and can involve certain Rho family GTPases (5, 7, 8). The role of
accessory factors in the TCF-independent pathway has yet to be
completely defined.
We have observed that maximal TCF-independent serum induction of the
SRE requires SRF and another transcription factor, C/EBP
(9, 10).
C/EBP
, which binds to the SRE overlapping and immediately 3' to SRF,
is a member of the basic leucine zipper family of transcription factors
(11, 12). The C/EBP
gene contains 3 in-frame methionines that give
rise to three translation products of 38, 35, and 20 kDa (13). (Values
are for the murine or rat genes; the human and avian genes are slightly
larger). The N-terminal half of p38 and p35 contains a strong
transactivation domain, whereas p20-C/EBP
lacks this transactivation
domain and acts as an inhibitor of transcription. Many fibroblast cell
types, such as NIH 3T3 cells, express both the long (35 kDa) and short
(20 kDa) forms of C/EBP
at approximately equal levels (14). Both p35
and p20 homodimers as well as p35-p20 heterodimers bind to the SRE with
equivalent affinity. Moreover, in transient transfection assays
p20-C/EBP
completely blocks serum induction of the c-fos
SRE, whereas p35-C/EBP
potentiates activity approximately
20-30-fold (10). Interestingly, transactivation by p35-C/EBP
is
dependent upon SRF binding to the c-fos SRE. Moreover, we
have shown that C/EBP
is able to bind SRF through protein-protein
interactions in vitro (10). Taken together, these results
suggest the requirement for SRF binding in p35-C/EBP
transactivation
of the SRE reflects some physical interaction between these two
transcription factors that is essential for transactivation.
In this study we confirm that SRF and C/EBP
associate in
vivo using both a mammalian two-hybrid assay for demonstrating
protein-protein interactions and coimmunoprecipitation studies. The
domains of each protein that are sufficient for their interaction
in vivo are comparable to the domains previously identified
in vitro (10) and include the DNA binding domain of SRF
(amino acids 133-265) and the C terminus of C/EBP
(amino acids
152-297) common to p35 and p20. Strikingly, the association of SRF and
p35-C/EBP
but not p20 C/EBP
is dramatically stimulated by
activated Ras. Furthermore, mutation of the threonine within a MAP
kinase consensus motif PGTP in the C terminus of C/EBP
eliminates
the response to Ras. These results indicate that C/EBP
-SRF
protein-protein interactions may be one target for TCF-independent
signaling pathways to the SRE.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
HeLa cells (kindly provided
by Dr. R. Chalkley, Vanderbilt University) were maintained in
Dulbecco's modified Eagle's medium and nutrient mixture F-12 (Life
Technologies, Inc.) containing 10% donor calf serum with iron (Life
Technologies, Inc.), 0.24% sodium bicarbonate, 25 units of penicillin
G sodium/ml, and 25 mg of streptomycin sulfate/ml. NIH 3T3 fibroblasts,
from the American Type Culture Collection, were grown in Dulbecco's
modified Eagle's medium with 10% calf serum (Colorado Serum Company),
0.22% sodium bicarbonate, and 4 mM
L-glutamine, penicillin, and streptomycin. COS-7 cells
(kindly provided by Dr. S. Hann, Vanderbilt University) were maintained
in the same medium as described for NIH 3T3 fibroblasts except that
10% fetal bovine serum (Hyclone) instead of calf serum was added.
HeLa and COS-7 cell transfections were performed using the calcium
phosphate technique (15). Cells were plated at a density of 5 × 105 cells/dish 1 day before transfection. The cells were
exposed to the CaPO4-DNA precipitate for 8 h. The
medium was removed and replaced with complete medium for an additional
36 h before harvesting. NIH 3T3 cell transfections were performed
using a CalPhos Maximizer (CLONTECH) as described
by the manufacturer. Cells at 60-70% confluence were exposed to the
CaPO4-DNA precipitate for 8 h before the medium was
removed and replaced with complete medium for 24 h. Cells were
then serum-deprived for 36-40 h in Dulbecco's modified Eagle's medium supplemented as above except containing 0.5% calf serum before
harvesting. Cell extracts were prepared, and chloramphenicol acetyl
transferase (CAT) assays were performed on extracts containing equivalent cell protein as described previously (16).
Two-hybrid Assay--
The pGAL4, pVP16, and pG5CAT plasmids used
are from the Mammalian MATCHMAKER Two-Hybrid Assay Kit
(CLONTECH). pGAL4-SRF and pGAL4-SRF(133-265) were
constructed by polymerase chain reaction amplification of a 1563-bp
fragment (encoding amino acids 1-508 of SRF) or a 420-bp fragment
(encoding amino acids 133-265 of SRF), respectively, from pGEM3.5
(gift of R. Treisman) as described previously (10). The amplified
fragments with EcoRI ends were inserted into the
EcoRI site of pGAL4. pVP16-LIP was constructed by digestion
of pRSETA-LIP (14) with BamHI, treatment with DNA polymerase
I at 4 °C to generate blunt ends, and subsequent digestion with
HindIII to release a 582-bp fragment. This fragment was
inserted into a pVP16 plasmid, digested with MluI, treated
with DNA polymerase I at 4 °C, and digested with HindIII.
CMV-LAP was a gift of U. Schibler (University of Geneva, Geneva,
Switzerland), and CMV-RasV.12 was a gift of E. Ruley (Vanderbilt University).
Coimmunoprecipitation--
COS-7 cells were harvested in
phosphate-buffered saline containing 0.1 mM sodium vanadate
and collected by low speed centrifugation. Cells were resuspended in
antibody lysis buffer (10 mM Tris, pH 7.5, 1 mM
EDTA, 50 mM NaCl, 0.25% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 µg of aprotinin/ml, 0.1 mM sodium vanadate, 10 mM sodium molybdate, and
10 mM
-glycerol phosphate) and lysed by sonication with
a microtip on setting 2 and 20% duty cycle for 10 s. After
clarification by centrifugation at 12,000 × g for 10 min, the cell extract was incubated with T7 tag antibody-agarose beads
(Novagen) for 2 h. The beads were collected by low speed centrifugation and washed 3 times with antibody lysis buffer. All steps
were done at 4 °C. pcDNA3.1/His-SRF was constructed by cloning
the full-length 1563-bp fragment obtained from polymerase chain
reaction amplification of pGEM3.5, as described above, into EcoRI digested pcDNA3.1/ HisC (Invitrogen).
Immunoblots--
The samples from the coimmunoprecipation
described above were analyzed by electrophoresis on an SDS-12%
polyacrylamide gel for 3 h at 160 V. The gel was equilibrated in
transfer buffer (33 mM Tris base, 192 mM
glycine, 20% methanol) for 15 min before the transfer of proteins to
Immobilon-P membrane (Millipore Corp.). After transfer, an immunoblot
was performed as described previously (10), except a 1:2000 dilution of
anti-C/EBP
C-terminal peptide antibody (Santa Cruz Biotechnology)
and a 1:5000 dilution of goat anti-rabbit antibody (Roche Molecular
Biochemicals) were used. The secondary antibody was detected using
SuperSignal Chemiluminescent Substrate (Pierce).
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RESULTS |
C/EBP
and SRF Associate in Vivo--
To extend the validity of
the C/EBP
-SRF protein interactions previously demonstrated by GST
pulldown assays in vitro (10), we have also developed a
mammalian "two-hybrid" system to monitor the possible association
of SRF and p35-C/EBP
(also called LAP) or p20-C/EBP
(also called
LIP) in vivo. Initially, we prepared a construct in which
the DNA binding domain of Gal4 (amino acids 1-80) is fused to SRF.
When pGal4-SRF is transfected into HeLa cells along with pG5CAT
(CLONTECH), a CAT reporter gene-driven by five
copies of the Gal4 DNA binding site linked to the E1B promoter, at best
only a 1.5-fold increase transcription is observed with the average of
four experiments being 1.1-fold (Fig. 1). Thus, although SRF contains a C-terminal transactivation domain, it is
not particularly potent in this assay. Other investigators have also
observed very low activity with Gal4-SRF (17). However, a much larger
increase in transactivation is observed when pCMV-LAP (encoding
p35-C/EBP
) is cotransfected with pGal4-SRF and the pG5CAT reporter.
At the highest level of p35-C/EBP
, an average 7.5-fold increase in
CAT activity was observed with up to a 15-fold increase in some
experiments. This increase is not observed when p35-C/EBP
alone is
expressed with pG5CAT as expected, because Gal4 sites, not C/EBP
sites, are upstream of the promoter driving the CAT gene. The results
presented in Fig. 1 indicate that SRF, when affixed to the DNA through
a Gal4 DNA binding domain, can recruit p35-C/EBP
to the promoter
whereby the transactivation domain of p35-C/EBP
, either alone or in
concert with the transactivation domain of SRF, results in a strong
increase in CAT activity. In the strictest sense, this approach does
not constitute a two-hybrid assay, because only one hybrid protein,
Gal4-SRF, was employed. Because p35-C/EBP
intrinsically contains a
strong activation domain, we did not need to append another. However,
because the approach follows the concept of the two-hybrid assay and
because we do in one instance (Fig. 4) use two-hybrid proteins Gal4-SRF and VP16-p20-C/EBP
, we will for simplicity sake refer to this experimental approach as the two-hybrid assay.

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Fig. 1.
SRF and p35-C/EBP
associate in vivo. HeLa cells were
transfected with 6 µg of pG5CAT either alone or with 2 µg of
pGal4-SRF and/or the indicated amount of pCMV-LAP (encoding
p35-C/EBP ). Total DNA in each transfection was adjusted to 16 µg
with pUC19. Cells were harvested 48 h after transfection, and CAT
activity was measured as described in Ref. 16. Data are the average of
four determinations in duplicate; average standard error was
33%.
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Next we extended the two-hybrid system to examine possible mitogenic
regulation of SRF-p35-C/EBP
interactions in NIH 3T3 cells. In these
experiments we used a Ras protein with an activating mutation at codon
12 (Ras.V12) to simulate a variety of growth-promoting signals. As
shown in Fig. 2A, dramatic
increases (190-240-fold) in pG5CAT reporter gene expression are
observed when pGal4-SRF and pCMV-LAP (encoding p35-C/EBP
) are
cotransfected with an expression construct for activated Ras, compared
with a 10-13-fold increase in the absence of Ras. This result suggests
that the interaction of SRF and p35-C/EBP
could be regulated
in vivo by a Ras-dependent signaling pathway. It
is also possible that activated Ras promotes the "unmasking" of a
dormant transactivation domain in p35-C/EBP
as has been observed in
some cell types. Because p35-C/EBP
, when overexpressed, activates
the c-fos SRE nearly equivalently in quiescent as well as
stimulated cells (10), it does not appear that the transactivation
domain of p35-C/EBP
necessarily requires unmasking by a
signal-dependent pathway in NIH 3T3 cells. However, to
address this possibility directly we repeated the experiment in Fig.
2A with a Gal4-LAP construct. pGal4-LAP activated pG5CAT but
no stimulation by activated Ras was observed (data not shown). In Fig.
2B we demonstrate that the DNA binding domain of SRF (amino acids 133-265) is sufficient for both the interaction with
p35-C/EBP
in vivo and stimulation of this interaction by
activated Ras, although the magnitude of the effect is somewhat
smaller, particularly in the case of activation by Ras (note the
difference in y axis scales in Fig. 2, A and
B). Nonetheless, given that the DNA binding domain of SRF is
instrumental in TCF-independent serum activation of the SRE (5, 7),
this result is highly significant.

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Fig. 2.
Ras stimulates the interaction of SRF and
p35-C/EBP in vivo.
A, NIH 3T3 cells were transfected with 5 µg of pG5CAT
alone, with 2 µg of pGAL4-SRF, or with 2 µg of pGAL4-SRF and 1 or 2 µg of pCMV-LAP in the absence or presence of 2 µg of an activated
Ras expression vector. Total DNA in each transfection was adjusted to
11 µg with pUC19. Cells were serum-deprived for 40 h prior to
harvesting. CAT activity was measured as described in Ref. 16.
B, same as A except that 2 µg of
pGAL4-SRF(133-265) were used. Data are the average of four-six
determinations in duplicate; average standard error in A was
33% and in B was 30%.
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The Association of SRF and C/EBP
Is Stimulated by Activated Ras
in Vivo--
Although the two-hybrid system is a standard and well
accepted approach for analyzing protein-protein interactions in
vivo, we cannot exclude the possibility that the stimulation by
activated Ras observed in Fig. 2 is because of other DNA binding
factors and/or general transcription factors that have been modified to make them more responsive to the effects of SRF/p35-C/EBP
on the
promoter. We therefore sought direct evidence, via
coimmunoprecipitation, that SRF and p35-C/EBP
associate in
vivo and that their interaction is stimulated by activated Ras.
COS cells were transfected with an expression vector for a 6×
histidine-tagged version of SRF carrying the
10 (T7 tag) epitope
sequence in the presence or absence of pCMV-LAP (encoding
p35-C/EBP
). Because endogenous COS cell C/EBP
proteins are larger
in size (42 and 45 kDa, similar to the human C/EBP
gene,
NFIL6), the exogenous p35 rat C/EBP
is easily
distinguished (compare Fig. 3,
lanes 8 and 9). Because p35-C/EBP
is
overexpressed, to avoid overexposure of the p35-C/EBP
signal, the
film exposure in lane 8 is shorter than in lane 9 (10 versus 60 s). This difference is reflected in the
correspondingly lower signal of endogenous p42/45 and p20-C/EBP
in
lane 8 compared with lane 9. Cell lysates were
incubated with T7 tag antibody-agarose beads, and the
immunoprecipitated proteins were subjected to immunoblotting with
C/EBP
antibody. As shown in Fig. 3, p35-C/EBP
coprecipitated with
tagged SRF on the antibody beads but only when activated Ras was
included in the cotransfections (lane 5). We are aware of
the caveats of using overexpressed proteins in this type of assay,
because the high level of expression may promote their fortuitous
association. However, in this case nonspecific interactions are
rendered less likely by the fact that the SRF-p35-C/EBP
interaction is only observed in the presence of activated Ras.

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Fig. 3.
p35-C/EBP
coimmunoprecipitates with SRF but only in the presence of
activated Ras. COS cells were transfected with 10 µg of
pcDNA3.1SRF (lanes 1, 2, 4, and
5) and 10 µg of pCMV-LAP (lanes 2,
3, 5, 6, and 8) in the
presence or absence of 2 µg of an activated Ras expression construct
as indicated. Cells were harvested 40 h posttransfection, and
whole cell lysates were analyzed by immunoblotting with C/EBP
antibody (lanes 8 and 9) or incubated with T7 tag
antibody (T7tag Ab)-agarose beads followed by immunoblotting
of precipitated proteins (lanes 1-7).
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The Association of SRF and p35-C/EBP
, but Not SRF and
p20-C/EBP
, Is Stimulated by Activated Ras in Vivo--
In Fig. 2 we
determined that the DNA binding domain of SRF is sufficient for
association with C/EBP
in vivo, in agreement with the GST
pulldown experiments in vitro (data not shown). We also
wanted to determine the domain of C/EBP
sufficient for interaction with SRF in vivo. Because p20-C/EBP
binds to SRF in GST
pulldown assays (10), we proceeded to determine whether it would also interact with SRF in vivo. Because p20-C/EBP
lacks a
transactivation domain, we used a chimeric VP16-p20-C/EBP
protein in
the two-hybrid assay shown in Fig. 4.
Gal4-SRF was able to recruit VP16-p20-C/EBP
to the pG5CAT promoter
resulting in a 9-fold activation in transcription (Fig. 4A).
We consistently required more VP16-p20-C/EBP
expression construct to
obtain the same degree of activation as observed with p35-C/EBP
.
However, it is difficult to determine if this is reflective of a weaker
interaction between Gal4-SRF and p20-C/EBP
because we do not know
the relative potencies of transactivation domains, although VP16 is
generally considered to be quite strong. VP16-p20-C/EBP
consistently
gave a small (3-fold) increase in transcription in the absence of
Gal4-SRF. Because there are no DNA binding sites for p20-C/EBP
in
the Gal4-dependent promoter, we are unsure of the mechanism
for this increase. Because no increase is observed with VP16 alone, it
may involve some protein-protein interaction between p20-C/EBP
and a
component of the initiation machinery, which although weak, leads to a
detectable increase in transcription because of the extremely strong
nature of the VP16 transactivation domain. However, the key finding is
that activated Ras completely failed to stimulate the association of Gal4-SRF and VP16-LIP in the two-hybrid assay (Fig. 4B). In
fact, a 3-4-fold inhibition was observed so that the level of
transactivation by VP16-p20-C/EBP
was no different than that
observed upon expressing a high level of VP16 alone. These data suggest
that selectively stimulating the physical association of p35-C/EBP
,
but not p20-C/EBP
, with SRF may be a mechanism for
Ras-dependent signaling pathway(s) to increase
transcriptional activity of the c-fos SRE.

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Fig. 4.
The association of SRF and
p35-C/EBP , but not SRF and
p20-C/EBP , is stimulated by Ras in
vivo. A, NIH 3T3 cells were transfected with
5 µg of pG5CAT alone or with 2 µg of pGAL4-SRF, 1 or 2 µg of
pCMV-LAP, 5 µg of VP16-p20-C/EBP , or 10 µg of VP-16 as
indicated. Total DNA in each transfection was adjusted to 19 µg with
pUC19. Cells were serum-deprived for 40 h prior to harvesting. CAT
activity was measured as described in Ref. 16. B, same as
A except that 2 µg of an activated Ras expression vector
were included. Data are the average of 6-16 determinations. Average
standard error was 52% for A and 40% for
B.
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Ras-stimulated Association with SRF Requires Threonine 235 in
C/EBP
--
Mitogen-activated protein kinases play pivotal roles in
mediating signal transduction upon activation of GTPases such as Ras. The mitogen-activated protein kinases are proline-directed kinases, recognizing the consensus motif PX(S/T)P. Such a consensus motif, PGTP,
is found in the C terminus of C/EBP
(common to LAP and LIP) and is
conserved among C/EBP
genes from all species. Moreover, Nakajima
et al. (23) found that phosphorylation of threonine 235 (numbering for human gene) is induced by cotransfecting Ras.V12 in NIH
3T3 cells. Nakajima et al. (23) kindly provided us with a
site-directed mutant of human C/EBP
(NFIL6) in which they
had substituted alanine for threonine 235. We used an expression
construct for this mutant NFIL6 protein to determine if Ras-activated
association with SRF in the two-hybrid assay might require
phosphorylation of Thr235. Substitution of Ala for
Thr235 resulted in only a small (but reproducible)
reduction in NFIL6 interaction with Gal4-SRF in the unstimulated state
(Fig. 5). However, stimulation by
activated Ras was completely abolished with the mutant NFIL6; in fact,
activated Ras was slightly inhibitory. These results suggest that
Ras-activated association of SRF with C/EBP
requires phosphorylation
of Thr235 by a Ras-dependent mitogen-activated
protein kinase.

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Fig. 5.
Ras-activated association with SRF requires
threonine 235 in C/EBP . NIH 3T3 cells
were transfected with 5 µg of pG5CAT alone, 2 µg of pGAL4-SRF, or 2 µg of pGAL4-SRF and 1 or 2 µg of pCMV-NFIL6 or pCMV-NFIL6(235T-A)
as indicated in the absence or presence of 2 µg of an activated Ras
expression vector. Total DNA in each transfection was adjusted to 11 µg with pUC19. Cells were serum-deprived for 40 h prior to
harvesting. CAT activity was measured as described in Ref. 16. Data are
the average of four determinations in duplicate; average standard error
was 24%.
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DISCUSSION |
In this report we have presented evidence that C/EBP
and SRF
associate in vivo and this protein-protein interaction is
regulated by Ras-dependent signaling pathway(s). These
transcription factors interact in vivo through the DNA
binding domain of SRF, which is known to be required for
TCF-independent signaling to the SRE (5, 7) and the C terminus of
C/EBP
common to p20 and p35-C/EBP
. Both the two-hybrid assay in
mammalian cells and in vivo coimmunoprecipitations were used
to show that activated Ras dramatically stimulates the association of
p35-C/EBP
and SRF. These data support the involvement of a physical
association of C/EBP
and SRF in transcriptional activation of the
SRE, which is stimulated by Ras-dependent signaling pathways. However, future development of a mutant C/EBP
unable to
bind SRF will be necessary to confirm the role of C/EBP
-SRF protein-protein interactions in SRE function.
Interestingly, activated Ras completely failed to stimulate the
association of Gal4-SRF and VP16-p20-C/EBP
in the two-hybrid assay;
in fact, a 3-4-fold inhibition was observed. Unfortunately, we have
been unable to confirm the lack of Ras-stimulated association of SRF
and p20-C/EBP
in the coimmunoprecipitation studies because of an
unacceptably high level of nonspecific binding of p20-C/EBP
(or
VP16-p20-C/EBP
) to the antibody-agarose beads. We presume this is
because of the highly charged nature of p20-C/EBP
and are currently
working on developing satisfactory precautions to prevent this.
Nonetheless, our observations with the two-hybrid assay suggest a new
model for SRE function in which protein-protein interactions with the
DNA binding domain of SRF are a critical target for TCF-independent
mitogenic regulation because they are key to determining which isoform
of C/EBP
stably occupies the SRE. Our data indicate that p35 and
p20-C/EBP
are recruited to the SRE not only by binding to the DNA,
which is not a high affinity site, but also by protein-protein
interactions with SRF. If SRF and p20 homodimers occupy the SRE, no
transactivation occurs. When SRF and p35 homodimers or a p35/20
heterodimer occupy the SRE, transactivation results. Mitogenic
signals that activate Ras dramatically enhance the interaction of SRF
selectively with p35-C/EBP
, ultimately resulting in efficient transactivation.
This mechanism is compatible with the fact that both p35 and p20 are
located in the nucleus in similar amounts in serum-deprived or
-stimulated NIH 3T3 cells. Moreover, the distribution of homodimers and
heterodimers comprising C/EBP
does not appear to change in gel shift
assays performed with nuclear extracts from serum-deprived or
-stimulated cells (data not shown), suggesting that direct regulation
of p20-C/EBP
or p35-C/EBP
DNA binding activity is unlikely.
In vivo footprinting experiments have not found detectable changes in protein occupancy of the SRE upon serum stimulation (18).
However, the exchange of p35-C/EBP
for p20-C/EBP
would be
compatible with this observation in that both proteins share the same
DNA binding domain and thus would be expected to exhibit extremely
similar, if not identical, footprints on SRE DNA.
The Ras-dependent signaling pathway(s), which selectively
promote the interaction of p35-C/EBP
with SRF, have yet to be
defined. Although Raf was the first effector protein located downstream of Ras to be identified, it is now clear that there are multiple Ras
effector proteins (for review, see Ref. 19). Moreover, Ras can activate
another family of small GTPases, the Rho family GTPases (for review,
see Ref. 20). Recent studies on MAP kinase signaling pathways have
uncovered the presence of at least three distinct MAP kinase pathways
that can be activated by Ras and Rho family GTPases. The terminal
kinases in these three pathways are the extracellular signal-regulated
kinases, Jun N-terminal kinases/stress-activated protein kinases, and
p38 kinases (for review, see Refs. 21 and 22). The effect of Ras on
p35-C/EBP
-SRF protein-protein interactions was not observed with the
Thr235-Ala C/EBP
mutant, suggesting that phosphorylation
of threonine 235 strongly enhances the interaction of p35-C/EBP
with
SRF. Threonine 235 is contained in a MAP kinase consensus sequence conserved among C/EBP
genes from all species, and this site is known
to be an in vitro target for partially purified MAP kinase (23). It will be interesting to investigate which of the terminal MAP
kinase(s) may phosphorylate C/EBP
at Thr235 in response
to mitogenic signals in vivo and identify the upstream kinases that link this phosphorylation event to Ras activation.