From the Transcription Laboratory, Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom
Received for publication, March 26, 2001, and in revised form, April 27, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of the transcription factor serum
response factor (SRF) is dependent on Rho-controlled changes in
actin dynamics. We used pathway-specific inhibitors to compare the
roles of actin dynamics, extracellular signal-regulated kinase (ERK)
signaling, and phosphatidylinositol 3-kinase in signaling either
to SRF itself or to four cellular SRF target genes. Serum,
lysophosphatidic acid, platelet-derived growth factor, and phorbol
12-myristate 13-acetate (PMA) each activated transcription of a
stably integrated SRF reporter gene dependent on functional RhoA
GTPase. Inhibition of mitogen-activated protein kinase-ERK kinase
(MEK) signalling reduced activation of the SRF reporter by all stimuli
by about 50%, except for PMA, which was effectively blocked.
Inhibition of phosphatidylinositol 3-kinase slightly reduced reporter
activation by serum and lysophosphatidic acid but substantially
inhibited activation by platelet-derived growth factor and PMA.
Reporter induction by all stimuli was absolutely dependent on actin
dynamics. Regulation of the SRF (srf) and vinculin
(vcl) genes was similar to that of the SRF reporter gene;
activation by all stimuli was Rho-dependent and required
actin dynamics but was largely independent of MEK activity. In
contrast, activation of fos and egr1 occurred independently of RhoA and actin polymerization but was almost completely dependent on MEK activation. These results show that at
least two classes of SRF target genes can be distinguished on the basis
of their relative sensitivity to RhoA-actin and MEK-ERK signaling pathways.
Serum response factor
(SRF)1 is a transcription
factor that controls many "immediate-early" genes whose
transcription is induced by extracellular signals and many genes
constitutively expressed in muscle (for references see Ref. 1). The
activity of SRF is regulated both by cellular signal transduction
pathways and by its interaction with other transcription factors. At
the immediate-early fos and egr1 promoters, for
example, SRF forms a ternary complex with members of the ternary
complex factor (TCF) family of mitogen-activated protein
kinase-regulated Ets domain proteins (Ref. 2; for review see Ref. 3).
It remains unclear whether all SRF-controlled immediate-early gene
promoters bind TCF, however, because the TCF recognition site is simple
and can be located at variable distances from that of SRF (4). SRF also
exhibits functional cooperation with a number of other, constitutively
active, transcription factors including Sp1, ATF6, GATA4, Nkx2.5, and
the myogenic regulatory factors (5-10).
The signaling pathways impinging on SRF and its TCF partners at
immediate-early promoters have been extensively studied.
Transcriptional activation by the TCF proteins is potentiated by
signal-induced phosphorylation of a conserved C-terminal activation
domain (3, 11). Promoter mutant and TCF expression studies suggest that TCF binding is required to link the fos and egr1
promoters to the Ras-Raf-MEK-ERK signaling pathway (12-16). Consistent
with this, the specific MEK inhibitor PD98059 (17, 18) inhibits fos induction by a number of stimuli (19, 20). By contrast, serum stimulation potentiates SRF activity via a signaling pathway involving the Rho GTPase (21). In transfection assays, both the
serum-induced activity of SRF and fos reporter genes and the constitutive activity of certain muscle-specific promoters are strongly
dependent on functional Rho (21-23). Phosphatidylinositol 3-kinase
(PI-3K) has also been implicated in signaling to SRF via both
Rho-dependent and -independent mechanisms, although this is
not detectable in all cell types (24-27).
Recent studies have shown that Rho GTPases activate SRF via their
ability to induce depletion of the G-actin pool
(28-30).2 Although this
pathway is required for serum-induced transcription of the cellular
SRF, vinculin, and cytoskeletal actin genes, it contributes little to
activation of fos or egr1 (28). This suggests that the efficiency of Rho-actin signaling to SRF is dependent on
promoter context and that other signal pathways must control the
activity of SRF target genes not responsive to Rho-actin signaling. Here we use pathway-specific inhibitors to investigate the roles of
Rho-actin signaling, MEK-ERK, and PI-3K in activation of an SRF
reporter gene by different stimuli. We compare the results to those
obtained with a panel of endogenous SRF target genes. Our results
define two classes of SRF target gene controlled by Rho-mediated actin
dynamics and MEK-ERK signaling, respectively.
Cell Lines, Transfections, and General Methods--
SRE.FosHA
cells are NIH3T3 cell-derivative cells carrying an integrated 3D.AFos
HA reporter (21, 31). NIH3T3 cells in a 6-well plate were transiently
transfected using LipofectAMINE (Life Technologies, Inc.) according to
the manufacturer's recommendations; 6 µl of LipofectAMINE, 0.05 µg
of LexOP2tkLuc, 0.02 µg ofr NLexElkC, 0.1 µg of MLVLacZ, and 0.65 µg of MLV128 RNase Protection Assays--
RNA preparation and RNase
protection assays were as described (13, 21). GAPDH and 3D.AFos probes
were as described (21, 28). Other probes were: fos, a
199-nucleotide probe spanning 5'-flanking region and part of exon 1, nucleotides 540-738 (GenBank accession number V00727), generating a
185-nucleotide protected fragment; egr1, a 348-nucleotide
probe spanning 5'-flanking region and part of exon1, nucleotides
1348-1661 (GenBank accession number M22326), generating a
267-nucleotide protected fragment; pre-srf, a 343-nucleotide
probe spanning the exon 5-intron 5 boundary, nucleotides 8181-8450
(GenBank accession number AB03837), generating a 276-nucleotide
protected fragment (exon 5-intron 5 precursor) and a 192-nucleotide
protected fragment (exon 5 mRNA); pre-vcl, a
443-nucleotide fragment spanning the exon 3-intron 3 boundary, nucleotides 413-769 (GenBank accession number L13299) generating a
357-nucleotide protected fragment (exon 3-intron 3 precursor) and a
108-nucleotide protected fragment (exon 3 mRNA). For quantitation of RNase protection assays, images were obtained using a PhosphorImager (Molecular Dynamics). Protected fragments were quantified after background subtraction with ImageQuant software and normalized to the
GAPDH signal.
Immunoblotting and Antibodies--
For Western blot analysis
4 × 105 cells were plated per 60-mm dish,
serum-starved for 24 h, pretreated with inhibitors as required, and then stimulated with different agents. The cells were rinsed twice
with ice-cold phosphate-buffered saline and lysed into ice-cold lysis
buffer (20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 40 mM Stimuli, Inhibitors, and Toxins--
Stimuli were used at the
following concentrations: fetal bovine serum (Life Technologies, Inc.),
15%, LPA (Sigma), 10 µM; PDGF-BB (Calbiochem), 25 ng/ml;
PMA (Sigma), 50 ng/ml; cytochalasin D (Calbiochem), 2 µM;
and jasplakinolide (Molecular Probes), 0.5 µM. The
inhibitors were used as follows: U0126 (Promega), 10 µM; LY294002 (Calbiochem), 20 µM; wortmannin (Sigma), 200 nM; and latrunculin B (Calbiochem), 0.5 µM.
UO126, LY294002, and wortmannin pretreatments were for 30 min, and
latrunculin B treatments were for 60 min. Cycloheximide and anisomycin
(both Sigma) were used at 10 µg/ml, and C2IN-C3/C2II chimeric toxin
(32) and toxin B were kindly provided by H. Barth and K. Aktories
(Freiburg University). C2IN-C3 and C2II proteins were added directly to
the tissue culture medium at 0.4 µg/ml final concentration 5 h
prior to stimulation; toxin B was added to 5 ng/ml 60 min before
stimulation. At these times more than 90% of the cells were rounded up.
RhoA-dependent Activation of an Integrated SRF Reporter
by Different Stimuli--
Although responsive to serum, Fos reporter
genes controlled by the minimal fos SRE are unresponsive to
receptor-tyrosine kinase activation and PMA stimulation upon transient
transfection into NIH3T3 cells (21, 33). To test the possibility that
SRF reporter genes maintained in stable transfectants might respond to
a greater range of stimuli, we examined the NIH3T3 cell line SRE.FosHA
(31). These cells contain the SRF reporter gene 3D.AFos, which
comprises the human c-fos transcription unit controlled by a
chimeric promoter comprising a cytoskeletal actin TATA region and three
SRF binding sites. Reporter activity was evaluated by RNase protection
at various times following stimulation of serum-deprived cells with serum, LPA, PDGF, or PMA. The reporter gene showed a robust response to
stimulation with each agent (Fig.
1A). Activation by PMA, but not the other stimuli, required protein kinase C activation because it
was blocked by a prolonged PMA pretreatment and by the protein kinase C
inhibitor GF109203X (data not shown). Maximal reporter RNA accumulation
following serum, LPA, and PMA stimulation occurred ~60 min after
stimulation, followed by a slow decline; in contrast, PDGF-induced
reporter RNA was maximal at 30 min and declined rapidly thereafter
(Fig. 1A). Similar results were obtained with two further NIH3T3 cell clones carrying the 3D.AFos reporter (data not shown).
Activation of a transfected SRF reporter gene by serum and LPA requires
functional RhoA (21); therefore we next investigated the involvement of
RhoA in activation of the integrated SRF reporter gene. We used two
different toxins to inactivate Rho family GTPases: the chimeric toxin
C2-C3, which ADP-ribosylates and inactivates RhoA (32), and
Clostridium difficile toxin B, which glucosylates and
inactivates the Rac1, Cdc42, and Rho GTPases (34, 35). The cells were
treated with toxin for a period sufficient to induce rounding up of the
entire cell population (data not shown) and then stimulated as before.
Activation of the integrated SRF reporter gene by serum, LPA, PDGF, and
PMA was completely inhibited in cells pretreated either with C2-C3
toxin (Fig. 1B) or toxin B (Fig. 1C). In
contrast, activation of the reporter by cytochalasin D, which alters
actin dynamics directly by interacting with actin, was not affected
(data not shown).
Differential Dependence of SRF Activation on MEK and PI-3K
Signaling--
All the stimuli are strong activators of the ERK
pathway; therefore we next investigated the contribution of this
pathway to activation of the SRF reporter gene. The cells were
pretreated for 30 min with the specific MEK inhibitor U0126 (36) before stimulation and analysis of reporter activity as before. Serum- and
LPA-induced transcription was reduced by almost 50%, whereas activation by PDGF was reduced by 60%; only induction by PMA was reduced to background levels (Fig.
2A). To examine the efficacy of the inhibitor, we measured ERK activation by immunoblotting using an
antiserum specific for the activated form of ERK1/2. U0126 treatment
completely blocked ERK activation by all the stimuli except serum,
where a low level of activation persisted at late times; the inhibitor
did not affect PI-3K activity (Fig. 2C). Similar results
were obtained using another MEK inhibitor, PD98059 (18). The ability of
serum, LPA, and PDGF to activate the SRF reporter gene is thus
substantially independent of ERK signaling (see "Discussion").
We next investigated whether reporter activity was dependent on PI-3K,
which has previously been implicated in SRF activation in transfected
HeLa cells (24). Cells were pretreated with the PI-3K inhibitor
LY294002, then stimulated, and analyzed as before. The inhibitor had
variable effects according to the stimulus. Serum induction was
unaffected, and LPA induction was reduced by some 20%; in contrast,
PDGF-induced reporter activity was reduced by 70%, and PMA induction
was substantially blocked (Fig. 2B). Similar results were
obtained using another PI-3K inhibitor, wortmannin (data not shown). To
evaluate the activation of PI-3K in response to the various stimuli, we
examined phosphorylation of Akt, a downstream target of the PI-3K
pathway, using an antiserum specific for Akt Ser(P)473.
Serum and PDGF strongly induced Akt phosphorylation, which was blocked
by LY294002; in contrast LPA very weakly induced Akt phosphorylation, whereas induction by PMA was not detectable (Fig. 2D,
upper panels). The efficiency of Akt activation did not
correlate with reporter activity (compare Fig. 1A and
2D, upper panels). Moreover, LY294002 treatment
did not affect ERK activation by any of the stimuli (Fig.
2D, lower panels). The apparent failure of PMA
stimulation to activate Akt phosphorylation suggests that, at least in
this case, inhibition of SRF activation by LY294002 reflects a
requirement for basal PI-3K activity.
Activation of SRF by All Stimuli Requires Actin
Polymerization--
We previously showed that serum and LPA induction
of an SRF reporter gene is absolutely dependent on signal-induced
changes in actin polymerization (28). Because activation of this
reporter by PDGF and PMA exhibits a different requirement for MEK and
PI-3K signaling compared with serum and LPA, we next investigated
whether activation by these stimuli is also dependent on signal-induced actin polymerization. Serum-starved reporter cells were pretreated for
60 min with 0.5 µM latrunculin B, which inhibits actin
polymerization by sequestering G-actin monomer (37), prior to
stimulation and analysis of reporter RNA activity. Latrunculin B
treatment completely blocked transcriptional induction by all stimuli
including PDGF and PMA (Fig.
3A; U0126 ± latrunculin
B allows comparison with Fig. 6). To confirm that latrunculin treatment
did not affect the activation of the ERK and PI-3K pathways, we
performed immunoblotting experiments with the activated ERK and Akt
Ser(P)473 antibodies. Latrunculin B treatment did not
inhibit activation of either ERK or PI-3K by any stimuli (Fig.
3B).
Activation of TCF Elk-1 Requires MEK but Not Actin
Polymerization--
All the stimuli tested potentiate transcriptional
activation by members of the ternary complex factor family of Ets
domain proteins. The SRF reporter gene does not contain consensus TCF binding sites, but we nevertheless considered the possibility that its
enhanced ability to respond to stimuli when integrated into cellular
DNA might reflect TCF binding. To test this idea, we evaluated the
effects of the inhibitors on TCF activation using a TCF Elk-1 reporter
system. The cells were transfected with a LexA operator-controlled
reporter plasmid and an expression vector encoding a LexA/Elk-1 fusion
protein (11). Following treatment with the various inhibitors, cells
were stimulated, and reporter gene activity was measured. Activation of
the Elk-1 reporter by all stimuli was reduced to background level by
U0126 pretreatment but was not affected by pretreatment with
latrunculin B (Fig. 4). Although Elk-1
reporter activation by PDGF and LPA was reduced some 50% by LY294002
pretreatment, LY294002 had no substantial effect on ERK activation by
these stimuli (compare Fig. 4 with Fig. 2D).
Taken together, these results provide strong support for the notion
that TCF is not involved in activation of the integrated SRF reporter
gene. The availability of inhibitors specific for the signal
transduction pathways that regulate SRF and TCF activation provides a
simple way to evaluate the potential contributions of these
transcription factors to immediate-early gene transcription.
Kinetics of Vinculin and srf Activation by Extracellular
Stimuli--
We previously demonstrated that serum-induced
transcription of various SRF target genes exhibits a differential
sensitivity to actin dynamics; induction of the genes encoding vinculin
(vcl), cytoskeletal actin (actb), and SRF
(srf) was sensitive to latrunculin B, like the SRF reporter
gene, whereas induction of fos transcription was not (28).
Having established the contributions of different signaling pathways to
activation of the SRF reporter gene, we set out to determine their
contribution to SRF target gene activation. The kinetics of
transcriptional activation of fos and egr1 are well established (38, 39); however, although it is clear that growth
factors activate vcl and srf gene expression at
the transcriptional level (40, 41), the kinetics of this have not been
investigated in detail. The high basal levels and stabilities of the
srf and vcl mRNAs allow only strong and
prolonged transcriptional changes to be reliably measured by
quantitation of mRNA and preclude the use of the mRNA level as
a measure of transcription rate. To circumvent these problems, we
developed an RNase protection assay that allows simultaneous
measurement of the levels of both mRNA and unspliced precursor
transcripts of each gene, by use of RNA probes spanning the
srf exon 5-intron 5 and vcl exon 3-intron 3 borders.
Serum stimulation of NIH3T3 cells led to rapid and transient appearance
of srf and vcl mRNA precursors. Increased
srf precursor RNA level was observed 15 min following
stimulation, reached a maximal level at 30 min, and declined over a 2-h
period; vcl precursor RNA was not detectable until 30 min
following stimulation and declined thereafter (Fig.
5A, compare pre-srf
and pre-vinc). In contrast, accumulation of srf
and vcl mRNAs proceeded gradually throughout the 2-h
period following stimulation, although that of vcl was
delayed, consistent with the delayed appearance of its precursor (Fig.
5A, compare pre-srf with srf and
pre-vinc with vinc). Serum-induced
transcriptional activation of both genes was insensitive to inhibition
of protein synthesis by cycloheximide or anisomycin, and these
treatments also did not affect the delayed onset of vcl
transcription (Fig. 5A). We previously showed that srf and vcl mRNAs accumulate in response to
treatment with the actin binding drugs jasplakinolide, cytochalasin D,
and swinholide (28). Use of the precursor-specific probes demonstrated
that Jasplakinolide treatment induced transient accumulation of
srf and vcl precursor RNA with kinetics similar
to those of serum stimulation (Fig. 5B). Similar results
were obtained with cytochalasin D and swinholide (data not shown). LPA
and PMA activated srf and vcl transcription with
similar kinetics to serum stimulation, whereas PDGF-induced activation
was much more transient, reverting to prestimulation levels after
1 h (Fig. 5C). Taken together, these data establish
that the srf and vcl genes are rapidly and transiently induced at the transcriptional level by the stimuli under
investigation. Moreover, the kinetics of srf and
vcl precursor accumulation, apart from the delayed
appearance of the vcl precursor, resemble those of the SRF
reporter gene (compare Figs. 1A and 5).
SRF Target Genes Differ in Their Sensitivity to Actin Dynamics,
MEK, and PI-3K Signaling--
We used latrunculin B pretreatment to
examine sensitivity of the different SRF target genes to alterations in
actin dynamics. Induction of srf and vcl
transcription by all stimuli was effectively blocked by latrunculin B
(Fig. 6A, upper
panels). In contrast, activation of fos and
egr1 was strikingly less sensitive to the inhibitor;
egr1 transcription was essentially unaffected, and fos induction was reduced by only 30-40%; a small residual
activation of these genes observed in U0126-treated cells (see below)
was also sensitive to latrunculin treatment (Fig. 6A,
lower panels). Next we evaluated the contribution of MEK-ERK
signaling to transcriptional activation of the different genes using
U0126 pretreatment. The inhibitor reduced activation of the
srf and vcl genes by serum, LPA, and PDGF by
40-50%, with srf being slightly less sensitive than
vcl; only in the case of PMA did U0126 block induction (Fig. 6A, top panels). U0126 treatment did not abolish
the delay in accumulation of vcl precursor (data not shown).
In contrast, U0126 treatment effectively prevented induction of
fos and egr1 transcription by all of the stimuli,
although a residual fos induction by serum was detectable
(Fig. 6A, lower panels). Thus with respect to
these treatments, the behavior of the srf and vcl
genes is similar to that of the SRF reporter, whereas that of
fos and egr1 resembles that of the TCF
reporter.
We also examined the effect of the PI-3K inhibitors LY294002 and
wortmannin on activation of the various target genes, because this
treatment also differentially affects activation of the SRF and TCF
reporters. Transcriptional induction of the srf and
vcl genes was sensitive to LY294002; induction by PDGF and
PMA was effectively blocked, and induction by LPA was reduced some
50%, whereas serum induction was either slightly impaired
(srf) or not affected (vcl) (Fig. 6B).
In contrast, activation of fos and egr1
transcription by all stimuli was insensitive to LY294002, with
fos transcription actually showing a slight enhancement
(Fig. 6B). As with MEK-ERK and RhoA-actin signaling, PI-3K
thus makes qualitatively distinct contributions to srf and
vcl compared with fos and egr1.
SRF Target Genes Exhibit Differential Dependence on Rho
GTPases--
Finally, we used C2-C3 toxin and toxin B treatment to
investigate the dependence of each of the target genes upon functional RhoA. Activation of srf and vcl transcription by
all stimuli was blocked in cells treated with C2-C3 toxin; in contrast,
egr1 and fos induction was substantially less
sensitive to C2-C3 toxin, with egr1 unaffected and
fos reduced by up to 50% (Fig.
7, left panel). A similar
result was obtained when cells were treated with toxin B; induction of
srf and vcl transcription by all stimuli was
completely sensitive to toxin treatment, whereas activation of
egr1 and fos was at most only partially affected
(Fig. 7, right panel). In the latter case toxin B treatment
caused ~50% reduction in serum- and PDGF-induced fos and
egr1 transcription but substantially reduced activation by
LPA; PMA induction was not affected. Thus only those SRF target genes
whose activation is critically dependent on actin dynamics exhibit a
requirement for Rho GTPase activity.
Recent studies have led to the identification of a number of
inhibitors and toxins specific for signaling molecules that regulate the expression of cellular immediate-early genes, including kinases, small GTPases, and cytoskeletal components (for references see Refs.
28, 42, and 43). Here we have used inhibitors specific for Rho GTPases,
actin dynamics, MEK, and PI-3K to investigate signaling to the SRF
transcription factor and four of its cellular target genes in response
to different stimuli. Our results define two types of SRF target gene,
illustrated in Fig. 8. One class, which
includes srf and vcl, behaves in a fashion
similar to that of an SRF reporter gene: regulation of these genes
requires functional Rho and actin polymerization but is only partially
dependent on MEK activity. Regulation of the second class, which
includes fos and egr1, occurs largely
independently of functional Rho and actin dynamics but is instead
critically dependent on MEK-ERK signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(11) were used per well. The cells were lysed in 200 µl of reporter lysis buffer, and luciferase activity was determined
by standard methods. DNA manipulations and plasmid DNA preparation were
by standard methods. RNase protection probes were synthesized from
appropriate derivatives of pCR2.1TOPO (T7; pre-vcl), pGEM-T
(SP6; pre-srf), pSP65 (SP6; egr1), and pSP64
(SP6, fos). LexOP2tkLUC comprises the LexA operator and
HSVtk promoter sequences from LexOP2tkCAT (3, 11) inserted into the
XhoI site of pGL3Basic (Promega).
-glycerophosphate, 50 mM NaF, 0.1 mM vanadate, 1 mM phenylmethylsulfonyl
fluoride, and 1 µg/ml of leupeptin, pepstatin, and aprotinin).
Following clarification, equal amounts of lysate were resolved by
SDS-PAGE, transferred onto polyvinylidene difluoride membrane
(Immobilon-P, Millipore), and probed with the following antibodies:
phospho-ERK, anti-phospho ERK1(T202,Y204) monoclonal E10 (New England
Biolabs, 9106S); pan-ERK (Transduction Laboratories, E17120); Akt
anti-Ser(P)473 (New England Biolabs, 9271S); and Akt (New
England Biolabs, 9272S). Horseradish peroxidase-conjugated anti-rabbit
or anti-mouse goat antibodies were from DAKO; ECL detection reagents
were from Amersham Pharmacia Biotech. Western blots were stripped in
0.1 M glycine, pH 2.5, 0.1% SDS for 30 min and reprobed
with the appropriate antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Activation of SRF reporter genes by different
stimuli. A, integrated 3D.AFos reporter gene. SRE.FosHA
cells were seeded at 4 × 105/6-cm dish and after
24 h in medium containing 10% serum transferred to medium
containing 0.5% serum for 36 h before stimulation with 15% fetal
calf serum (SER), 10 µM LPA, 25 ng/ml PDGF, or
50 ng/ml phorbol 12 myristate 13-acetate (TPA) for the times
indicated on the figure. Transcripts of the reporter and a control
gene, GAPDH, were analyzed by RNase protection; nuclease-resistant
fragments derived from the two RNAs are indicated on the figure. At
peak accumulation, relative transcript levels expressed as a percentage
of those induced by serum at 60 min (means ± S.E., three
independent experiments) were as follows: LPA, 55.7 ± 5.3; PDGF,
29.4 ± 4.5; and PMA, 42.9 ± 3.4. Similar results were
obtained with two further cell lines containing the 3D.AFos reporter
(data not shown). B, inhibition of SRF reporter activity by
C2-C3 toxin. Serum-deprived cells were pretreated for 5 h with
C2-C3 toxin as described in Experimental Procedures before stimulation
and analysis for 3D.AFos reporter and GAPDH reference transcripts.
Transcript levels were reduced to background in each case. Protected
fragments are indicated. C, inhibition of SRF reporter
activity by toxin B. Serum-deprived cells were pretreated for 1 h with
Toxin B before stimulation and analysis as in part
(B).
View larger version (72K):
[in a new window]
Fig. 2.
Dependence of SRF-linked signaling pathways
on PI-3K and MEK. A, effect of the MEK inhibitor U0126
on SRF reporter gene activity. Serum-deprived SRE.FosHA cells were
pretreated for 30 min with 10 µM U0126 and then
stimulated as indicated on the figure. SRF 3D.AFos reporter and GAPDH
transcripts were quantitated by RNase protection. The transcript levels
at 30 min, expressed as percentages of those in untreated cells, were
as follows (means ± S.E., three independent experiments): fetal
calf serum (SER), 54.2 ± 6.0; LPA, 53.6 ± 3.2;
PDGF, 36.4 ± 7.5; PMA (TPA), 10.2 ± 0.5. B, effect of the PI-3K inhibitor LY294002 on SRF reporter
gene activity. Serum-deprived SRE.FosHA cells were pretreated for 30 min with 20 µM LY294002 and then stimulated as indicated
on the figure. SRF 3D.AFos reporter and GAPDH transcripts were
quantitated by RNase protection. The transcript levels at 30 min,
expressed as percentages of those in untreated cells were as follows
(means ± S.E., three independent experiments): fetal calf serum
(SER), 108 ± 8.3; LPA, 83.2 ± 3.2; PDGF,
35.3 ± 3.4; PMA (TPA), 16.0 ± 1.0. C,
U0126 does not affect PI-3K activation by the stimuli tested.
Serum-deprived cells were pretreated for 30 min with 10 µM U0126 and stimulated for the times indicated. Whole
cell lysates were prepared, fractionated by SDS-PAGE, and transferred
to polyvinylidene difluoride membrane before analysis by immunoblotting
for diphospho-ERK and total ERK (top panels) and Akt
Ser(P)473 and total Akt (bottom panels).
D, LY294002 does not affect ERK activation by the stimuli
tested. Serum-deprived cells were pretreated for 30 min with 20 µM LY294002 and stimulated for the times indicated. Whole
cell lysates were prepared, fractionated by SDS-PAGE, and transferred
to polyvinylidene difluoride membrane before analysis by immunoblotting
for Akt Ser(P)473 and total Akt (top panels) and
diphospho-ERK and total ERK (bottom panels).
View larger version (46K):
[in a new window]
Fig. 3.
Activation of SRF by all stimuli requires
actin polymerization. A, SRF reporter activity is
inhibited by the G-actin sequestering drug latrunculin B but not by
U0126. Serum-deprived SRE.FosHA cells were pretreated for 60 min with
0.5 µM latrunculin B (LB), 30 min with 10 µM U0126 (U), or both (U LB). SRF
3D.AFos reporter and GAPDH transcripts were quantitated by RNase
protection. Reporter activity in LB treated cells was reduced to
background level in three independent experiments. For relative
transcript levels in U0126-treated cells see the Fig. 2A
legend. B, neither ERK nor PI-3K activation is affected by
latrunculin B. Serum-deprived cells were pretreated for 30 min with 0.5 µM latrunculin B and stimulated for the times indicated.
Whole cell lysates were prepared, fractionated by SDS-PAGE, and
transferred to polyvinylidene difluoride membrane before analysis by
immunoblotting for Akt Ser(P)473 and total Akt (top
panels) and diphospho-ERK (bottom panels). Total ERK
levels are shown in Fig. 2D.
View larger version (18K):
[in a new window]
Fig. 4.
TCF Elk-1 activation is insensitive to
latrunculin B but inhibited by U0126. NIH3T3 cells were
transfected with an expression plasmid encoding the chimeric
transactivator NLexElkC together with Lex operator-controlled
luciferase reporter gene, maintained in 0.5% fetal calf serum for
24 h and then stimulated following a 30-min pretreatment with 20 µM LY294002, 10 µM U0126, or 0.5 µM latrunculin B as indicated. The data are normalized to
the serum response, which is taken as 100; the error bars
indicate S.E. from three independent transfections; where not shown,
the S.E. was always less than 20% of the experimental value.
Un., untreated; SER, fetal calf serum; TPA,
phorbol 12-myristate 13-acetate.
View larger version (54K):
[in a new window]
Fig. 5.
Transient kinetics of srf
and vcl gene activation in NIH3T3 cells.
A, serum activation and independence of new protein
synthesis. Serum-deprived cells were stimulated with 15% serum for the
indicated times after pretreatments with protein synthesis inhibitors
or before analysis with probes specific for srf exon
5-intron 5 (top panels) or vcl exon 3-intron 3 (bottom panels) each together with GAPDH reference probe.
Chx, 10 µg/ml cycloheximide; An, 10 µg/ml
anisomycin. Protected fragments from srf precursor
(pre-srf), srf mRNA (srf),
vcl precursor (pre-vinc), vcl mRNA
(vinc), and GAPDH are indicated. B, activation by
jasplakinolide. Serum-deprived cells were stimulated for the indicated
times with serum or the the F-actin stabilizing drug jasplakinolide
(0.5 µM). RNA was prepared and analyzed as in
A. C, activation by LPA, PDGF, and PMA
(TPA). Serum-deprived cells were stimulated for the
indicated times with LPA, PDGF, and PMA. RNA was prepared and analyzed
as in A.
View larger version (78K):
[in a new window]
Fig. 6.
SRF target genes differ in their requirement
for cellular signaling pathways. A, differential
dependence on actin polymerization and MEK activity. Serum-deprived
cells were stimulated for the indicated times after 30-min
pretreatments with latrunculin B (LB), U0126 (U),
or both (U LB). RNA was prepared and analyzed using probes
specific for srf exon 5-intron 5 (top panel),
vcl exon 3-intron 3 (middle panel), or
egr1 plus fos (bottom panel) each
together with GAPDH reference probe. The protected fragments from
srf precursor (pre-srf), srf mRNA
(srf), vcl precursor (pre-vinc),
vcl mRNA (vinc), egr1 and
fos, and GAPDH are indicated. Transcript levels at 30 min
expressed as percentages of those in untreated cells were as follows
(means ± range or S.E.). In U0126-treated cells: fetal calf serum
(SER): pre-srf 59.4 ± 1.5 (n = 2), pre-vcl 42.7 ± 7.3 (n = 2), egr1 3.8 (n = 1),
fos 13.4 ± 2.0 (n = 2); LPA:
pre-srf 65.8 ± 3.5 (n = 2),
pre-vcl 41.2 ± 3.4 (n = 2), egr1,
~1.0 (n = 1), fos 4.8 ± 3.3 (n = 2); PDGF: pre-srf 59.4 ± 1.0 (n = 2), pre-vcl 56.3 ± 16.2 (n = 2),
egr1 ~1.0 (n = 1), fos 2.3 ± 1.6 (n = 2); PMA (TPA):
pre-srf 15.3 ± 2.4 (2), pre-vcl 5.4 ± 3.8 (n = 2), egr1 ~1.0 (n = 1), fos 0.4 ± 0.3 (n = 2). In
Latrunculin B-treated cells, fetal calf serum (SER):
pre-srf 5.1 ± 1.0 (n = 2),
pre-vcl 1.5 ± 1.1 (n = 2),
egr1 100.5 ± 0.7 (n = 2),
fos 69.3 ± 7.4 (5); LPA, pre-srf 3.6 ± 0.6 (2), pre-vcl 0.3 ± 0.2 (n = 2),
egr1 66.2 (n = 1), fos 55.9 ± 7.4 (n = 5); PDGF, pre-srf 3.5 ± 1.0 (n = 2), pre-vcl 1.8 ± 0.3 (n = 2), egr1 122.6 ± 6.1 (n = 2), fos 71.4 ± 5.0 (5), PMA,
pre-srf 11.1 ± 5.3 (n = 2),
pre-vcl 2.8 ± 0.2 (n = 2),
egr1 82.5 (n = 1), fos 74.7 ± 12.2 (n = 5). B, differential dependence
on PI-3K. Serum-deprived cells were stimulated for the indicated times
after 30-min pretreatments with 20 µM LY294002
(LY) or 0.2 µM wortmannin (W). RNA
was prepared and analyzed using probes specific for srf exon
5-intron 5 (top panel), vcl exon 3-intron 3 (middle panel), or egr1 plus fos
(bottom panel) each together with a GAPDH reference probe.
The protected fragments are indicated as for A. The
transcript levels at 30 min expressed as percentages of those in
untreated cells (LY294002/wortmannin; means ± range or S.E.) were
as follows. fetal calf serum (SER): pre-srf
64.5/58.1 (n = 1), pre-vcl 82.3 ± 9.1/96.0 ± 2.7 (n = 2); egr1 94.7 (LY;
n = 1), fos 134.8 (n = 2);
LPA, pre-srf 36.0/48.0 (n = 1),
pre-vcl 63.0 ± 7.0/30.6 ± 10.9 (n = 2); egr1 87.7 (LY; 1), fos
90.0 (LY; n = 1); PDGF, pre-srf 15.0 ± 7.3 (n = 2)/32.7 (n = 1),
pre-vcl 8.7 ± 6.2/7.1 ± 5.1 (n = 2) egr1 82.2 (LY; n = 1), fos
132.8 (LY; n = 1); PMA, pre-srf 4.3/10.0
(n = 1), pre-vcl 14.6/5.8 (n = 1) egr1 109.1 (LY; n = 1), fos
245.3 (LY; n = 1). Un., untreated.
View larger version (87K):
[in a new window]
Fig. 7.
Activation of the srf and
vcl genes but not fos or
egr1 is RhoA-dependent.
Serum-deprived cells were pretreated for 5 h with C2-C3 toxin
(left panels) or for 1 h with toxin B (right
panels) as described under "Experimental Procedures" before
stimulation and analysis as in Fig. 6. The protected fragments are
indicated on the figure. Pre-srf and pre-vcl
transcript levels were reduced to background in both cases; for
egr1 and fos transcript levels (C2-C3/toxin B
expressed as percentages of untreated signal at 30 min) were as
follows: fetal calf serum (SER): egr1 88.4/66.9,
fos 62.5/47.3; LPA: egr1 102.3/33.0,
fos 45.8/19.0; PDGF: egr1 97.7/67.2,
fos 50.8/50.1; PMA (TPA): egr1
108.4/122.8, fos 77.7/93.6. Un., untreated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
[in a new window]
Fig. 8.
Extracellular signals control at least two
distinct classes of SRF target gene. Outline signal transduction
pathways controlled by the small GTPases Ras and Rho are shown with
known SRF targets in italics. Downstream pathway components
are boxed. ERK indicates the Raf-MEK-ERK pathway
previously shown to control TCF activity in response to mitogenic
stimuli; Actin dynamics indicates the actin treadmilling
cycle, which controls SRF activity and is regulated by the RhoA
effector systems ROCK-LIM kinase-cofilin and Diaphanous (Refs. 28 and
29; O. Geneste, J. Copeland, and R. Treisman, manuscript in
preparation; J. Copeland and R. Treisman, manuscript in
preparation). Pathway specific inhibitors U0126 and latrunculin
are indicated. Dotted arrows indicate that signal
transmission through each pathway may also respond to (i) changes in
Ras and Rho GTP loading induced by cell cycle/growth cues and
cytoskeletal events respectively and (ii) cross-talk between the
pathways. TCF binding to the fos and egr1
promoters is known to be important for signal transduction (2, 12, 15);
however, it remains unclear whether TCF plays a role in regulation of
srf or vcl. For discussion see text.
SRF reporter activity is critically dependent on Rho GTPase and actin polymerization, whether induced via activation of serpentine receptors (serum and LPA), receptor tyrosine kinases (PDGF), or intracellular activation of protein kinase C (PMA). Signaling to SRF by PDGF and PMA differs in several ways from the other stimuli. First, only PDGF- and PMA-induced reporter activation requires PI-3K activity. PMA treatment does not induce activation of PI-3K itself, at least as assessed by Akt phosphorylation; therefore it would appear likely that SRF activation by PMA, and probably PDGF, requires only basal PI-3K activity. Consistent with this idea, expression of activated PI-3K p110 does not activate the SRF reporter gene in our cells.3 Second, PMA-induced reporter activation, which unlike the other stimuli results from the activation of protein kinase C, is substantially blocked by inhibition of MEK. This observation is consistent with a model in which SRF can be activated by both MEK-dependent and -independent routes, with only the former being activated by PMA, although further studies are necessary to confirm this. Finally, although PMA and PDGF induction of the SRF reporter gene (and cellular srf and vcl) is absolutely dependent on functional RhoA, previous studies indicate that these stimuli rapidly decrease rather than increase GTP loading of RhoA in NIH3T3 cells (44),4 suggesting that activation of SRF by these agents requires only basal RhoA activity. We propose that PDGF and PMA might act to stabilize a pool of F-actin whose assembly requires basal RhoA activity; PDGF is a strong activator of Rac in NIH3T3 cells, and one way this could be achieved is by Rac-dependent activation of the actin stabilizer LIM kinase (45). Further work will be necessary to clarify the connection between PDGF and PMA-induced signaling and actin dynamics.
Our data show that the SRF target genes srf and
vcl behave like the SRF reporter, requiring Rho-actin but
not MEK-ERK signaling, whereas activation of fos and
egr1 requires MEK-ERK but not Rho-actin signaling. At
present it remains unclear whether all SRF target genes fall into these
two classes. In principle further classes of SRF target gene might
exist, perhaps dependent on both RhoA-actin and MEK-ERK signaling or
regulated by other signaling pathways with or without input from
RhoA-actin signals. Among immediate-early genes, the junB
gene exhibits similar signaling requirements to fos and
egr1.3 However, there is as yet insufficient
data to classify other SRF targets. Several SRF-controlled
muscle-specific promoters are Rho-dependent (22, 23), and
at least the smooth muscle -actin and SM22 promoters are dependent
on alterations in actin polymerization (30), suggesting that they might
fall into the srf-vcl class. The role of MEK-ERK
signaling in the expression of such muscle-specific SRF target genes
has not been resolved; however, the failure of the MEK inhibitor
PD98059 to block differentiation of C2 skeletal myoblasts suggests that
MEK-ERK signaling may not be essential for expression of SRF target
genes in these cells (46, 47). It is also intriguing to note that the
cyr61-related immediate-early gene CTGF exhibits similar signaling
requirements to srf and vcl (48, 49); however, it
remains to be confirmed whether CTGF actually is an SRF target. We are
currently comparing signaling requirements for activation of different
SRF target genes.
Our results extend previous characterization of signaling to SRF target promoters. Previous transfection studies have clearly established the functional significance of the SRF sites present in the srf and vcl promoters (50-52). In addition to signal inputs through the SRF site, LPA-induced activation of the srf promoter involves a Ras-dependent signaling input through an adjacent Sp1 site, whereas FGF-induced activation involves both the Sp1 and a Rho family-dependent input via a nearby Ets motif (53, 54). However, signal-regulated srf and vcl transcription is absolutely dependent on functional RhoA and actin polymerization; therefore cooperating signals that act through other elements in these promoters must be insufficient for activation of transcription. Moreover, because neither the vcl nor srf promoters require active MEK, it is unlikely that they contain sequences directly regulated by ERK. Vcl transcription exhibits a delayed onset compared with that of srf and the SRF reporter gene; this does not reflect a requirement for new protein synthesis, and its basis is currently under investigation. The role of PI-3K in SRF target gene activation is less clear. Although experiments involving activated and inhibitory PI-3K mutants have implicated PI-3K in activation of SRF target genes in some signaling systems, at least some of these effects can be attributed to inhibition of Ras-Raf-MEK-ERK signaling rather than SRF itself (24-27, 55-57). In our cells, the SRF target genes tested respond in distinct ways to inhibition of PI-3K; the srf and vcl genes respond in a similar way to the SRF reporter, although the effects of the inhibitor on these genes are less marked, whereas activation of the fos or egr1 genes by all stimuli is unimpaired.
We found that in NIH3T3 cells signal-induced activation of the endogenous fos and egr1 genes occurs largely independently of RhoA; a similar finding has been reported using Rat-1 cells (58). These findings contrast with our own previous transient transfection experiments and those of others, in which fos activation exhibits a strong dependence on functional RhoA (21). One potential explanation for this discrepancy is that transiently transfected promoters are somehow more sensitive to the Rho-actin pathway than their chromosomal counterparts; however, this would appear unlikely because a transfected fos gene is insensitive to latrunculin.3 An alternative explanation can be based on the observation that in fibroblasts ERK activation is partially dependent on functional RhoA (21, 59); perhaps the presence of a large number of transfected fos gene templates is sufficient to render ERK signaling limiting, with the result that the dependence of ERK signaling upon RhoA would then become significant. We also found that the stably transfected SRF reporter gene was more responsive to PDGF- and PMA-induced signaling than in transient transfection assays. The reason for this is unclear but again might reflect reporter copy number; transfected reporters may have a relatively high basal level of activity, and if signal strength by PDGF and PMA, but not serum and LPA, is limiting, the transfected reporter might appear less sensitive to PDGF and PMA. Further experiments will be required to resolve these issues, which caution against the use of transfected, high copy, reporter systems.
In this work we have identified four SRF target genes that are either
sensitive to actin dynamics and independent of MEK-ERK signaling or
vice versa (Fig. 8). How might such mutually exclusive linkage of
different signaling pathways to SRF-dependent promoters be
achieved? We previously suggested that promoter-specific combinatorial interactions between SRF and other transcription factors might control
the sensitivity of SRF to signaling via actin dynamics (28). The
results described here suggest a refinement of this model, in which the
physical interactions between SRF and different cofactors responsible
for actin-dependent signaling and MEK-ERK signaling
respectively are mutually exclusive. Several observations suggest that
the TCF proteins are good candidates for factors controlling signaling
specificity at SRF target promoters (Fig. 8). First, they are direct
targets for MEK-ERK signaling (3). Second, the SRF binding sites in
actin-dependent promoters such as vcl and
srf do not have obvious TCF sites associated with them, whereas SRF sites in MEK-ERK-dependent promoters such as
fos and egr1 do (2, 15). Third, expression of
inactive forms of TCF can interfere with RhoA-dependent
signaling to SRF reporter genes (21). It should be borne in mind,
however, that SRF also functionally cooperates with several other
transcription factors including SP1, GATA4, Nkx2.5, the myogenic
factors, and ATF6 (5-10); moreover, SRF sites are frequently
associated with AP1/ATF sites, which are also targets for signaling
pathways (60). Combinatorial interactions between SRF and such other
factors might also therefore constrain its sensitivity to Rho-actin
signaling. We are currently studying signaling to a number of different
SRF-controlled promoters to elucidate the role of TCF and other
transcriptional regulators in the control of signaling to SRF.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Holger Barth and Klaus Aktories (Freiburg) for generous gifts of C2-C3 toxin and toxin B, John Copeland for the egr1 RNase protection probe, Ross Thomas for LexOP2tk LUC, and members of the laboratory, Caroline Hill, and Ian Kerr for useful discussions and comments on the manuscript.
![]() |
FOOTNOTES |
---|
* 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.
Supported by a predoctoral award from the Overseas Research
Student Scheme of the United Kingdom Committee of Vice Chancellors and Principals.
§ To whom correspondence should be addressed: Transcription Laboratory, Rm. 401, Imperial Cancer Research Fund Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, UK; Fax: 44-20-7269-3093.
Published, JBC Papers in Press, May 7, 2001, DOI 10.1074/jbc.M102678200
2 O. Geneste, J. Copeland, and R. Treisman, manuscript in preparation.
3 D. Gineitis, unpublished data.
4 R. Grosse, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: SRF, serum response factor; TCF, ternary complex factor; LPA, lysophosphatidic acid; PDGF, platelet-derived growth factor; PMA, phorbol 12-myristate 13-acetate; PI-3K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase-ERK kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAGE, polyacrylamide gel electrophoresis.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Arsenian, S.,
Weinhold, B.,
Oelgeschlager, M.,
Ruther, U.,
and Nordheim, A.
(1998)
EMBO J.
17,
6289-6299 |
2. | Shaw, P. E., Schroter, H., and Nordheim, A. (1989) Cell 56, 563-572[Medline] [Order article via Infotrieve] |
3. | Treisman, R. (1994) Curr. Opin. Gen. Dev. 4, 96-101[Medline] [Order article via Infotrieve] |
4. | Treisman, R., Marais, R., and Wynne, J. (1992) EMBO J. 11, 4631-4640[Abstract] |
5. | Zhu, C., Johansen, F. E., and Prywes, R. (1997) Mol. Cell. Biol. 17, 4957-4966[Abstract] |
6. |
Durocher, D.,
Charron, F.,
Warren, R.,
Schwartz, R. J.,
and Nemer, M.
(1997)
EMBO J.
16,
5687-5696 |
7. | Chen, C. Y., Croissant, J., Majesky, M., Topouzis, S., McQuinn, T., Frankovsky, M. J., and Schwartz, R. J. (1996) Dev. Genet. 19, 119-130[CrossRef][Medline] [Order article via Infotrieve] |
8. | Sartorelli, V., Webster, K. A., and Kedes, L. (1990) Genes Dev. 4, 1811-1822[Abstract] |
9. |
Belaguli, N. S.,
Sepulveda, J. L.,
Nigam, V.,
Charron, F.,
Nemer, M.,
and Schwartz, R. J.
(2000)
Mol. Cell. Biol.
20,
7550-7558 |
10. |
Moore, M. L.,
Wang, G. L.,
Belaguli, N. S.,
Schwartz, R. J.,
and McMillin, J. B.
(2001)
J. Biol. Chem.
276,
1026-1033 |
11. | Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73, 381-393[Medline] [Order article via Infotrieve] |
12. | Graham, R., and Gilman, M. (1991) Science 251, 189-192[Medline] [Order article via Infotrieve] |
13. | Hill, C. S., Wynne, J., and Treisman, R. (1994) EMBO J. 13, 5421-5432[Abstract] |
14. | Kortenjann, M., Thomae, O., and Shaw, P. E. (1994) Mol. Cell. Biol. 14, 4815-4824[Abstract] |
15. | McMahon, S. B., and Monroe, J. G. (1995) Mol. Cell. Biol. 15, 1086-1093[Abstract] |
16. | Hill, C. S., and Treisman, R. (1995) EMBO J. 14, 5037-5047[Abstract] |
17. |
Alessi, D. R.,
Cuenda, A.,
Cohen, P.,
Dudley, D. T.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
27489-27494 |
18. | Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7686-7689[Abstract] |
19. | Price, M. A., Cruzalegui, F. H., and Treisman, R. H. (1996) EMBO J. 15, 6552-6563[Abstract] |
20. |
Lazar, D. F.,
Wiese, R. J.,
Brady, M. J.,
Mastick, C. C.,
Waters, S. B.,
Yamauchi, K.,
Pessin, J. E.,
Cuatrecasas, P.,
and Saltiel, A. R.
(1995)
J. Biol. Chem.
270,
20801-20807 |
21. | Hill, C. S., Wynne, J., and Treisman, R. (1995) Cell 81, 1159-1170[Medline] [Order article via Infotrieve] |
22. |
Carnac, G.,
Primig, M.,
Kitzmann, M.,
Chafey, P.,
Tuil, D.,
Lamb, N.,
and Fernandez, A.
(1998)
Mol. Biol. Cell
9,
1891-1902 |
23. |
Wei, L.,
Zhou, W.,
Croissant, J. D.,
Johansen, F. E.,
Prywes, R.,
Balasubramanyam, A.,
and Schwartz, R. J.
(1998)
J. Biol. Chem.
273,
30287-30294 |
24. | Wang, Y., Falasca, M., Schlessinger, J., Malstrom, S., Tsichlis, P., Settleman, J., Hu, W., Lim, B., and Prywes, R. (1998) Cell Growth Differ. 9, 513-522[Abstract] |
25. |
Wei, L.,
Zhou, W.,
Wang, L.,
and Schwartz, R. J.
(2000)
Am. J. Physiol.
278,
H1736-H1743 |
26. |
Poser, S.,
Impey, S.,
Trinh, K.,
Xia, Z.,
and Storm, D. R.
(2000)
EMBO J.
19,
4955-4966 |
27. | Reif, K., Nobes, C. D., Thomas, G., Hall, A., and Cantrell, D. A. (1996) Curr. Biol. 6, 1445-1455[Medline] [Order article via Infotrieve] |
28. | Sotiropoulos, A., Gineitis, D., Copeland, J., and Treisman, R. (1999) Cell 98, 159-169[Medline] [Order article via Infotrieve] |
29. | Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S. A., and Alberts, A. S. (2000) Mol. Cell 5, 13-25[Medline] [Order article via Infotrieve] |
30. |
Mack, C. P.,
Somlyo, A. V.,
Hautmann, M.,
Somlyo, A. P.,
and Owens, G. K.
(2001)
J. Biol. Chem.
276,
341-347 |
31. | Alberts, A. S., Geneste, O., and Treisman, R. (1998) Cell 92, 475-487[Medline] [Order article via Infotrieve] |
32. |
Barth, H.,
Hofmann, F.,
Olenik, C.,
Just, I.,
and Aktories, K.
(1998)
Infect. Immun.
66,
1364-1369 |
33. | Wagner, B. J., Hayes, T. E., Hoban, C. J., and Cochran, B. H. (1990) EMBO J. 9, 4477-4484[Abstract] |
34. | Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Just, I.,
Wilm, M.,
Selzer, J.,
Rex, G.,
von Eichel-Streiber, C.,
Mann, M.,
and Aktories, K.
(1995)
J. Biol. Chem.
270,
13932-13936 |
36. |
Favata, M. F.,
Horiuchi, K. Y.,
Manos, E. J.,
Daulerio, A. J.,
Stradley, D. A.,
Feeser, W. S.,
Van Dyk, D. E.,
Pitts, W. J.,
Earl, R. A.,
Hobbs, F.,
Copeland, R. A.,
Magolda, R. L.,
Scherle, P. A.,
and Trzaskos, J. M.
(1998)
J. Biol. Chem.
273,
18623-18632 |
37. | Coue, M., Brenner, S. L., Spector, I., and Korn, E. D. (1987) FEBS Lett. 213, 316-318[CrossRef][Medline] [Order article via Infotrieve] |
38. | Greenberg, M. E., and Ziff, E. B. (1984) Nature 311, 433-438[Medline] [Order article via Infotrieve] |
39. | Lau, L. F., and Nathans, D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1182-1186[Abstract] |
40. | Misra, R. P., Rivera, V. M., Wang, J. M., Fan, P. D., and Greenberg, M. E. (1991) Mol. Cell. Biol. 11, 4545-4554[Medline] [Order article via Infotrieve] |
41. | Ben-Ze'ev, A., Reiss, R., Bendori, R., and Gorodecki, B. (1990) Cell Regul. 1, 621-636[Medline] [Order article via Infotrieve] |
42. | Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Biochem. J. 351, 95-105[CrossRef][Medline] [Order article via Infotrieve] |
43. | Aktories, K. (1997) Trends Microbiol. 5, 282-288[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Sander, E. E.,
ten Klooster, J. P.,
van Delft, S.,
van der Kammen, R. A.,
and Collard, J. G.
(1999)
J. Cell Biol.
147,
1009-1022 |
45. | Edwards, D. C., Sanders, L. C., Bokoch, G. M., and Gill, G. N. (1999) Nat. Cell Biol. 1, 253-259[CrossRef][Medline] [Order article via Infotrieve] |
46. |
Sarbassov, D. D.,
and Peterson, C. A.
(1998)
Mol. Endocrinol.
12,
1870-1878 |
47. |
Cuenda, A.,
and Cohen, P.
(1999)
J. Biol. Chem.
274,
4341-4346 |
48. | Reiser, C. O., Lanz, T., Hofmann, F., Hofer, G., Rupprecht, H. D., and Goppelt-Struebe, M. (1998) Biochem. J. 330, 1107-1114[Medline] [Order article via Infotrieve] |
49. |
Hahn, A.,
Heusinger-Ribeiro, J.,
Lanz, T.,
Zenkel, S.,
and Goppelt-Struebe, M.
(2000)
J. Biol. Chem.
275,
37429-37435 |
50. |
Spencer, J. A.,
and Misra, R. P.
(1996)
J. Biol. Chem.
271,
16535-16543 |
51. |
Belaguli, N. S.,
Schildmeyer, L. A.,
and Schwartz, R. J.
(1997)
J. Biol. Chem.
272,
18222-18231 |
52. |
Moiseyeva, E. P.,
Weller, P. A.,
Zhidkova, N. I.,
Corben, E. B.,
Patel, B.,
Jasinska, I.,
Koteliansky, V. E.,
and Critchley, D. R.
(1993)
J. Biol. Chem.
268,
4318-4325 |
53. | Spencer, J. A., and Misra, R. P. (1999) Oncogene 18, 7319-7327[CrossRef][Medline] [Order article via Infotrieve] |
54. |
Spencer, J. A.,
Major, M. L.,
and Misra, R. P.
(1999)
Mol. Cell. Biol.
19,
3977-3988 |
55. | Jhun, B. H., Rose, D. W., Seely, B. L., Rameh, L., Cantley, L., Saltiel, A. R., and Olefsky, J. M. (1994) Mol. Cell. Biol. 14, 7466-7475[Abstract] |
56. | Hu, Q., Klippel, A., Muslin, A. J., Fantl, W. J., and Williams, L. T. (1995) Science 268, 100-102[Medline] [Order article via Infotrieve] |
57. |
Yamauchi, K.,
Holt, K.,
and Pessin, J. E.
(1993)
J. Biol. Chem.
268,
14597-14600 |
58. |
Beltman, J.,
Erickson, J. R.,
Martin, G. A.,
Lyons, J. F.,
and Cook, S. J.
(1999)
J. Biol. Chem.
274,
3772-3780 |
59. |
Kumagai, N.,
Morii, N.,
Fujisawa, K.,
Nemoto, Y.,
and Narumiya, S.
(1993)
J. Biol. Chem.
268,
24535-24538 |
60. | Wang, Y., and Prywes, R. (2000) Oncogene 19, 1379-1385[CrossRef][Medline] [Order article via Infotrieve] |