From the Department of Biochemistry and Genetics, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, United Kingdom
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ABSTRACT |
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Serum response elements (SREs) play important
roles in transforming extracellular signals into specific nuclear
responses. The SRE-binding protein, serum response factor (SRF), plays
a pivotal role in this process. Several transcription factors have been
shown to interact with SRF and thereby create distinct complexes with
different regulatory potentials. The ETS domain transcription factor
Elk-1 is one such protein and serves to integrate distinct mitogen-activated protein kinase cascades at SREs. Elk-1 uses a short
hydrophobic surface presented on the surface of an -helix to
interact with SRF. In this study we have used site-directed mutagenesis
to identify residues in SRF that comprise the Elk-1 binding surface.
The Elk-1 binding surface is composed of residues that lie on a
hydrophobic surface-exposed groove located at the junction of the MADS
box and C-terminal SAM motif. Different residues are implicated in
interactions between SRF and the transcription factor Fli-1, indicating
that although some overlap with the Elk-1 binding surface occurs, their
interaction surfaces on SRF are distinct. Our data are consistent with
the hypothesis that the SRF DNA-binding domain acts as docking site for
multiple transcription factors that can bind to small surface-exposed
patches within this domain.
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INTRODUCTION |
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The serum response element (SRE)1 within the c-fos promoter has been studied extensively and has developed into a paradigm for how immediate-early genes are regulated. The SRE is bound by the serum response factor (SRF) both in vitro and in vivo (reviewed in Refs. 1-3) and thus plays a pivotal role in regulating transcriptional activation of the c-fos promoter via this element. Multiple diverse extracellular signals are transduced through the SRE including both mitogenic stimuli (e.g. epidermal growth factor) and stress stimuli (e.g. UV light) (reviewed in Ref. 3). At least two alternative pathways exist to transduce these signals via SRF (4, 5). One pathway goes through a ternary complex composed of the SRE, SRF, and a member of the ternary complex factor (TCF) subfamily of ETS domain transcription factors (reviewed in Refs. 2 and 3). This pathway is responsive to the majority of mitogenic and stress stimuli. The second pathway acts via a TCF-independent mechanism and is responsive to alternative serum constituents such as lysophosphatidic acid and upstream activators such as the Rho family of GTPases (6, 7).
The TCF-dependent pathways have been extensively studied.
In this complex, the TCF component is the recipient of signals
transduced through the mitogen-activated protein kinase cascades (Refs.
8-13 and reviewed in Refs. 14 and 15). The TCFs use both protein-DNA (by their ETS domain) and protein-protein interactions to bind to the
binary SRF·SRE complex (reviewed in Ref. 2) as predicted by the
"grappling hook" model (1). A short region of the TCF Elk-1 (the B
box) has been demonstrated to be sufficient for interaction with SRF
(16). Upon binding to SRF, this region is thought to adopt an inducible
-helical structure in which hydrophobic amino acids play a major
role (17). The minimum region of SRF required for this interaction with
the TCFs in vitro maps to its minimal core DNA-binding
domain (coreSRF) (18). Experiments using chimeric
transcription factors have localized the TCF binding surface to the
C-terminal half of this domain (18, 19). Moreover, the core domain is
sufficient for correct dimerization, sequence-specific DNA binding, and
protein-induced DNA bending by SRF (reviewed in Refs. 2 and 20).
Recently the structure of this domain has been solved (21) and shown to
adopt a novel fold in which the N-terminal portion (MADS box) makes all
the DNA contacts. Dimerization is mediated by elements within the MADS
box and additional contributions from the C-terminal extension (the SAM
motif).
In addition to binding TCFs, the core DNA-binding domain of SRF is
sufficient to form DNA complexes with or bind directly to other
transcription factors including myogenic basic helix-loop-helix proteins (22), the ETS-domain protein Fli-1 (23, 24) and p65/NF-B
(25). Moreover, a higher order complex involving SRF, Phox, and
TFII-I/SPIN and the c-fos SRE has also been identified (26).
These diverse transcription factors show little homology with the Elk-1
B box, implying that they use alternative interaction motifs and
different surfaces on SRF.
The aim of this study was to identify the TCF interaction surface on SRF. The recent elucidation of the structure of the minimal DNA-binding domain of SRF (21) permits both improved experimental design and interpretation of the biochemical data. By using an extensive mutagenic approach, we have identified a surface-exposed patch on SRF which is involved in interactions with Elk-1. The transcription factor Fli-1 interacts with a different surface on SRF which overlaps the Elk-1 binding region. Taken together our data are consistent with a model in which the SRF DNA-binding domain acts as a docking site with interaction surfaces for multiple transcription factors involved in diverse cellular processes.
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MATERIALS AND METHODS |
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Plasmid Constructs-- The following plasmids were constructed for making proteins by in vitro transcription/translation. pAS197 encodes the ETS DNA-binding domain and B box of Elk-1 (Elk1-168; amino acids 1-168) (17). pAS3052 encodes the N-terminally truncated zebrafish Fli-1 protein, Fli-219-451 (Fli-1 amino acids 219-451). pT3G-SRF encodes the DNA-binding domain of SRF (coreSRF; amino acids 132-222) (18).
pAS30 (Y195D), pAS31 (F197D) (28), pAS432 (V187A), pAS626 (S189A), pAS627 (E190A), pAS640 (H193A), pAS641 (T191A/H193A), pAS431 (V194A), pAS430 (T196A), pAS625 (V194A/T196A), pAS493 (T199A), pAS629 (R200A), pAS642 (T196A/R200A), pAS646 (Q203A/T207A), pAS429 (K201A), pAS643 (V194A/K201A), pAS494 (L202A), pAS630 (Q203A), pAS428 (P204A), pAS427 (I206A), pAS495 (T207A), pAS644 (T196A/T207A), pAS635 (V194E), pAS491 (T196E), pAS492 (T196K), pAS628 (A198D), pAS632 (T199D), pAS637 (Q203E), pAS636 (I206D), pAS631 (T207D), pAS634 (T210D), and pAS633 (Q216E) encode mutant derivatives of coreSRF from pT3G-SRF with the indicated site-directed mutations. pAS433, pAS434, pAS435, pAS439, and pAS52 encode point mutant derivatives of METcoreSRF from pAS37 (29) with site-directed mutations L155A, L155E, Y158A, T166A, and K154E/V144K, respectively. Mutations were introduced by a two-step PCR protocol using a mutagenic primer and two flanking primers as described previously (30). pAS489 encodes Elk-1-168 with the mutation L158P and was also constructed using this two-step PCR protocol. Details of mutagenic primers can be supplied upon request. The following plasmids were used for protein expression in Escherichia coli. pAS77 encodes the GST-B box fusion (Elk-1 amino acids 139-168) (16). pAS58 encodes GSTcoreSRF (SRF amino acids 132-222) (16). Plasmids encoding the mutant GSTcoreSRF proteins E190A (pAS647), H193A (pAS656), T196A (pAS660), V194E (pAS650), Y195D (pAS651), T196K (pAS652), and Q203E (pAS653) were constructed by replacement of the EcoRI fragment in pAS58 with a PCR product (COR3 C-terminal primer; Ref. 16) derived from the corresponding pBluescript-based plasmids described above. pAS76 contains the c-fos SRE cloned into the circular permutation vector pBEND2 (20).Protein Production-- In vitro transcription and translation of SRF, Elk-1, and Fli-1 derivatives were carried out with TNTTM-coupled reticulocyte lysate system (Promega) according to the manufacturer's recommendations. 35S-Labeled proteins were analyzed by electrophoresis through 0.1% SDS-12% polyacrylamide gel electrophoresis before visualization and quantification of bands representing intact proteins by phosphorimaging (Fuji BAS-1500 phosphorimager and TINA 2.08e software).
GST fusion proteins were purified from E. coli as described previously (16). The purity and concentration of protein samples were estimated by SDS-polyacrylamide gel electrophoresis.Gel Retardation and GST Pull-down Assays-- Pull-down assays for protein-protein interactions with GST-B box and GSTcoreSRF derivatives were carried out essentially as described previously (16).
Gel retardation assays were performed in 12-µl reaction volumes as described previously (28). Assays were carried out on the c-fos SRE (oligonucleotides ADS134 and ADS135; top strand, 5'-CTAGCTTACACAGGATGTCCATATTAGGACATCTGCGTCAGCAGG-3') or the CECI site (oligonucleotides ADS341 and ADS342; top strand, 5'-CGCGTGAGCCGGAAATGTGATCAACTATTTATAGATA-3'). The CArG box SRF-binding sites are underlined, and the Elk-1-binding sites (ets motifs) are shown in bold. The CECI site (combined ets and CArG) consists of the S30 ets motif identified by site selection (31) and the N10 site (29). Elk-1 binds to the ets motif in this site with higher affinity than to the c-fos SRE (31). Protein-DNA complexes were resolved by electrophoresis through nondenaturing 5% polyacrylamide (30% acrylamide, 0.8% bisacrylamide) gels cast in either 0.5× Tris/borate/EDTA (TBE) or 1× TBE for circular permutation assays. Relative DNA binding affinities were calculated by phosphorimager analysis of protein-DNA complexes (Fuji BAS1500; TINA 2.08e software). Experiments were carried out to achieveCircular Permutation Analysis--
For circular permutation
analysis, DNA fragments were produced by appropriate restriction enzyme
digestion of PCR products derived from pAS76 (containing the SRE) and
purified as described previously (20, 32). Curve fitting and apparent
DNA bend angles were calculated as described previously (32). Bend
angles are quoted as the average of three independent experiments.
Standard deviations (n 1) of bend angles are in the
range 0.5-1.6°. To show direct visual comparisons of data obtained
from proteins that give rise to complexes of differing mobilities, the
data were normalized for the fastest mobility complex
(µM) (32).
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RESULTS |
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Experimental Rationale-- A mutagenic approach was used to identify residues in SRF that are involved in binding Elk-1. Amino acids were either changed to alanines (removing potential interacting side chains) or to charged residues. The latter approach relies on introducing amino acids that will disrupt protein-protein contacts and therefore the residues need not be in direct contact with Elk-1 but might instead be in very close proximity to residues comprising the binding surface. Both these approaches have been used successfully in independent studies with virtually identical results to map surface residues on TATA binding protein which bind to components of the transcription preinitiation complex (33, 34). The majority of the mutations introduced were at single amino acid residues. Individual residues in the core DNA-binding domain of SRF could have multiple roles in addition to protein-protein interactions including DNA binding, dimerization, or protein-induced DNA bending. A combination of assays were therefore used to assess the function of mutant proteins in both the presence and absence of DNA.
Mutations were introduced into the minimal DNA-binding domain of SRF (coreSRF) and tested for binding to the truncated Elk-1 protein (Elk-1-168) which contains the ETS DNA-binding domain and the B box SRF-binding motif. These truncated proteins are sufficient for complex formation in both the presence and absence of DNA (16, 17). The results of these assays are presented below and summarized in Table I.
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DNA Binding by SRF Mutants-- Initially the mutant SRF proteins were tested for their ability to bind to the CArG (CC{A + T rich}6GG) box in the c-fos SRE. The majority of the mutant proteins bound the SRE with similar efficiency to the wild-type protein (Fig. 1; Table I). However, the mutants V194A/T196A, T199A, L202A, and T199D exhibited a reduced affinity for the SRE (Fig. 1, lanes 8, 9, 14, and 27). Binding of the mutants P204A, F197D, A198D, and I206D was severely reduced and not detectable by this assay (Fig. 1, lanes 16, 25, 26, and 29).
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Ternary Complex Formation with Elk-1 and CArG Boxes-- The ability of the mutant SRF proteins to form ternary DNA-bound complexes with Elk-1 and the c-fos SRE was tested by gel retardation analysis. Protein-protein interactions with SRF are essential for efficient recruitment of Elk-1 into ternary complexes on this site (16). Assays were carried out using concentrations of SRF that give equal amounts of binary SRF·SRE complex. The efficiency of ternary complex formation was virtually identical for all the mutant SRF proteins tested (Fig. 2B) with the exception of the mutants V194E, T196E, and T196K which exhibited a reduced efficiency of complex formation (Fig. 2B, lanes 19, 21 and 22). In the case of V194E no ternary complex was detectable.
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Protein-Protein Interactions between Elk-1 and SRF Mutant Proteins in the Absence of DNA-- Direct protein-protein interactions between SRF and Elk-1 were investigated using the GST pull-down assay. Although Elk-1 and SRF are usually found as DNA-bound complexes, complexes can be detected in vitro in the absence of DNA using this assay (16). First, SRF mutants were translated in vitro as 35S-labeled proteins and tested for binding to an immobilized protein consisting of GST fused to the Elk-1 B box (GST-B box) (16). Initially, a series of SRF derivatives with mutations in the N terminus of the MADS box were tested. However, none of the tested mutations reduces the binding of SRF to the B box (Table I; data not shown). Further SRF derivatives were tested that harbor mutations in the C-terminal end of the MADS box or within the SAM domain. Of the alanine substitution mutants, the single mutants E190A, T199A, and P204A (Fig. 4A lanes 3, 9, and 16; Fig. 4B) and the double mutants V194A/T196A, T196A/T207A, and Q203A/T207A (Fig. 4A lanes 8, 19, and 21; Fig. 4B) display large decreases in binding to GST-B box (<50% wild-type binding). The residues Val-194, Thr-196, and Gln-203 probably play the major roles in the reductions observed in the double alanine mutants as single mutations in these residues also cause reductions in SRF binding to GST-B box (Fig. 4A; lanes 6, 7, and 15; Fig. 4B). Furthermore, the mutant protein I206A shows a moderate reduction in binding the Elk-1 B box (Fig. 4B, lane 7; 51% WT). The lack of binding by the P204A mutant is probably due to a structural change in SRF (see "Discussion"). However, the residues Glu-190, Val-194, Thr-196, Thr-199, and Ile-206 are directly implicated in binding to Elk-1 which correlates well with their proposed role in ternary complex formation on the CECI site (Fig. 3).
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Protein-induced DNA Bending Is Unaltered in SRF Mutant Proteins-- SRF-mediated DNA bending is observed in the ternary SRF·Elk-1·SRE complex (20). The magnitude of protein-induced DNA bending may affect the ability of SRF to recruit Elk-1 into ternary complexes. Several residues in SRF have been implicated in protein-induced DNA bending (32), including His-193 which is located in the vicinity of several residues investigated in this study (Fig. 8; see Ref. 21). To rule out an effect of the important residues identified in this study on protein-induced bending and hence ternary complex formation, the DNA-bending properties of several mutant proteins were investigated using the circular permutation assay. In this assay, changes in the mobility of protein-DNA complexes as the binding site location is moved with respect to DNA ends is indicative of protein-induced DNA bending. Moreover, this assay has the added advantage in that gross changes in protein structure would also become apparent.
Wild-type SRF induces an apparent DNA bend of 72° (Fig. 6A; 32). Similarly, the SRF mutants V194A/T196A, V194E, and T196E induce apparent DNA bends of comparable magnitude (68°-70°; Fig. 6, B-D). These mutant proteins also bind DNA with a similar efficiency to wild-type SRF (Fig. 1). Collectively, these results demonstrate that DNA binding by the SRF mutants V194A/T196A, V194E, and T196E is unaltered and that the differences in ternary complex formation with Elk-1 (Figs. 2 and 3) and Fli-1 (see below) are attributable to changes in direct protein-protein interactions.
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Complex Formation with the Transcription Factor Fli-1--
In
addition to Elk-1, the core DNA-binding domain of SRF has also been
demonstrated to form complexes with p65/NFB (25) and Fli-1 (23, 24).
The series of SRF mutant proteins were tested for interaction with
these transcription factors to determine whether their interaction
surfaces on SRF differ. First, interactions between p65/NF
B and the
SRF mutant proteins were analyzed by the GST pull-down assay in which
the DNA-binding domain of p65/NF
B was immobilized as a GST fusion
protein. In contrast to the reductions in binding to the Elk-1 B box
observed in several of the mutant SRF proteins (Fig. 4), no large
decreases in binding to p65/NF
B were observed (data not shown).
These results imply that different residues comprise the p65/NF
B and
Elk-1 binding surfaces on SRF.
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DISCUSSION |
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Protein-protein interactions play a key role in the formation of
the ternary nucleoprotein which forms between SRF, Elk-1, and the
c-fos SRE (Refs. 16, 18, and 19 and reviewed in Ref. 2). The
interaction surface on Elk-1 is provided by residues in the conserved B
box which is predicted to inducibly form an -helix upon interaction
with SRF (17). In this study we have used site-directed mutagenesis to
identify residues which comprise the Elk-1 binding surface on SRF.
The Elk-1 Binding Surface--
Residues in SRF were mutated to
either alanine or charged residues. A reduction of interaction upon
introduction of an alanine residue was taken as evidence for a direct
role of the amino acid in binding to Elk-1. Moderate effects by
introduction of charged residues suggest that the residues are close to
(but not necessarily part of) the Elk-1-binding motif, whereas severe
effects indicate that the residues are probably within the binding
surface. Based on these criteria, Glu-190, Val-194, Thr-196, Thr-199,
and Ile-206 are identified as residues that are important in forming
the binding surface, and Tyr-195, Gln-203, and Thr-207 are located
close to the binding surface. Significantly, these residues form a
continuous surface-exposed patch, whereas residues that are not thought
to be part of this surface (including Thr-210, Gln-216, Val-187, Ser-189, Arg-200, and Lys-201) are located outside this patch (Fig.
8, B-D). Other
residues such as Ile-206 whose mutation causes moderate changes in
Elk-1 binding are located close to this patch (Fig. 8E). The
total loss of Elk-1 binding exhibited by the mutant V194E is consistent
with the central location of this residue in the binding surface (Fig.
8, C and D). This surface-exposed patch is
predominantly hydrophobic in character (Fig. 8B) and is
consistent with the observation that the residues in Elk-1 which play
important roles in SRF binding are hydrophobic in character and are
predicted to form a hydrophobic surface presented on the face of an
-helix (17). Together, our data are therefore consistent with a
model in which protein-protein interactions between Elk-1 and SRF are
mediated by the interaction of the hydrophobic surface of an
-helix
formed by the Elk-1 B box and a hydrophobic patch on the surface of
SRF. Recently, the structure of the related yeast MADS box protein Mcm1
in a ternary complex with DNA-bound MAT
2 has been solved (35). In
this complex, a surface-exposed hydrophobic groove forms the binding
surface for MAT
2. One end of this groove is centered on Val-69 with
other defining residues including Phe-72 and Thr-74 toward the opposite
end of the groove. The analogous residues in SRF (Val-194, Phe-197, and
Thr-199) all lie within the Elk-1 binding surface (Fig. 8,
B-D). Furthermore, MAT
2 inserts a
phenylalanine residue into the hydrophobic pocket surrounding Val-69.
As three aromatic residues in Elk-1 are critical for interaction with
SRF (17), it is tempting to speculate that one of these residues
occupies a similar position in the hydrophobic pocket surrounding
Val-194 in SRF. This scenario is consistent with the severe reduction
in Elk-1 binding caused by the introduction of a charged residue in
place of Val-194.
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Binding Surfaces for Other Transcription Factors--
Fli-1 (23,
24) and p65/NFB (25) can both form complexes with SRF although in
the former case, the biological significance is unknown. Testing of
these transcription factors in addition to Elk-1 for binding to the
panel of SRF mutants identified residues that appear to have different
roles in binding each protein, e.g. Thr-199 (Elk-1) and
Thr-196 (Fli-1). In contrast, other residues (e.g. Glu-190
and Val-194) play key roles in binding both Elk-1 and Fli-1. None of
the mutations caused dramatic reductions (or enhancements) in
interaction with p65/NF
B, indicating that the tested residues either
play different roles or that different residues comprise the p65/NF
B
binding surface. The observation that these proteins interact using
different surfaces is consistent with the observation that neither
Fli-1 nor p65/NF
B exhibits strong homology to the B box SRF
interaction motif of Elk-1. Further studies are required to fully
define the interaction epitopes of Fli-1 and p65/NF
B, but it is
interesting to note that Val-194 appears to form an important part of
each interaction surface and is associated within a hydrophobic cleft
in the protein (Fig. 8E). Furthermore, removal of the side
chain of the neighboring residue Thr-196 significantly enhances
(5-10-fold; Fig. 7; Table I) the formation of complexes between Fli-1
and SRE-bound SRF. This mutation removes a hydrophilic side chain and
increases the size of the hydrophobic groove on the surface of SRF
(Fig. 8, B and E). One possible role for this
residue may be to reduce potential interactions with Fli-1 in
vivo. Further experiments are required to address the possible
physiological significance of this observation.
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ACKNOWLEDGEMENTS |
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We thank Margaret Bell and Catherine Pyle for excellent technical and secretarial assistance and Bob Liddell for DNA sequencing and oligonucleotide synthesis. We are grateful to members of our laboratory for stimulating discussions and comments on the manuscript. We are grateful to Neil Perkins for reagents and Song Tan and Tim Richmond for communicating data prior to publication.
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FOOTNOTES |
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* This work was supported by the North of England Cancer Research Campaign and Medical Research Council Studentships (to A. G. W. and E. C. R.).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: Marie Curie Research Institute, The Chart, Oxted,
Surrey, RH8 OTL, United Kingdom.
§ Research Fellow of the Lister Institute of Preventative Medicine. To whom correspondence should be addressed. Tel.: 0044-191 222 8800; Fax: 0044-191 222 7424; E-mail: a.d.sharrocks{at}ncl.ac.uk.
1 The abbreviations used are: SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor; PCR, polymerase chain reaction; GST, glutathione S-transferase; SAM, SRF, ArgRI, Mcm1; MADS, Mcm1, ArgRI/AG, DEFA, SRF.
2 A. L. Brown, T. F. Schilling, A. Rodaway, T. Jowett, P. W. Ingham, R. Patient, and A. D. Sharrocks, submitted for publication.
3 E. C. Roberts, unpublished data.
4 Y. Ling and A. D. Sharrocks, unpublished data.
5 A. D. Sharrocks, unpublished data.
6 A. G. West and A. D. Sharrocks, unpublished data.
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REFERENCES |
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