c-Jun Enhancement of Cyclic Adenosine 3',5'-Monophosphate Response Element-Dependent Transcription Induced by Transforming Growth Factor-ß Is Independent of c-Jun Binding to DNA
Patrick Pei-chih Hu1,
Beth L. Harvat1,
Sara S. Hook,
Xing Shen,
Xiao-Fan Wang and
Anthony R. Means
Department of Pharmacology and Cancer Biology Duke University
Medical Center Durham, North Carolina 27710
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ABSTRACT
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Transforming growth factor-ß (TGFß) enhances
transcription from reporter genes regulated by a single consensus
cAMP-response element (CRE) upon transfection into the immortalized
human keratinocyte cell line, HaCaT. Whereas both CRE-binding protein
(CREB) and c-Jun present in extracts of unstimulated cells can complex
with a CRE in gel-shift experiments, TGFß treatment increases the
amount of c-Jun found in the complex. Overexpression of c-Jun is
sufficient to increase CRE and GAL4-CREB-dependent transcription and
mimics the stimulatory effects of TGFß on transcription from either
reporter gene. Surprisingly, although a portion of CREB in unstimulated
cells is phosphorylated on the activating serine residue, Ser-133, this
level of phospho-CREB is not altered by TGFß treatment. In fact, the
CREB-dependent transcriptional effects of TGFß or c-Jun do not
require phosphorylation of Ser-133, although CREB-binding protein (CBP)
is required as evidenced by the observation that the adenoviral
oncoprotein E1A can block the effects of both agents. c-Jun enhancement
of CRE or GAL4-CREB-dependent transcription neither requires the
DNA-binding nor N-terminal domains of c-Jun. Collectively, these
results are consistent with a model in which signaling pathways
initiated by TGFß can stimulate CREB-dependent transcription by
increasing the cellular concentration of c-Jun, which participates in
activation of the CBP-containing transcription complex.
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INTRODUCTION
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Transforming growth factor-ß (TGFß) is a multifunctional
cytokine whose action is critical for complex biological processes such
as embryonic development, wound healing, and immune system function.
Loss of responsiveness to TGFß signaling is associated with diseases
such as fibrosis, arthritis, and cancer. Many of the effects of TGFß
involve transcriptional induction of genes whose products induce growth
arrest in late G1 of the cell cycle (reviewed in Refs. 1, 2) or promote an increase in extracellular matrix deposition.
Growth arrest is mediated by an inhibition of cyclin-dependent kinase
(CDK) activity due to either increased synthesis of CDK inhibitors
(3, 4, 5, 6, 7, 8) or a decrease in expression of the cdk activating
phosphatase cdc25A (9). TGFß induces expression of two CDK
inhibitors, p15Ink4b and p21WAF1/Cip1. Effects
of TGFß on extracellular matrix (ECM) deposition are due to induction
of both ECM structural genes such as type I collagen and inhibitors of
ECM degradation such as plasminogen activator inhibitor (PAI-I). The
coactivators CREB-binding protein (CBP) and p300 are required not only
for TGFß-mediated transcription of p15 and p21 (10) but also for
induction of PAI-1 (11, 12, 13).
In searching for additional genes that may mediate TGFß
effects, Wong et al. (14) found that TGFß increases c-Jun
expression. The induction of c-Jun, in turn, may serve to increase
transcription of PAI-1 and type I collagen, since these genes are
activated by AP-1 elements within their promoters (15, 16) and c-Jun is
a common component of AP-1 protein complexes. Although it is unclear
exactly how c-Jun activates transcription, it is known to bind to the
coactivators p300 and CBP. The N-terminal transactivation domain of
c-Jun interacts with the KIX (CREB-binding) domain of p300/CBP (17),
while the C-terminal leucine zipper domain can interact with the C/H2
domain of CBP (18).
Although the cAMP response element (CRE, consensus sequence TGACGTCA)
has been defined by its ability to bind to and be stimulated by the
transcription factor CRE-binding protein (CREB) (19), c-Jun itself
binds to the CRE-like element in its promoter that positively
autoregulates transcription of the c-Jun gene (20). TGFß induction of
a reporter gene regulated by the c-Jun promoter in HaCaT cells requires
the CRE-like site, which only differs from the consensus CRE by a
single base pair (TGACATCA). Studies in
Drosophila have shown that the ability of the TGFß
ortholog decapentaplegic to stimulate transcription of
Ultrabithorax (Ubx) is absolutely dependent on
the presence of a consensus CRE present in the Ubx promoter (21).
Together these observations raised the question of whether TGFß might
stimulate transcription of genes containing consensus CREs in mammalian
cells and, if so, whether this response involved c-Jun.
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RESULTS
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TGFß Stimulates Formation of a c-Jun/CRE Complex and c-Jun
Potentiates CRE-Mediated Transcription in HaCaT Cells
To evaluate the ability of TGFß to affect CRE-mediated
transcription through increasing c-Jun levels, we first demonstrated by
electrophoretic mobility shift assay (EMSA) (Fig. 1A
) that both CREB and c-Jun, but not
ATF-2 (data not shown), can complex with a consensus CRE (TGACGTCA) in
lysates from untreated HaCaT cells. While no effect of TGFß
treatment on the complex was observed in the first hour, lysates from
cells with longer TGFß treatment (3 h) demonstrated an increased
amount of complex binding to the probe, most of which could be
supershifted by the c-Jun antibody (Fig. 1B
). The increase in complex
formation occurred coordinately with increased c-Jun levels in the
cells.

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Figure 1. TGFß Increases c-Jun Protein Synthesis and
Incorporation into the CRE Complex
HaCaT nuclear lysates from untreated cells or cells treated with 100
pM TGF-ß for 1 h (A) or 3 h (B) were incubated
with a radioactively labeled CRE probe and EMSA was performed. CREB
complexes were supershifted with -CREB (Santa Cruz Biotechnology, Inc.), and c-Jun complexes were shifted with
-c-Jun (Santa Cruz Biotechnology, Inc.). Western
analysis for c-Jun from the nuclear lysates are below the EMSA (B)
results.
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TGFß treatment of HaCaT cells for 24 h caused a 5- to 9-fold
increase in transcription from a luciferase reporter gene driven by one
copy of a consensus CRE (1xCRE, Fig. 2A
, left panel). We questioned
whether the increase in c-Jun in response to TGFß could account for
the TGFß-inducible transcription from the CRE. Increasing amounts of
cotransfected c-Jun raised levels of transcription and essentially
mimicked the TGFß response (Fig. 2A
, right panel).
Expression of c-Jun also increased transcription of a truncated
c-Fos promoter-CAT reporter gene in which the only known response
element is the CRE (Fos
80) (Fig. 2C
). Thus, overexpression of c-Jun
not only activates a consensus CRE connected to a reporter gene in an
artificial context, but also activates a CRE in the context presented
by the c-Fos promoter.

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Figure 2. TGFß, c-Jun, and c-Jun Mutants Stimulate
CRE-Mediated Transcription
A, Luciferase activity in HaCaT cell extracts was measured after
transient cotransfections of a reporter plasmid containing only a
minimal TATA+Initiator without the consensus 1xCRE vs.
the reporter plasmid with the consensus CRE inserted (left
panel) or the 1xCRE reporter plasmid with increasing amounts of
c-Jun cDNA (right panel). HaCaT cells were transfected
using diethylaminoethyl-dextran as described in Materials and
Methods, and then treated with TGFß for 2022 h before
harvesting. Transfection efficiency was normalized with
ß-galactosidase activity. B, Luciferase activity from transient
cotransfections using the 1xCRE reporter plasmid and 1 µg of either
wild type c-Jun or c-Jun with mutations in the DNA-binding domain
( 270) or N-terminal activation domain (TAM-67) in the absence
(white bars) or presence (black bars) of
TGFß. Vector refers to the parent CMV-driven expression vector for
the c-Jun constructs. C, CAT assay measuring amount of c-Fos promoter,
Fos- 80CAT, activated by overexpressed wild-type c-Jun, 270, or
TAM-67. Identical transfections were performed as those described in
panel B for luciferase assays. D, The c-Jun cDNAs that were used are
depicted with their various functional domains.
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TGFß-Induced CREB-Dependent Transcription Does not Require
Phosphorylation of CREB at Ser-133
Because CREB is also in complex with the CRE in HaCaT cells, we
questioned whether in addition to increasing c-Jun in the complex,
TGFß treatment could also alter CREB transcriptional activity.
Phosphorylation of CREB on Ser-133 is known to enhance its
transactivating function by mediating recruitment of the coactivator
CBP (22). However, in HaCaT cells, Ser-133 phospho-CREB is readily
detected in the absence of stimulus. In multiple experiments, ionomycin
treatment of HaCaT cells for 1 h induced an increase (5- to
8-fold) in the phosphorylated form of CREB (Fig. 3A
), consistent with the reported
calcium-mediated phosphorylation of CREB (23, 24, 25). In contrast, TGFß
treatment altered CREB phosphorylation minimally, if at all (no change
to 2-fold, Fig. 3A
). Similar results were observed during a time course
of TGFß treatment from 0 to 24 h (data not shown). To confirm
that a portion of CREB was phosphorylated on Ser-133, HaCaT cells were
metabolically labeled by incubation with
[32P]orthophosphate for 4 h in the
absence or presence of TGFß. CREB was recovered by gel
purification and subjected to two-dimensional peptide mapping.
Autoradiograms of the peptide map in the absence (left) or
presence (right) of TGFß are shown in Fig. 3B
. In each
case, the predominant 32P-labeled peptide corresponded to
the one known to contain Ser-133 (25). The degree of labeling of the
Ser-133-containing peptide did not change in response to TGFß
treatment for 1 h (Fig. 3B
) or between 10 min and 4 h (data
not shown). The fact that it is possible to metabolically radiolabel
Ser-133 within a 4-h time period shows that the
phosphorylation-dephosphorylation of Ser-133 in HaCaT cells is in
dynamic equilibrium. TGFß treatment does not change this equilibrium.
Thus, the effect of TGFß on transcription of the 1xCRE-reporter gene
does not involve a change in the steady state level of
phospho-CREB.

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Figure 3. TGFß Does not Stimulate CREB Phosphorylation
A, Analysis of phosphorylated and total CREB levels by immunoblot after
1 h of treatment with 100 pM TGF-ß (ß) or 1.5
µM ionomycin (I). B, Two-dimensional tryptic peptide
mapping of 32P-metabolically labeled phospho-CREB isolated
from HaCaT cells in the absence or presence of TGFß treatment for
1 h. Similar results were obtained from cells treated for 10 min
or 4 h.
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To examine whether TGFß can stimulate CREB-dependent transcription, a
GAL4-CREB construct lacking the bZip DNA-binding/dimerization domain of
CREB was cotransfected into HaCaT cells with a 5x(GAL4)-luciferase
reporter plasmid. As shown in Fig. 4A
, this reporter gene is responsive to TGFß. To investigate whether
Ser-133 phosphorylation was required for TGFß to stimulate
transcription, GAL4-CREB, in which Ser-133 was mutated to Ala, was
transfected into HaCaT cells. The S133A mutant was still responsive to
TGFß (Fig. 4A
), albeit somewhat less so than wild-type CREB. These
results suggest that the phosphorylation of Ser-133 is not essential
for the TGFß effect on GAL4-CREB transcription. On the other hand,
overexpression of c-Jun potentiated transcription by GAL4-CREB and
markedly decreased the fold induction in response to TGFß (Fig. 4B
).
Overexpression of c-Jun by itself had no effect on the 5xGAL4 reporter,
which contains no apparent c-Jun binding sites, suggesting that the
c-Jun effect on GAL4-CREB-mediated transcription may be independent of
c-Jun binding directly to DNA. To test this idea, we cotransfected a
c-Jun DNA binding mutant (
70) that previously had been shown to have
a dominant negative effect on c-Jun/AP-1-mediated transcription (26)
and determined that it could activate GAL4-CREB transcription in a
manner equivalent to wild-type c-Jun (Fig. 4B
). The latter observation
prompted us to test whether DNA binding was necessary for c-Jun
stimulation of transcription using the 1xCRE reporter. As shown in Fig. 2B
, the
270 DNA-binding mutant activated the CRE-containing
transgene nearly as efficiently as wild- type c-Jun. Similar results
were obtained using the truncated c-Fos promoter construct (Fig. 2C
).
Collectively, these experiments suggest that the role of c-Jun in the
CRE complex could be to mediate protein-protein interactions rather
than to participate directly in DNA binding.

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Figure 4. Phosphorylation of Ser-133 on CREB Is not Essential
for TGFß or c-Jun Stimulation of Transcription Driven by a GAL4-CREB
Fusion Protein Whereas p300/CBP May Be Required
A, Luciferase activity from HaCaT cells cotransfected with a 5xGAL4
reporter gene and 2 µg of either GAL4-wild-type CREB or
GAL4-CREB-S133A. Both CREB cDNAs lack the bZip DNA binding/dimerization
domain. Transfections were performed as in Fig. 2 in the presence or
absence of TGFß. B, HaCaT cells were cotransfected with 1 µg of
wild type c-Jun cDNA, mutant c-Jun that cannot bind DNA ( 270), or
the N-terminal deletion mutant TAM-67, GAL4-CREB, and the 5xGAL4
reporter. C, Transient cotransfections were performed using wild- type
E1A or mutant E1A ( 236), which cannot bind CBP, with the 5xGAL4
reporter gene and GAL4-CREB. D, HaCaT cells were transfected with
wild-type E1A or mutant E1A ( 236), the 5xGAL4 reporter gene,
GAL4-CREB-S133A, and c-Jun. E, HaCaT cells were transfected with
wild-type c-Jun cDNA, TAM-67, or TAM-67 zip and either the 1xCRE
reporter or the 5xGAL4 reporter gene and GAL4-CREB.
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TGFß and c-Jun Transcriptional Responses Blocked by E1A
A protein complex that could be influenced by c-Jun might include
CBP, as both CREB and c-Jun are known to bind to this transcriptional
coactivator. To determine whether CBP was necessary for TGFß
regulation of GAL4-CREB, we performed similar experiments as shown in
Fig. 4
, A and B, but in the presence of E1A, an adenoviral oncoprotein
that binds to and inactivates p300/CBP (27, 28, 29, 30, 31, 32). Both basal and
TGFß-induced transcription from either GAL4-CREB or GAL4-CREB-S133A
could be abrogated by coexpression of wild-type E1A but not by a mutant
of E1A that is unable to bind CBP,
236 E1A (Fig. 4C
). Similarly,
the ability of c-Jun to potentiate CREB-mediated transcription could be
blocked by cotransfection of E1A but not
236 E1A (Fig. 4D
). Thus,
the effects of TGFß and c-Jun on transcription of GAL4-CREB require
CBP.
c-Jun binds to two regions of CBP. The N-terminal end of c-Jun
associates with the KIX domain of CBP (17). On the other hand, the
C-terminal leucine zipper domain of c-Jun associates with the C/H2
domain of CBP (18). To determine the relative importance of the two CBP
binding regions of c-Jun on CRE and GAL4-CREB-mediated transcription,
we evaluated the effects of a mutant form of c-Jun from which the N
terminus (residues 3122) had been deleted. As shown in Fig. 2B
, TAM-67 markedly stimulates transcription of the 1xCRE reporter gene and
mimics the effect of TGFß, even though its expression level detected
by immunoprecipitation-Western analysis appears to be slightly
lower than the other Jun constructs (data not shown). In addition,
TAM-67 is a potent agonist of the reporter gene regulated by the
truncated c-Fos promoter, Fos
80CAT (Fig. 2C
). Finally, the
GAL4-CREB reporter gene is also markedly stimulated by TAM-67 in either
the absence or presence of TGFß as demonstrated in Fig. 4B
. It should
be pointed out that the data in Fig. 4B
were obtained using 1 µg of
TAM-67 cDNA. Increasing the amount of this mutant causes an additional
increase in transcription and completely mimics the effects of TGFß.
Conversely, a mutant of TAM-67 from which the leucine zipper has been
deleted was unable to stimulate transcription from either the CRE or
GAL4-CREB reporter gene (Fig. 4E
). Full-length c-Jun lacking only the
leucine zipper was also inactive with these reporter genes (data not
shown). These data suggest that the interaction of the leucine zipper
region of c-Jun with the C/H2 region of CBP could be involved in the
stimulation of CREB/CBP-dependent transcription.
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DISCUSSION
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Our data show that c-Jun overexpression in HaCaT cells can
stimulate CRE and CREB-mediated transcription in a manner that requires
CBP, presumably through recruitment of c-Jun to CBP. Bannister et
al. (17) showed that transactivation by a Jun/CBP complex requires
phosphorylation of Ser63 and Ser73 in c-Jun, and Hocevar et
al. (33) recently demonstrated in a fibrosarcoma HT1080-derived
cell line that TGFß signals can activate Jnk, the kinase responsible
for the phosphorylation of Jun. It was surprising, therefore, that when
the N terminus of c-Jun was deleted (residues 3122; TAM-67), the
mutant c-Jun activated transcription from the CRE or through GAL4-CREB
even better than wild-type c-Jun. Since stimulation of the CRE and
GAL4-CREB is inhibited by E1A and therefore appears to utilize
endogenous CBP, we hypothesized that the activation by TAM-67 was due
to the recently reported interaction of the leucine zipper domain of
c-Jun with the C/H2 domain of CBP (18). In support of this, we found
that a C-terminal truncation of full-length c-Jun or TAM-67 to remove
the leucine zipper resulted in proteins that could not stimulate
transcription. Thus, the leucine zipper is required for the CRE/CREB
response, even in the context of GAL4-CREB
bzip where Jun cannot form
a heterodimer on the DNA. We cannot rule out, however, that the leucine
zipper may mediate essential interactions with other, as yet
unidentified, proteins involved in CRE transactivation. The
dispensability of Ser63/73 phosphorylation in certain situations is
supported by recent findings that a LexA-c-Jun fusion protein in which
both residues are mutated to Leu is transcriptionally active in a
CBP-dependent manner during calcium influx (34).
Additionally, while the amount of c-Jun increases in complex with the
consensus CRE upon TGFß treatment as measured by EMSA, Jun does not
appear to bind to DNA directly since the DNA binding mutant activates
CRE-dependent transcription as well as wild-type Jun does. This finding
is in direct contrast to numerous reports in which DNA binding mutants
or the TAM-67 mutant of c-Jun act as dominant negatives for
Jun-mediated transcription on AP-1 sites (35, 36, 37, 38). Both DNA binding and
the Jnk phosphorylation sites are necessary for transcriptional
activity by c-Jun as a heterodimer either with c-Fos on AP-1 sites or
with ATF-2 on CRE-like sites (17, 39). Indeed, we originally used the
DNA-binding and transactivation domain mutant c-Jun constructs in an
attempt to block the Jun effects on the consensus CRE, to demonstrate
that c-Jun directly participates in TGFß stimulation. Instead, we
uncovered a novel mechanism by which Jun activation of CRE-mediated
transcription requires the leucine zipper, but neither DNA binding nor
the N-terminal phosphorylation sites for JNK. Unfortunately, these
results precluded using the mutant constructs as proof of principle
that TGFß signals to the CRE through up-regulation of Jun. However,
it is striking that cotransfection of either full-length CREB or the
TGFß-activated transcription factor Smad 3, which can bind to
and thus titrate out endogenous Jun from a GAL4-CREB DNA-bound complex,
acts to squelch TGFß enhancement of GAL4-CREB-mediated transcription
(B. L. Harvat, unpublished results). Cotransfection of the various
Jun constructs and the 1xCRE reporter in HeLa cells gave identical
results as they did in HaCaT cells (B. L. Harvat, unpublished
observations), suggesting that the CBP-dependent mechanism that depends
on the leucine zipper of Jun, but not DNA-binding or Jnk
phosphorylation, may be used by other epithelial-type cells. Additional
signals may also impact the CBP/Jun complex proposed here, as we have
observed a similar ability of c-Jun to substitute for ionomycin
stimulation of CRE and GAL4-CREB-mediated transcriptional activation in
HaCaT cells (B .L. Harvat and A. R. Means, unpublished
observations).
As a transcriptional cointegrator, p300/CBP binds to multiple
transcription factors that, in turn, bind to their respective DNA
promoter elements (reviewed in Ref. 40). Genes containing DNA binding
sites for three of these factors, CREB, nuclear factor-
B, and
AP-1, are TGFß responsive in HaCaT cells (14, 41, 42). In addition,
TGFß induces transcription of p15 and p21 CDK inhibitors through
Sp1-binding elements and, although direct association between p300/CBP
and Sp1 has not been demonstrated, the up-regulation of these genes is
blocked by E1A, implicating involvement of p300/CBP (10). The
mechanisms by which TGFß stimulates transcription through these
p300/CBP-associated factors are still unclear. However, it seems
possible that the proposed increase in c-Jun-bound p300/CBP could have
similar effects on other TGFß-responsive genes whose transcription is
p300/CBP dependent.
TGFß is also known to signal through the phosphorylation and nuclear
translocation of Smad-2 and Smad-3 proteins (reviewed in Refs. 2, 43), which can then bind to response elements in DNA, recruit p300 and
CBP (11, 12, 13, 44), and stimulate transcription. Overexpression of Smad-3
and Smad-4 greatly increases TGFß-independent transcription of
plasminogen activator inhibitor (PAI-1), a known TGFß target (45),
presumably because overexpression can force localization in the nucleus
and binding to DNA in the absence of Smad 3 phosphorylation by TGFß
receptor. We investigated the ability of overexpressed Smad-3 and
Smad-4 to similarly stimulate GAL4-CREB and CRE-dependent transcription
or enhance the c-Jun effect, even though our CRE reporters lack
Smad-binding elements in the promoters. In contrast to c-Jun,
overexpression of Smad-3 and Smad-4 did not substitute for the ability
of TGFß to stimulate CRE/CREB-mediated transcription, but rather
squelched transcription (P. Hu., B. L. Harvat., X.-F. Wang, and
A.R. Means, unpublished observations). The most direct interpretation
of these results would be that Smads are unable to participate in the
formation or stabilization of the transcription complex on consensus
CRE or GAL4-CREB elements in the absence of DNA binding sites for Smads
and thus cannot stimulate Jun activity or affinity for the complex.
However, because of their ability to bind to p300/CBP (11, 12, 13, 44) or
Jun (46, 47), when overexpressed, Smads can squelch transcription by
binding and sequestering these proteins that appear essential for
CRE/CREB activity. In contrast, in the context of promoters that
contain both Smad-binding elements and a CRE, such as the Jun promoter
(14) or the Drosophila ultrabithorax promoter (48),
Smads may stabilize the CRE-dependent complex and thus contribute
to Jun activation of these promoters.
c-Jun stimulation of GAL4-CREB-mediated transcription suggests that
CREB has a role in directing the TGFß response. We know that TGFß
treatment does not significantly alter the amount of CREB
phosphorylated on Ser-133 in HaCaT cells. Indeed, in untreated HaCaT
cells, a substantial fraction of CREB is phosphorylated on Ser-133,
suggesting that a constitutive phospho-CREB/CBP association might
exist. Furthermore, since Ser-133 of endogenous CREB can be
metabolically phosphorylated within a 4-h labeling period, there must
be a continual turnover of Ser-133 phosphorylation. This suggests there
may be a dynamic equilibrium of CBP/CREB association at CRE sites in
HaCaT cells. If so, such transient complexes could be stabilized by the
elevated levels of c-Jun that occur as a rapid response to TGFß. In
our experiments, CREB cannot recruit c-Jun through heterodimerization,
since the leucine zipper domain is missing in the GAL4-CREB constructs.
However, CREB association with CBP through the KIX domain could recruit
CBP-bound c-Jun to the transcription complex via the association of
c-Jun with another region of CBP. The C/H2 region is the only other
region of CBP that has been shown to bind Jun to date, and we also have
seen that GST-CBP-C/H2 region fusion proteins can pull down Jun from
HaCat lysates (B. L. Harvat, unpublished results). Since TGFß
can also stimulate transcription mediated by CREB-S133A, and this
transcription can be prevented by E1A, it is possible that either lower
affinity contacts of CREB with CBP or interaction of CREB with
components of transcription factor IID (TFIID) (49, 50, 51) acting
as an indirect bridge between CREB and CBP may still allow the mutant
CREB to recruit c-Jun through CBP. An equally plausible possibility is
that the increase in c-Jun bound to CBP upon TGFß treatment could
lead to stabilization of a CREB/CBP complex, even without an increase
in Ser-133 phosphorylation, and subsequently increase CRE-dependent
transcription. In addition, we cannot rule out the possibility that
other cell type-specific accessory factors or TGF-ß-mediated
phosphorylation of Jun may be involved in these transcriptional
effects. At any rate, the question of a universal role for the
CREB-binding/KIX domain in CBP in integrating signals for
proliferation, as opposed to growth arrest or differentiation, is an
interesting one. c-Jun has an important role in regulating keratinocyte
proliferation and differentiation (52), and keratinocytes may be
particularly sensitive to c-Jun levels. Finally, the discovery that
c-Jun can stimulate CBP-mediated transcription in a manner that is
independent of its ability to bind DNA represents a novel mechanism by
which signal transduction pathways can be integrated through CBP to
influence transcription.
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MATERIALS AND METHODS
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Plasmids/Constructs
For 1xCRE-luc, a consensus CRE (5'-GCT CTC TGA CGT CAG GCA
AT-3') was subcloned into pgl2-basic (Promega Corp.,
Madison, WI). Insertion of only one copy of the CRE was verified by
sequencing. Tam67
zip was created by inserting two stop codons in the
Tam67 construct in place of the codons for amino acids K283 and K285,
which are at the 5'-end of the leucine zipper. The mutagenesis was done
using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, La Jolla, CA) and the following primers:
sense, 5'-GGAGGAATAAGTGTAAACCTTG-3', and
antisense, -5'-CAAGGTTTACACTTATTCCTCC-3'. The
underlined nucleotides are the ones that were changed from
the wild-type sequence.
Cell Culture
HaCaT cells were grown in
-MEM (Life Technologies, Inc., Gaithersburg, MD), containing 10% FBS, 2 mM
L-glutamine (Life Technologies, Inc.)
and 50 U/ml Penicillin-G and 50 µg/ml Streptomycin Sulfate
(Life Technologies, Inc.).
EMSA
EMSAs were performed using 13 µl of nuclear extracts
prepared from cells untreated or treated with 100 pM
TGFß1 for 1 h or 3 h. The 1xCRE (5'-GCT CTC TGA CGT CAG GCA
AT-3') construct was radiolabeled with 32P-
ATP with T4
Polynucleotide kinase and purified using a spin column
(QIAGEN, Chatsworth, CA). Gel shift conditions are
exactly as described in Yingling et al. (41). For supershift
analysis of CREB or c-Jun, we used 2 µl of anti-CREB (24H24-X) and
c-Jun (KM-1-X), respectively (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Cotransfections and Luciferase Assays
HaCaT cells were plated in six-well plates either at 175,000
cells per well and grown overnight or at 50,000 cells per well and
grown for 2 days. The media were then replaced with
-MEM containing
100 µM chloroquine. The indicated amounts of DNA were
diluted to a total volume of 225 µl in Hanks solution, mixed with 75
µl of diethylaminoethyl-dextran, and added dropwise to
each well. Cells were incubated with the DNA mix for 3 h, then
rinsed once in PBS, glycerol shocked for 2 min, rinsed again, and
incubated in growth media overnight. The following day, cells were
treated with either 100 pM TGFß or left untreated and
harvested 2024 h later. Soluble extracts were prepared, centrifuged
at 14,000 rpm for 5 min at 4 C, and analyzed for luciferase activity
using either a Berthold or Labsystems Luminoskan luminometer.
ß-Galactosidase activity was assayed using the substrate,
chlorophenolred-ß-D-galactospyranoside (Roche Molecular Biochemicals, Nutley, NJ), and determining the
optical density at 575 nm. The results were used to normalize the
luciferase activity for transfection efficiency. Each transfection
experiment was repeated two to five times. Individual transfections
within a single experiment were done independently in duplicate or
triplicate as were the luciferase and ß-galactosidase assays for each
sample lysate.
pCREB Western
HaCaT cells were plated and grown overnight in culture media.
The next day, cells were treated with TGFß or ionomycin for 1 h
or left untreated. Cells were harvested as previously described (53) in
boiling 2x sample buffer. The samples were then subjected to
immunoblot analysis using phospho-specific (Dr. D. Ginty) and total
CREB rabbit polyclonal antibodies (Upstate Biotechnology, Inc., Lake Placid, NY).
In Vivo Labeling and 2-D Electrophoresis
Exponentially growing HaCaT cells (2 x 106)
were washed in phosphate-free DMEM (Life Technologies, Inc.) and preincubated 12 h. The media were then aspirated,
and approximately 2 mCi of [32P]orthophosphate (ICN Biochemicals, Inc., Cleveland, OH) in 2 ml of fresh media
were added. Cells were labeled for 4 h, during which TGFß was
added for either 10 min, 1 h, or 4 h before harvesting or
cells were left untreated. Cells were then washed and lysed in
Universal Lysis Buffer containing 50 mM Tris-HCl (pH 7.5),
150 mM NaCl, 0.5% Nonidet P-40, 50 mM NaF, 1
mM sodium orthovanadate, 0.2 mM sodium
molybdate, 20 mM ß-glycerophosphate, 1 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and
protease inhibitors.
32P-labeled proteins were separated by SDS-PAGE and
transferred to Immobilon (Millipore Corp., Bedford, MA).
The membrane was rinsed in H2O and exposed at -80 C for 30
min to visualize labeled proteins. After excision, the bands were
immediately incubated in 0.5% PVP-360 in 100 mM acetic
acid for 30 min at 37 C. The bands were then washed five times with
H2O and twice with 50 mM
NH4HCO3. Two hundred microliters of 50
mM NH4HCO3 were added to cover the
membrane, and digestion was started with 10 µl of 1 mg/ml
TPCK-treated trypsin (Worthington Biochemical Corp., Freehold, NJ). Two hours later an additional 10
µl of trypsin were added and incubated overnight. After removing the
membrane and lyophilizing the sample, 50 µl of performic acid (900
µl formic acid, 100 µl H2O2) were added for
1 h on ice; 1 ml of H2O was added and the sample was
lyophilized. Cherenkov counts were determined and the sample was
resuspended in a small volume of H2O. Similar to the
procedure used by Sun et al. (25), 12 µl (
20005000
cpm) were loaded on a TLC plate and electrophoresed for 20 min at 1,000
V by use of the Hunter Thin Layer Electrophoresis System (HTLE-7000,
CBS Scientific Co., Delmar, CA), followed by TLC with
n-butanol-pyridine-acetic acid-water in volume ratios of
0.375, 0.25, 0.075, and 0.30, respectively. Autoradiography was used to
visualize the phosphopeptides.
 |
ACKNOWLEDGMENTS
|
---|
We thank R & D Systems (Minneapolis, MN) for
supplying TGFß1. We are grateful to Dr. R. Maurer for the GAL4-CREB
and GAL4-CREB 133A DNA constructs; Dr. D. Ginty for his phospho-serine
133-CREB antibody; Dr. S. Shenolikar and H. Quan for purified CREB; Dr.
T. Curran and Dr. M. Birrer for the c-Jun expression plasmids; Dr. M.
Gilman for the c-Fos
80 construct; and Dr. J. Nevins for E1A
plasmids. We are grateful to Carolyn Wong and Elissa M. Rougier-Chapman
for sharing unpublished information as well as technical advice. We
would like to thank members of the Wang and Means laboratories for
helpful discussions, Charles Mena and Yong Yu for excellent technical
assistance, and Tso-Pang Yao for critical reading of this
manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Anthony R. Means, Department of Pharmacology, Duke University Medical Center, C238 Levine Science Research Center, Durham, North Carolina 27710.
S.S.H. is supported by a predoctoral fellowship from the Department of
the Army DAMD1797-17101. X.-F. W. is a Leukemia Society
Scholar. This work was supported by NIH grants to A. R. M.
(HD-07503; GM-33976) and X.-F. W. (DK-45746).
1 These authors contributed equally to this work. 
Received for publication June 2, 1999.
Revision received September 16, 1999.
Accepted for publication September 21, 1999.
 |
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