[Cancer Research 60, 276-281, January 15, 2000]
© 2000 American Association for Cancer Research
Heregulin Induces Expression, DNA Binding Activity, and Transactivating Functions of Basic Leucine Zipper Activating Transcription Factor 41
Amjad H. Talukder,
Ratna Vadlamudi,
Mahitosh Mandal and
Rakesh Kumar2
The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
 |
ABSTRACT
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Heregulin ß1 (HRG), a combinatorial ligand for human epidermal growth
factor receptor 3 and human epidermal growth factor receptor 4
receptors, is a regulatory secretory polypeptide with distinct
biological effects such as growth stimulation, differentiation,
invasiveness, and migration in breast cancer cells. The mechanism
underlying the diverse functions of HRG is not well established, but it
is believed to be dependent on the induced changes in expression of
specific cellular gene products, their modification, or both. The
binding of basic leucine zipper transcription factors to the cAMP
response element is known to activate a variety of gene products with a
role or roles in growth regulation. In the studies presented here, we
identified basic leucine zipper activating transcription factor (ATF) 4
as one of the HRG-inducible gene product. We demonstrated that HRG
stimulation of human cancer cells induces expression of ATF4 mRNA and
protein, ATF4 DNA binding activity, and ATF4 transactivating function.
Consistent with its role as a transcriptional activator, HRG-stimulated
ATF4 protein stimulated the transcription from an artificial promoter
with three tandem ATF sites or from a naturally occurring promoter with
ATF4 sites such as E-selectin. We also demonstrated a preferential role
of the HRG-stimulated mitogen-activated protein kinase pathway, but not
the phosphatidylinositol 3'-kinase pathway, in supporting the observed
increase in ATF4 DNA binding activity and transcription from E-selectin
promoter in HRG-stimulated cells. Because ATF4 binding sites are
present in a variety of growth-regulating cellular genes, these
findings suggest that the stimulation of ATF4 expression and its
transactivating functions may constitute an important mechanism of
HRG-mediated regulation of putative genes with diversified functions.
The present study is the first demonstration of regulation of
expression and transactivation ability of ATF4 by any polypeptide
growth factor.
|
Introduction
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Growth factors and their receptors play an essential role in the
regulation of epithelial cell proliferation. It has been demonstrated
that abnormalities in growth factor expression and action contribute to
the progression and maintenance of the malignant phenotype. For
example, c-erbB2 encodes
HER2,3
the overexpression of which is frequently associated with an aggressive
clinical course and increased metastasis in human breast cancer
(1)
. Recently, additional members, HER3 and HER4, have
been added to the HER2 family because these receptors also share
sequence homology with the tyrosine kinase domain of HER1
(2)
. The family receptors can be transactivated by
receptor-receptor interaction in a ligand-dependent manner and thus can
use more than one pathway to execute their biological functions
(2, 3, 4)
. In addition, accumulating evidence suggests that
the progression of human breast cancer cells may be regulated by
heregulin, a combinatorial ligand for HER3 and HER4 receptors.
Recently, we and others (5, 6, 7, 8, 9)
have demonstrated that HRG
activation of breast cancer cells (in the absence of HER2
overexpression) also promotes the development of more aggressive
phenotypes in breast cancer cells. Although these observations suggest
that ligand-driven activation of HER receptors may play an important
biological role or roles in the progression of breast cancer cells to a
malignant phenotype, the nature of the pathways by which HRG signals
are relayed to the nucleus in breast cancer cells remains poorly
understood.
It is widely believed that the induction of a set of early
growth-responsive (also known as immediately early) genes, in the
absence of de novo protein synthesis, may constitute the
first step in the cellular molecular response to extracellular signals.
Transcription of protein-coding genes is one of the major regulatory
steps in gene expression. Among the major regulatory elements that
contribute to transcriptional regulation of extracellular signals are
the CRE and activator protein 1 sequence motifs. The CRE element
(TGACGTCA) is defined as the ATF binding site. The process of
transcription involves physical interactions of sequence-specific DNA
binding transcriptional activations with general transcription factor
directly, indirectly, or both (10)
. It is increasingly
accepted that the CRE site is recognized by a family of the
bZIP-containing proteins known as CREBs or ATFs. The bZIP DNA-binding
proteins need to dimerize for productive DNA binding (11)
.
The dimeric bZIP factors are characterized by a bipartite 
-helical
structure: the basic NH2-terminal half of the
motif interacts with sequence-specific DNA; whereas the COOH-terminal
half of the motif dimerizes to form a leucine zipper (11
, 12)
. The bZIP factors not only homodimerize but also
heterodimerize with specific bZIP proteins by the leucine zipper
(12)
. Because binding sites for ATF factors are present in
several growth-regulating cellular promoters (13)
, they
are believed to be involved in different regulatory circuits, allowing
cells to integrate signals from distinct pathways. In addition, the
activity of ATF is also regulated by coactivators such as CREB-binding
protein/p300 family members (14)
. In the present study, we
have investigated the possible involvement of ATF proteins in the
action of HRG in breast cancer cells. We report that recombinant HRG
up-regulates the expression of bZIP transcription factor ATF4, its DNA
binding activity, and the ability of ATF4 to transactivate the target
genes in HRG-responsive cancer cells.
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Materials and Methods
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Cell Cultures and Reagents.
Human breast cancer MCF-7 cells (8)
and colon cancer cell
lines LS174T, CaC02, and FET (15)
were maintained in
DMEM-Ham’s F-12 (1:1) supplemented with 10% FCS. Recombinant HRG was
purchased from Neomarkers, Inc., and anti-ATF4 Ab was purchased from
Santa Cruz, Inc.
Cell Extracts and Immunoprecipitation.
Cell lysates containing equal amounts of protein were resolved by
SDS-PAGE, transferred to nitrocellulose, and probed with the
appropriate Abs. An equal number of cells were metabolically labeled
for 12 h with 100 µCi/ml
[35S]methionine in methionine-free medium
containing 2% dialyzed fetal bovine serum in the absence or presence
of HRG. Cell extracts (equal perceptible trichloroacetic acid counts)
were immunoprecipitated with the desired or control Ab, resolved on a
SDS-PAGE gel, and analyzed by autoradiography (16)
.
RT-PCR and Northern Hybridization.
The sequences of the following primers were obtained from the Clontech:
(a) Tax, Tax-responsive enhancer element-binding protein
107; (b) PUF, c-Myc purine-binding transcription factor PUF;
and (c) Mac, Merkes-related protein. The RT-PCR for ATFD and
others genes was done according to the manufacturer’s instructions
using access RT-PCR systems (Promega). The forward primer sequence for
ATF4 was 5'-AATGGCTGGC'rGTGGATGGG'I''I'GGTCA-3', and the reverse primer
sequence was 5'-GATCATGGCAACGTAAGCAGTGTAGTCTG-3'. Human
E-selectin-specific primers were purchased from R&D Systems. Total
cytoplasmic RNA was analyzed by Northern hybridization using an ATF4
cDNA probe (17)
or a RT-PCR-generated 436-bp fragment.
Ribosomal 28S RNA and 18S RNA were used to assess the integrity of the
RNA.
Nuclear Extract Preparation and Electrophoresis Mobility Shift
Assay.
Nuclear extracts were prepared as described previously
(18)
. The synthetic oligonucleotides corresponding to the
consensus sequence of human CRE (5'-AGAGATTGCCtGACGTCAGAGAGCTAG-3'),
activator protein 1 (5'-CGCTTGATGACTCAGCCGGAA-3'), and nuclear factor
B (5'-AGTTGAGGGGACTTTCCCAGG-3') were purchased from Santa Cruz, Inc.
Oligonucleotides were end-labeled with 32P by
using the end-labeling kit (Boehringer Mannheim). To confirm the
ATF4 protein in the DNA-protein complex, 5-µg extracts were incubated
for 10 min with or without antisera recognizing different nuclear
factors.
Promoter Assays.
Dr. T. Hai (Ohio State University, Columbus, OH) generously provided
pEC (ATF)3-CAT (19)
and
E-selection-CAT (20)
. Cells were serum starved in low
serum medium (0.1% serum) for 48 h before transfection.
Serum-starved cells were transiently cotransfected with ATF4 plasmid
and a control vector using Lipofectamine (Life Technologies, Inc.). CAT
activity was measured 48 h after transfection using a CAT assay
kit (Promega).
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Results
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Identification of ATF4 as a HRG-inducible Gene.
To better understand the mechanism of HRG action in breast cancer
cells, we screened MCF-7 cells for HRG-inducible genes using the Atlas
cDNA Gene Array (Clontech). Total RNA was isolated from control and
HRG-treated cells, and cDNAs were generated by reverse transcriptase in
the presence of [-32P]dCTP and hybridized to
gene array filters. This screening resulted in the identification of
bZIP transcription factor ATF4 as a HRG-inducible gene in breast cancer
cells (Fig. 1A
). To validate the results of RT-PCR screening, Northern
blot analysis was performed using a 406-bp PCR probe specific for ATF4.
Data in Fig. 1B
demonstrate that HRG increased the
steady-state levels of the 1.6-kb mRNA of ATF4 by 2–4-fold in MCF-7
cells, with maximal induction between 10 and 24 h after HRG
treatment. Similar results were obtained when the Northern
hybridization was performed using a human ATF4 cDNA (data not
shown). Because there was no precedent of growth
factor-inducible up-regulation of ATF4, the experiment was
independently repeated five times, and similar results were obtained.
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Fig. 1. HRG regulation of ATF4 mRNA expression. A,
identification of ATF4 as a HRG-inducible gene. Tax,
Tax-responsive enhancer element binding protein 107;
PUF, c-Myc purine-binding transcription factor PUF;
Mac, Merkes-related protein; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase. B,
MCF-7 cells were treated with HRG for the indicated times. Total RNA
(20 µg) was analyzed by Northern blotting using a
PCR-generated 436-bp human ATF4 fragment as a probe. The blot was
reprobed with a glyceraldehyde-3-phosphate dehydrogenase cDNA probe.
Quantitation of ATF4 mRNA is shown in the bottom panel.
The results shown are representative of five independent experiments.
C, HRG induces ATF4 mRNA expression. MCF-7 cells were
treated with cycloheximide (50 µg/ml) or actinomycin D (10 µg/ml)
in the presence or absence of HRG (10 ng/ml) for 3 h. Total RNA
was isolated, and the levels of ATF4 mRNA were detected by Northern
blotting. Quantitation of ATF4 mRNA is shown in the bottom
panel. Results shown are representative of two independent
experiments.
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HRG Regulates ATF4 at the Pretranslational Level.
The observed HRG-mediated increase in ATF4 mRNA could be due to
increased synthesis of newly transcribed mRNA, enhanced stability of
ATF4 mRNA, or both. To delineate this possibility, we examined the
effect of actinomycin D, an inhibitor of transcription. Pretreatment of
cells with actinomycin D abolished the HRG-mediated induction of ATF4
mRNA, suggesting the need for continuous RNA synthesis in the
observed increased expression of ATF4 mRNA in HRG-treated cells (Fig. 1C
). To address the issue of translational regulation, we
used cycloheximide, a translational inhibitor. Treatment of cells with
cycloheximide by itself induced the expression of ATF mRNA (2–3-fold).
However, HRG treatment further induced the expression of ATF4 mRNA
compared with the level in cycloheximide-treated culture (compare
Lane 3 with Lane 4). These results suggest that
HRG regulates ATF4 expression at a pretranslational level.
HRG Induces the Expression of Newly Synthesized ATF4 Protein and
Its DNA Binding Activity.
To determine whether the observed increase in the level of ATF4 mRNA in
HRG-treated MCF-7 breast cancer cells was associated with an increase
in the expression of ATF4 protein, Western blot analysis was performed.
The results seen in Fig. 2A
demonstrate that treatment of MCF-7 cells with HRG was
accompanied by a significant increase in the steady-state level of -50
kDa ATF4 protein. The observed HRG-mediated increase in ATF4
expression was due to increased expression of newly synthesized ATF4 in
HRG-treated cells, as confirmed by metabolic labeling studies (Fig. 2B
). There was no effect of HRG on the half-life (<4 h) of
ATF4 protein as assessed by pulse-chase experiments (Fig. 2C
).
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Fig. 2. HRG up-regulates the level of ATF4 protein.
A, MCF-7 cells were treated with HRG for the indicated
times. Total lysates were subjected to SDS-PAGE and blotted with
anti-ATF4 Ab (middle panel). As an
internal control, the upper portion of same blot was probed with
anti-FAK Ab (top panel). Quantitation of ATF4 mRNA is
shown in the bottom panel. B, MCF-7 cells
were stimulated with HRG for 12 h and metabolically labeled with
[35S]methionine during the last 4 h before
harvesting. Cell lysates were immunoprecipitated with an anti-ATF4 Ab
and analyzed by SDS-PAGE, followed by fluorography. C,
MCF-7 cells were metabolically labeled with
[35S]methionine for 2 h and cultured further in the
presence or absence of HRG for the indicated times. Cell lysates were
immunoprecipitated with an anti-ATF4 Ab and analyzed by SDS-PAGE,
followed by fluorography. Results shown are representative of three
independent experiments. D, induction of ATF4 DNA
binding activity. Nuclear extracts were incubated with a
32P-labeled oligonucleotide containing the consensus CRE
element for 30 min at 37°C. When indicated, nuclear extracts were
preincubated with a 50-fold excess of unlabeled wild-type or mutated
oligonucleotide and also by ATF4 Ab (2 or 4 µl) for 15 min. An
arrow shows the specific DNA-protein complex with which
ATF4 Ab competed. Results shown are representative of three independent
experiments.
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To understand the physiological significance of ATF4 in HRG activity in
breast cancer cells, we investigated whether HRG signaling could
enhance the DNA binding of ATF4 to the CRE motif by using a gel-shift
assay. Data in Fig. 2D
show that HRG treatment for 30 min
was associated with a significant enhancement of DNA binding activity
of ATF4 because this activity could be competed by wild-type (Fig. 2D
, Wt oligo) unlabeled CRE oligonucleotide but not by
mutated (Fig. 2D
, Mt oligo) CRE oligonucleotide; the
activity was blocked by pretreatment of the nuclear extracts with a
well-characterized anti-ATF4 Ab (Ref. 17
; Fig. 2D
, the lower band is indicated by an arrow).
HRG Regulates ATF4 Expression and Its DNA Binding Activity in
Diversified Cell Types.
To determine whether the observed induction of ATF4 expression is an
effect restricted to HRG in MCF-7 cells or whether it could be
demonstrated in other HRG-responsive cells, we examined the effect of
HRG on ATF4 expression in HRG-responsive human colorectal LS174T,
CaCO2, and FET cells (15)
. As illustrated in Fig. 3
, HRG treatment was associated with a significant up-regulation of ATF4
mRNA expression in a number of HRG-responsive cells (Fig. 3A
) and also of ATF4 protein levels (Fig. 3B
).
Although HRG could induce ATF4 expression and its DNA binding activity
in a number of human cell lines, we have chosen human breast cancer
MCF-7 cells as a model system to further ascertain the potential
significance of ATF4 pathway in the regulation of HRG action.
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Fig. 3. HRG stimulates ATF4 expression in multiple cell lines.
A, FET, CaC02, and LS174T colon cancer cell lines were
treated with or without HRG for the indicated times, and ATF4 mRNA
expression was determined by Northern blotting. The blot was
sequentially reprobed with a glyceraldehyde-3-phosphate dehydrogenase
probe (GAPDH). B, lysates from control
and HRG-treated FET cells were analyzed by Western blotting using ATF4
mAb. Results shown are representative of two independent experiments.
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Regulation of ATF4 Transactivation Functions by HRG.
We next determined whether HRG could activate CRE site-driven
transcription using a CAT reporter system. MCF-7 cells were transiently
transfected with a CAT reporter driven by artificial ATF sites
[p(ATF4)3 CAT] and stimulated with or without
HRG. As illustrated in Fig. 4A
, HRG treatment stimulated the CAT reporter up to 3–5-fold
more than untreated control cells in a time-dependent manner. In
addition to activating the CAT reporter system driven by an artificial
promoter, HRG also stimulated the transcription from naturally
occurring promoters with ATF4 sites such as E-selection
(19)
, although to a lesser degree. In general, E-selectin
promoter was about 2.6 times less active, as compared to
(ATF4)3 promoter. This may be related to the fact
that MCF-7 cells do not express E-selectin, probably due to the
presence of the appropriate transcription factors in the appropriate
places to drive full expression of the promoter. Results in Fig. 4B
demonstrate that HRG activated the E-selectin promoter,
although to a lesser extent than did the synthetic ATF4 promoter. It is
possible that HRG-induced ATF4 protein may not interact efficiently
with the natural promoter due to its interactions with other factors.
Alternatively, the observed activation may be sufficient to regulate
the expression of E-selectin mRNA. The putative role of HRG-induced
ATF4 in the stimulation of E-selectin promoter was confirmed by
cotransfection studies using a ATF4 plasmid or control vector. As shown
in Fig. 4C
, cotransfection of MCF-7 cells with E-selectin
CAT with human pATF4-sense (Lanes 2 and 3), and
not with pATF4-antisense (Lane 4), resulted in a modest but
significant stimulation of promoter activity (Lane 3).
Because MCF-7 cells do not express E-selectin, we next examined whether
HRG-mediated activation of the E-selectin promoter could lead to
up-regulation of E-selectin mRNA in HUVECs. Results in Fig. 4D
demonstrate that HRG treatment of HUVECs was accompanied
by stimulation of E-selectin expression as determined by RT-PCR.
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Fig. 4. Regulation of ATF4 transactivation functions by HRG.
A and B, MCF-7 cells were transiently
transfected with p(ATF)3-CAT (A) or
E-selectin CAT (B) constructs, and CAT activity was
measured after 36 h of transfection. Some cultures were treated
with HRG for 4 or 24 h before lysis. These studies were
independently repeated four times with similar results. Quantitative
means of four experiments are shown in the bottom panel.
C, MCF-7 cells were transfected with E-selectin-CAT in
the absence (Lane 1) or presence of human ATF4-sense
(Lanes 2 and 3) or human ATF4-antisense
(Lane 4). Results shown are representative of three
separate experiments. D, HUVECs were treated with HRG or
tumor necrosis factor (as a positive control), and total RNA was
analyzed by RT-PCR for E-selectin mRNA expression (485-bp product).
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A Role of the MAPK Pathway in HRG-mediated ATF4 Transactivation.
To understand the nature of signaling pathways leading to ATF4
regulation, we next examined the potential involvement of MAPK and
PI-3K, two prototype signaling pathways that are widely activated by
growth factors. To demonstrate the functionality of these pathways,
MCF-7 cells were treated with or without HRG, and stimulation of PI-3K
and MAPK pathways was determined by thin-layer chromatography and
immunoblotting using phospho-p42/44 Ab, respectively (Fig. 5A
). In these studies, the activation of the MAPK and PI-3K
pathways was blocked by using well-characterized dominant-negative
mutants of MEKK (Ref. 21
; upstream regulator of MAPK) and
the p85 subunit of PI-3K (8)
, respectively. As illustrated
in Fig. 5, B and C
, there was no stimulatory
effect of HRG on ATF4 DNA binding activity (Fig. 5B
) and
transactivation function (Fig. 5C
) in cells transfected with
dominant-negative MEKK compared to control cells. In contrast, HRG
stimulated both DNA binding activity (Fig. 5B
, Lanes 5 and
6) and transactivation function (Fig. 5C
, Lanes 5
and 6) of ATF4 in cells expressing dominant-negative p85. In
brief, these results suggest a potential role of MAPK in regulation of
ATF4 transactivation in HRG-treated breast cancer cells.
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Fig. 5. Role of MAPK in regulation of ATF4 transactivation
functions by HRG. A, MCF-7 cells were treated for 30 min
with (+) or without (-) HRG, and cell lysates were assessed for the
activation of PI-3K (using thin-layer chromatography) or MAPK (using
phospho-MAPK Ab immunoblotting). B, cells were
transiently transfected with E-selectin CAT construct in the absence or
presence of dominant-negative mutants of MEKK and p85. Some cultures
were treated with HRG for 16 h before harvesting for
electrophoresis mobility shift assay. C, cells were
transiently transfected with E-selectin CAT construct in the absence or
presence of dominant-negative mutants of MEKK and p85. Some cultures
were treated with HRG for 16 h before lysis. CAT activity was
measured after a total of 36 h of transfection. These studies were
repeated two times with similar results. Quantitative means of four
experiments are shown in the bottom panel.
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Discussion
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HRG has been shown to modulate the growth, differentiation, and
motility of breast cancer cells. The multifunctional nature of HRG is
thought to be dependent on the induced changes in expression,
modification, or both of specific cellular gene products. Because we
identified the bZIP transcription factor ATF4 as one of the
HRG-inducible genes and because the binding sites for ATF4, the CRE
element, are commonly present in genes that regulate cell growth, we
explored the possible involvement of ATF protein in the action of HRG
using human breast cancer MCF-7 cells as a model system.
The results presented here indicate that treatment of human cells with
HRG induced significantly induced higher levels of ATF4 protein
expression. The expression and induction of ATF4 protein by HRG open a
whole new area of investigation regarding the role of bZIP
transcription factor in HRG activity. These observations, together with
the fact that ATF4 promoter itself contains CRE sites
(22)
, raise the possibility of an autoregulatory role of
increased ATF4 DNA binding activity in the induction of ATF4 expression
in HRG-stimulated cells.
Our conclusion that HRG is a very potent inducer of ATF4 expression is
supported by the following lines of evidence: (a) HRG
stimulated the expression of ATF4 mRNA as early as 3 h after
treatment; (b) elevated expression of ATF4 mRNA was
sensitive to the transcriptional inhibitor; (c) HRG-mediated
increased expression of ATF4 mRNA was accompanied by the enhancement of
ATF4 protein; and (d) HRG stimulated the expression of newly
synthesized 35S-labeled ATF4 protein. These
findings establish that ATF4 is a bona fide HRG-inducible
gene product. The induction of ATF4 expression by HRG was not a
phenomenon restricted to MCF-7 cells because HRG also induces the
expression of ATF4 mRNA and protein in other HRG-responsive human tumor
cell lines, such as colon carcinoma, FET, and CaCO2 cells
(15)
.
Another notable finding of this study is the potential role of
HRG-initiated signals in transcriptional activation of genes containing
CRE sites including ATF4 and E-selectin (19)
. The evidence
that cotransfection of human ATF4 has the capacity to activate
E-selectin promoter-driven transcription is of special interest because
it strongly suggests that HRG-induced ATF4 expression may contribute in
the modulation of expression of genes containing ATF4 binding sites
either directly, indirectly, or both by influencing the physical
interactions between ATF4 and its binding factors. Data from the
literature suggest that up-regulation of E-selectin expression in
endothelial cells may be closely associated with increased metastases
and invasion of human breast cancer cells (23)
. Because
HRG is a paracrine growth factor, our preliminary finding of
HRG-mediated stimulation of E-selectin expression in HUVECs implies
that HRG may use the ATF4 transcription factor to regulate the
expression of gene products with a role in breast cancer progression to
a more invasive phenotype.
Data from the literature suggest that in addition to transactivation,
ATF4 may also act as a transcriptional repressor, presumably due to
sequestration of factors and cofactors from the proximity of the target
promoter (10
, 11)
. In view of these earlier findings, it
seems reasonable to speculate that HRG may also use inducible
modification of ATF4 to potentially repress the expression of
unidentified cellular genes that negatively regulate the invasiveness
of breast cancer cells. Although we demonstrated the stimulation of
E-selectin as an example of transcription activation of a cellular gene
by bZIP transcription factor ATF4 in HRG-treated cells, our findings
may have a general implication for regulation of cellular genes with a
CRE motif by HRG-inducible signals. In the present study, we have not
identified the nature of ATF4 target genes in HRG-stimulated cells.
Another emerging concept from our present investigation is the
possibility that HRG regulation of ATF4 may provide an explanation for
multiple cellular functions of HRG because binding sites for the ATF4
factor are present in several growth-regulating cellular promoters and
because ATF4-interacting factors, particularly CREB-binding
protein/p300, are widely believed to be involved in integrating
regulatory signals from distinct pathways (13)
. Together,
these events may lead to differential regulation of genes that may
participate in the mediation of HRG in breast cancer cells.
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Acknowledgments
|
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We thank Joel F. Habener (Massachusetts General Hospital,
Boston, MA) for providing ATF4 cDNA and anti-ATF4 Ab; Tsonwin Hai
(Massachusetts General Hospital, Boston, MA) and James Whelan
(University of Tennessee, Knoxville, TN) for providing ATF4-CAT and
E-selectin-CAT constructs, respectively; Shizuo Akira (Hyogo College of
Medicine, Nishinomiya, Japan) for providing murine ATF4; Tucker
Collins for providing the E-selectin genomic clone; and Francois-Xavier
Claret for providing the MEKK constructs.
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FOOTNOTES
|
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by NIH Grants CA80066 and
CA65746 and the Breast and Ovarian Cancer Research Program of The
University of Texas M. D. Anderson Cancer Center (to R. K.). 
2 To whom requests for reprints should be
addressed, at Cell Growth Regulation Laboratory, Box 36, The University
of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard,
Houston, TX 77030. Phone: (713) 745-3558; Fax: (713) 745-3792; E-mail: rkumar{at}notes.mdacc.tmc.edu
3 The abbreviations used are: HER, human epidermal
growth factor receptor; HRG, heregulin ß1; ATF, activating
transcription factor; bZIP, basic leucine zipper; CRE, cAMP response
element; CREB, CRE-binding protein; MAPK, mitogen-activated protein
kinase; PI-3K, phosphatidylinositol 3'-kinase; Ab, antibody; RT-PCR,
reverse transcription-PCR; CAT, chloramphenicol acetyltransferase;
HUVEC, human umbilical vein endothelial cell; MEKK, MAPK kinase
kinase.
Received 10/ 1/99.
Accepted 11/30/99.
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ABSTRACT
Introduction
