From the Department of Biology and Molecular Biology, Institute, San Diego State University, San Diego, California 92182
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ABSTRACT |
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In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.
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INTRODUCTION |
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The activation of signal transduction pathways by growth factors stimulates transcription of certain genes (e.g. c-fos) that participate in the growth response. Among the regulatory regions in the promoter of the well studied c-fos gene that are responsible for conferring growth factor inducibility is the serum response element (SRE)1 (1). The c-fos SRE, which is comprised of a core sequence (CC(A/T)6GG) and a nearby Ets motif ((C/A)(C/A)GGA(A/T)), binds serum response factor (SRF) over the A/T-rich core sequence (2), as well as the ternary complex factors (TCF), Elk-1 or Sap-1, over the Ets motif. SRF and TCFs interact with each other as well their cognate DNA binding elements to form a ternary complex (3, 4). Growth factor-activated signal transduction pathways often converge on either the SRF or the TCF components of the ternary complex (1, 5). For example, all three MAP kinases, extracellular signal-regulated kinase (ERK), N-terminal jun kinase (JNK), and p38, phosphorylate the TCF, Elk-1, as part of the mechanism by which c-fos transcription is activated through the SRE (6). Although SRF itself is phosphorylated as a result of the activation of certain signaling pathways (e.g. pp90rsk and Ca/CaMKIV; Refs. 7 and 8), and this phosphorylation leads to increased c-fos transcription, there is no evidence that any of the MAP kinases can influence transcription by directly phosphorylating SRF. Accordingly, it is widely believed that MAP kinase-stimulated transcriptional induction through the c-fos SRE is dependent on Elk-1-, Sap-1-, or as yet unidentified TCFs.
In contrast to the c-fos promoter there are a number of
genes that possess critical SREs which do not have flanking- or nearby Ets motifs and therefore are not likely to bind Ets-related TCFs (4,
9-11); among them are several cardiac-specific genes that are induced
as part of the well studied hypertrophic growth program. For example,
the skeletal -actin (12, 13), cardiac
-actin (14), and atrial
natriuretic factor (ANF) genes (9) all possess such SREs that are
critical for induction by growth factors. Recent studies have shown
that the p38 pathway plays an important role in conferring growth
factor inducibility to these cardiac genes (15, 16). Accordingly,
induction by p38 presumably involves the SREs that are required for
growth factor-stimulated transcription. However, the only mechanism
presently known by which p38 can activate transcription through SREs
requires phosphorylation of Elk-1 by p38 (6). Thus, it is unclear how
p38 could enhance transcription through SREs in cardiac genes in an
Ets/TCF-independent manner. To address this question we used the ANF
gene in cardiac myocytes as a model system and asked first whether p38
pathway signals converge on the ANF SRE to confer inducibility and if
so, through what mechanism.
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MATERIALS AND METHODS |
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Cell Culture-- Primary ventricular myocytes, prepared from 1-4-day-old neonatal rats as described previously (9, 15, 17), were transfected (see below), plated onto fibronectin-coated plastic dishes, and then maintained for about 18 h in DMEM, 10% fetal bovine serum. The cultures were then washed briefly with medium, refed with serum-free, hormone-free DMEM containing 1 mg/ml bovine serum albumin, maintained for an additional 48 h and then extracted for reporter enzyme assays.
Transfections--
After isolation, myocardial cells were
resuspended at a density of 30 million cells/ml minimal medium (DMEM
(Sigma) containing 1 mg/ml bovine serum albumin) and transfections were
carried out as described previously (9, 15, 17). Briefly, for each transfection, 300 µl, or 9 million cells, were mixed with 15 to 30 µg of a reporter construct (e.g. ANF/Luc (9) or pG5E1bLuc (1, 18), 12 to 18 µg of pCH110 (SV40--galactosidase), which was
used for normalization, and in some experiments, 15 to 45 µg of an
MKK6, ATF6/Gal4, or Gal4 DNA-binding domain expression construct (see
below). The levels of plasmid used in each culture within an experiment
were equalized using empty vector DNA, such as pCMV6. Each 300-µl
aliquot was then electroporated in a Bio-Rad Gene Pulser at 500 V, 25 microfarads, 100
in a 0.2-cm gap cuvette, a protocol that allows
for the selective transfection of only cardiac myocytes (9, 15, 17).
This procedure results in an approximate 30% viability (9);
accordingly, the 3 million viable cells were plated into
fibronectin-coated 35-mm wells.
Reporter Enzyme Constructs--
The ANF/luciferase reporter
constructs used in this study include ANF 134 and ANF 65, which are
composed of ANF (134 to +65) or ANF (
65 to +65) driving luciferase
expression, respectively. ANF 134 is inducible, while ANF 65 displays
mostly basal promoter activity; both constructs display myocardial
cell-specific expression (9). The structures of ANF 65 (c-fos SRE), ANF 65 (ANF SRE), ANF 65 (M
c-fos SRE), and ANF 65 (M ANF SRE) are described
in Fig. 1. In each case the relevant synthetic oligonucleotide pairs (see below) were hybridized, ligated to form trimers, and cloned upstream of the minimal, cardiac-specific ANF promoter (ANF 65), as
described previously (9).
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Test Expression Constructs--
The following test expression
constructs were used: pCDNA3 MKK6 (Glu) (codes for activated MKK6,
also known as p38 MAPKK; from R. Davis, University of Massachusetts,
Worcester, MA), pCDNA3 MKK6 (K71R) (codes for a kinase-dead form of
MKK6; from R. Davis), RSV 3pK (Glu/Glu) (codes for a constitutively
active form of MAPKAP-K, or 3pK; from S. Ludwig, Wurzburg, Germay),
pCDNA3 p38 (codes for the native human p38; from J. Han, the
Scripps Institute, La Jolla, CA), pCGN AS-ATF6 (codes for an antisense
fragment of ATF6; Ref. 18), pSG424 ATF6(670)/Gal4 (codes for the
full-length, 670-amino acid ATF6 fused to the Gal4 DNA-binding domain;
prepared in the C. Glembotski laboratory as part of this study), pSG424 ATF6(1-273)/Gal4 (codes for the N-terminal 273 amino acids of ATF6
fused to the Gal4 DNA-binding domain; prepared in the C. Glembotski
laboratory as part of this study), pSG424 Gal4 (codes for the Gal4
DNA-binding domain; from M. Karin, University of California, San Diego,
La Jolla, CA), pG5E1bLuc (codes for 5X Gal4 sites cloned upstream of a
prolactin promoter driving luciferase expression; from R. Davis), pDCR
RacVal-12 (codes for activated Rac; from M. Cobb and J. Frost, University of Texas Southwestern Medical Center, Dallas, TX),
pCMV5
MEKK4 (codes for constitutively active
MEKK4;
from G. Johnson, University of Colorado, Denver, CO), pRK5
cdc42Leu-61 (codes for constitutively active cdc42; from A. Hall, University College London, London, United Kingdom). Preliminary
experiments using different concentrations of each construct verified
that optimal doses were chosen.
Reporter Enzyme Assays--
Transfected cells were maintained in
DMEM supplemented with 10% fetal bovine serum for approximately
16 h after electroporation. The cells were then washed thoroughly
and the medium was replaced with minimal medium. Unless otherwise
stated, 24 to 48 h later the cultures were extracted for reporter
enzyme assay. Luciferase and -galactosidase assays were performed as
described (9). Luciferase activity was measured for 30 s on a Bio
Orbit 1251 Luminometer (Pharmacia Biotech Inc., Piscataway, NJ). Data
are expressed as "Relative Luciferase (Rel Luc)" = arbitrary
integrated luciferase units/
-gal units, representative of at least
three independent experiments performed with two different plasmid
preparations, and represent the mean and S.E. of triplicate
cultures.
Preparation of Recombinant ATF6 and SRF-- The full-length cDNAs for human ATF6 (18) and SRF, the latter of which was obtained from E. Olson, University of Texas, were subcloned into pcDNA3.1 and used as templates for in vitro transcription/translation using TNT® T7 Quick Coupled Transcription/Translation System (Promega) ± [35S]Met, as per the manufacturer's instructions. The identities of the products were confirmed by observing the mobilities of the [35S]Met-labeled products after SDS-PAGE and by immunoprecipitating 35S-Met-labeled products with the anti-ATF6 (19) or anti-SRF (20) antisera followed by SDS-PAGE.
Electrophoretic Mobility Shift Assays-- EMSA was performed as described (9), with minor modifications. ANF SRE and MEF2C probes were prepared by T4 kinase end labeling of the relevant oligonucleotide pairs (see above). A typical binding assay contained 20,000 cpm of double-stranded probe and 5 to 10 µg of protein in 1 × binding buffer (30 mM NaCl, 0.1 mM EDTA, 8 mM Tris-HCl (pH 8.2), 8% glycerol, 1 nM dithiothreitol, 0.2 mM ZnCl2). After a 10-min preincubation of extract and 0.2 µg of nonspecific competitor (poly(dI-dC), Pharmacia Biotech Inc.) and test competitor oligonucleotide pairs, as indicated, the probe was added. Binding was allowed to proceed at room temperature for 30 min prior to separation of bound and free probe on a 4% native polyacrylamide (PA) gel (29:1 bis/acrylamide) high ionic strength buffer (50 mM Tris (pH 8.5), 380 mM glycine, 2.1 mM EDTA) at room temperature. DNA-protein complexes were detected by autoradiography. The autoradiograms of some gels in this article were scanned using a Molecular Dynamics Personal Densitometer and the resulting image was imported to Adobe Photoshop and Claris MacDraw Pro II for final figure preparation.
In Vitro Phosphorylation by p38 and MAPKAP
Kinase-3--
In vitro phosphorylation reactions with p38
and MAPKAP kinase-3 were carried out essentially as described
previously (21-23). Briefly, approximately 1 µg of either the active
form of p38 (obtained from Dr. J. Han) or the active form of MAPKAP
kinase-3 (obtained from Dr. Stephan Ludwig) were mixed with recombinant
ATF6, MEF2C, or HSP27 (StressGen), in 30 µl of kinase buffer (21) and
[-32P]ATP and allowed to incubate at 30 °C for 30 min. Approximately 1 µl of antisera specific for the protein in
question was then added to each reaction mixture and allowed to
incubate for 4 h at 4 °C after which protein A-Sepharose beads
were used to collect immune complexes, as described previously (15).
Immune complexes were then eluted from the beads using Laemmli sample
buffer and the eluted material analyzed by SDS-PAGE followed by
PhosphorImager analysis, as described in the legend to Fig. 3. In some
cases, the recombinant proteins were prepared in the presence of
[35S]methionine allowing them to be used as markers on
parallel lanes of a gel.
ERK, JNK, and p38 Assays--
For ERK and JNK assays, cultures
were treated for 30 min with control medium, or phenylephrine (10 µM), serum (10%), phorbol ester diburyate (100 nM), or endothelin-1 (10 nM), then extracted in
10 mM Tris (pH 7.6), 1% Triton X-100, 0.05 M
NaCl, 5 mM EDTA, 2 mM sodium
o-vanadate, and 20 µg/ml aprotinin. After brief
centrifugation, extracts to be tested for ERK activity were incubated
for 2 h at 4 °C with anti-ERK (raised against the C-terminal 16 amino acids of ERK-1; Santa Cruz SC-093) bound to Protein A-Sepharose (Pharmacia) and immune-complex kinase assays were carried out using the
appropriate substrates, as described previously (8, 15, 24). Briefly,
reactions were initiated by the addition of 1 µg of myelin basic
protein and 6 µM [-32P]ATP (5000 Ci/mmol) in a final volume of 30 µl of kinase buffer (20 mM HEPES (pH 7.4), 20 mM MgCl2, 20 mM
-glycerophosphate, 2 mM dithiothrietol,
20 µM ATP). After 30 min at 25 °C, the reactions were
terminated by the addition of Laemmli sample buffer and the phosphorylation level of substrate proteins was evaluated by SDS-PAGE followed by autoradiography and PhosphorImager analyses.
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RESULTS |
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Mapping p38-inducible Elements in the ANF Promoter--
To begin
determining how the p38 MAPK pathway participates in cardiac gene
induction, p38-inducible elements in the rat ANF promoter were mapped.
Primary myocardial cells were co-transfected with ANF
promoter/luciferase reporter plasmids (Fig.
1A) and a construct encoding
MKK6 (Glu), an activated form a p38 MAP kinase kinase (18, 21) which,
among the MAPKs, activates only p38 in cardiac myocytes (15, 16). In
previous studies it has been shown that nearly all the information
for optimal hormonal induction of ANF transcription resides in the
134-bp region just 5' of the transcriptional start site (9). Consistent
with those results, transcription from a reporter construct comprised
of the region spanning from 134 to +65 of the rat ANF 5'-flanking
sequence was strongly induced (approximately 6-fold) by MKK6 (Glu)
(Fig. 1B, Construct 1), similar to levels previously seen
using reporters comprised of 638 bp of ANF 5'-flanking sequence (15).
The induction of ANF 134 was completely blocked by SB 203580, a cell
permeable compound shown to selectively inhibit p38 (26). Truncating
ANF 134 by just 70 nt resulted in a construct, ANF 65, which had
completely lost p38 inducibility (Fig. 1B, Construct 3),
indicating that p38-inducible sequences must lie between
134 and
65
in the ANF 5'-flanking sequence.
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SRF and MEF2C Electrophoretic Mobility Shift Analysis-- An obvious candidate for another A/T-rich binding protein that could mediate p38 inducibility is MEF2C. MEF2C, an SRF-related transcription factor (30, 31), is found in high quantities in the heart, binds to A/T-rich sequences in cardiac genes (32, 33), and is required for proper cardiac development and gene expression (32-34). Furthermore, MEF2C is phosphorylated by p38 (22) in a manner that confers transcriptional inducibility in the cardiac context (15).
Thus, since the ANF SRE nearly fits the MEF2C consensus binding site ((C/T)T(A/T)(A/T)AAATA(A/G)), it seemed possible that MEF2C could be responsible for conferring p38-inducible ANF transcription. This hypothesis was investigated initially using electrophoretic mobility shift assays (EMSAs) and recombinant MEF2C or, as a control, recombinant SRF. However, when MEF2C was incubated with radiolabled oligonucleotides, there was no apparent binding to the ANF SRE probe, while the positive control, a muscle creatine kinase probe, bound MEF2C, as expected (34) (Fig. 2A). The competition profile showed that only the MCK MEF2C oligonucleotide was able to compete for MEF2C binding, with no competition obtained with either the c-fos SRE or the ANF SRE. The same result was obtained using ventricular nuclear extracts (not shown). The converse experiment showed that while recombinant SRF could bind specifically to the ANF SRE probe, and while the appropriate competition profile was obtained, SRF did not bind to the MCK MEF2C probe under these conditions (Fig. 2B).
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Phosphorylation Analysis--
Since MEF2C cannot bind to the ANF
SRE, the most likely candidate for conferring p38-mediated induction of
ANF through this element is SRF. This suggests an as yet
uncharacterized mechanism where SRF itself, and/or some other
non-Ets-related accessory protein, serves as the target for p38 and/or
p38-activated kinases. Accordingly, experiments were carried out to
evaluate whether SRF itself could serve as a downstream target for p38.
When recombinant SRF was incubated with p38 in the presence of
[-32P]ATP no apparent phosphorylation was observed
(Fig. 3A, lanes 2 and
3). Accordingly, the possibility that p38, or a downstream kinase, might phosphorylate a non-Ets-related SRF-binding partner was
considered. One such protein is ATF6, a 670-amino acid member of the
b-ZIP, ATF/CREB family recently shown to bind SRF, but not SREs, and to
enhance SRF-mediated transcription (19). Interestingly, when ATF6 was
incubated with p38 it was phosphorylated (Fig. 3A, lanes 6 and 8-10), consistent with a role for ATF6 in p38-mediated transcriptional induction.
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ATF6 Antisense-- To test whether ATF6 is involved in p38-activated transcription through the ANF SRE, an expression construct encoding ATF6 in the antisense orientation was employed. This construct has previously been used in HeLa cells to demonstrate the dependence of serum activation of SRF on ATF6 (19). Myocardial cells were co-transfected with MKK6 (Glu), various levels of the ATF6 antisense construct, and ANF 65 (ANF SRE). Interestingly, the ATF6 antisense construct (ATF6-AS) displayed a dose-dependent ability to inhibit MKK6-activated reporter expression from ANF 65 (ANF SRE) (Fig. 4), however, it had no inhibitory effect on the ability of MKK6 to activate ANF through an isolated Sp1-like element (not shown). These results indicate that overexpression of the ATF6 antisense transcript can inhibit p38-inducible transcription specifically through the ANF SRE, implying a role for ATF6 in this induction process.
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ATF6/Gal4 Trans-activation-- Further studies were carried out to assess whether ATF6 can serve as a p38-inducible transcriptional activator. ATF family members investigated to date commonly possess an N-terminal trans-activation domain, however, such a domain has not been identified in ATF6. Moreover, there is very little homology between ATF6 and other ATF family members in the N terminus of the protein. Accordingly, we tested whether ATF6 could confer transcriptional activation to a DNA-binding protein using a Gal4 trans-activation system (38). An expression construct was prepared wherein the Gal4 DNA-binding domain was fused to either the full-length, 670-amino acid ATF6, creating Gal4/ATF6 (670), or to an N-terminal fragment of ATF6 comprised of amino acids 1-273, creating Gal4/ATF6 (273) (Fig. 5A). Myocardial cells were then co-transfected with Gal4/ATF6 (670), or Gal4/ATF6 (273) and pG5E1bLuc, a luciferase reporter construct possessing five Gal4 upstream activating sequences driving luciferase expression (38), and either an empty vector control (CMV6) or MKK6 (Glu). When fused to the Gal4 DNA-binding domain, both the full-length and truncated forms of ATF6 were able to confer significant enhancement of trans-activation to the relatively inactive Gal4 DNA-binding protein (Fig. 5B, left panel). This result verified that ATF6 operates as a transcription factor in the cardiac context and that it is capable of responding to activation of the p38 MAPK pathway. Consistent with this hypothesis, recombinant ATF6 (273) was phosphorylated by p38 in vitro (Fig. 5B, left panel inset) and the relative levels of phosphorylation of ATF6 (273) and ATF6 (670) correlate with their relative abilities to trans-activate.
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DISCUSSION |
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In response to various stimuli, cardiac myocytes undergo an unusual growth program typified by increased cell size, enhanced sarcomeric organization, and augmented cardiac-specific gene expression, but no cell division. Recently, it was shown that many compounds that promote hypertrohic cardiac myocyte growth stimulate p38 and that the overexpression of MKK6 (Glu), which specifically activates p38 in cardiac myocytes, leads to the replication of all three features of this novel, hypertrophic growth program, including a robust activation of cardiac-specific transcription (15). In the present study it was shown that p38-mediated transcriptional induction of one such cardiac gene (ANF) requires a promoter proximal SRE. Since p38 had recently been demonstrated to phosphorylate and activate the cardiac myocyte-enriched transcription factor MEF2C (22), and since MEF2C was also shown to be activated by p38 in cardiac myocytes (15) and could conceivably bind to the ANF SRE, we first investigated the possibility that it was through MEF2C that p38 enhanced ANF transcription. However, the inability of MEF2C to bind to the ANF SRE led to the alternate hypothesis that p38-mediated trans-activation was SRF-dependent but occurred through an as yet unidentified mechanism.
The recent discovery that a new ATF family member, ATF6, which is present in the heart, binds to SRF and participates in SRF/SRE-enhanced transcription in response to serum (19), led us to evaluate whether ATF6 might serve as a convergence point for p38. When studied in vitro, ATF6 was phosphorylated by p38, while SRF was not. However, MAPKAP kinase-3 (23, 24), a kinase downstream of p38 (23) which is known to phosphorylate another ATF family member, cAMP response element-binding protein, in response to p38 activation (42), was incapable of phosphorylating ATF6 in vitro. Accordingly, it appears as though p38 might augment SRE-mediated ANF induction by directly phosphorylating ATF6. Our preliminary experiments have shown that ATF6 can bind to SRF but does not appear to bind to SREs, indicating that it is probable that SRF serves as an anchoring protein with which ATF6 can partner to confer p38-inducibility to ANF transcription.
To study ATF6 trans-activation in myocardial cells we developed a reporter system that takes advantage of the fact that DNA-binding domains and trans-activation domains of transcription factors can often times be interchanged. Accordingly, expression constructs were prepared which encoded chimeric proteins which possessed the Gal4/DNA-binding domain fused to either the full-length or an N-terminal fragment of ATF6. When these fusion proteins were expressed in cardiac myocytes, along with a luciferase reporter driven by the Gal4 upstream activating sequence, which binds the Gal4/DNA-binding protein, trans-activation by the overexpressed ATF6 could be studied in isolation from endogenous ATF6. It was found that either the full-length (670 amino acids) or the N-terminal 273 amino acids of ATF6 were capable of conferring trans-activation in response to MKK6 (Glu), p38, or several myocardial cell growth factors. This ATF6 trans-activation required kinase-active MKK6 and p38, but was unaffected by constitutively active MAPKAP kinase-3. Interestingly, ATF6 trans-activation was strongly stimulated by upstream activators of the p38 pathway, but was poorly stimulated by the ERK or JNK pathways.
In summary, this study has shown that ATF6 likely plays a key role in the induction of cardiac-specific genes, like ANF, during the hypertrophic growth program. Future studies devoted to determining the roles of ATF6 in the induction of other cardiac genes involved in hypertrophic growth will reveal a great deal about this novel SRF-mediated transcription mechanism. Additionally, it will be of interest to evaluate the roles of ATF6 in non-cardiac cell types in response to growth stimulation, as well as determining how ATF6 and other TCFs, such as Elk-1 or Sap-1, collaborate to regulate transcriptional induction through SREs that possess flanking Ets motifs.
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ACKNOWLEDGEMENTS |
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We thank Drs. Dafa Bar-Sagi, Melanie Cobb, Roger Davis, Jeff Frost, Alan Hall, Jiahuai Han, Gary Johnson, Michael Karin, Stefan Ludwig, Eric Olson, and Inder Verma for sharing reagents.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants NS/HL-25073 (to C. C. G.), HL-46345 (to C. C. G.), HL-56861 (to C. C. G.), and HL-54030 (to P. M. M.) and National Cancer Institute Grant CA 50329 to R. P.).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.
Present address: Dept. of Biological Chemistry, University of
California, Irvine, CA 92697.
§ This work was done during the tenure of a predoctoral research fellowship from the American Heart Association, California Affiliate.
¶ Present address: Dept. of Biological Sciences, Columbia University, 1212 Amsterdam Ave., MC2420, New York, NY 10027.
To whom correspondence should be addressed: Dept. of Biology,
San Diego State University, San Diego, CA 92182. Tel.: 619-594-2959; Fax: 619-594-6200. E-mail: cglembotski{at}sunstroke.sdsu.edu.
The abbreviations used are: SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor; ERK, extracellular signal-regulated kinase: JNK, N-terminal Jun kinase; ANF, atrial natriuretic factor; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; EMSA, electrophoretic mobility shift assay; MAPKAP, mitogen-activated protein kinase; bp, base pair(s).
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REFERENCES |
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