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At least 19 recordsLinked to original sources
PubMed1997

Interaction of ATF6 and serum response factor.

Serum response factor (SRF) is a transcription factor which binds to the serum response element (SRE) in the c-fos promoter. It is required for regulated expression of the c-fos gene as well as other immediate-early genes and some tissue-specific genes. To better understand the regulation of SRF, we used a yeast interaction assay to screen a human HeLa cell cDNA library for SRF-interacting proteins. ATF6, a basic-leucine zipper protein, was isolated by binding to SRF and in particular to its transcriptional activation domain. The binding of ATF6 to SRF was also detected in vitro. An ATF6-VP16 chimera activated expression of an SRE reporter gene in HeLa cells, suggesting that ATF6 can interact with endogenous SRF. More strikingly, an antisense ATF6 construct reduced serum induction of a c-fos reporter gene, suggesting that ATF6 is involved in activation of transcription by SRF. ATF6 was previously partially cloned as a member of the ATF family. The complete cDNA of ATF6 was isolated, and its expression pattern was described.

Activating Transcription Factor 6↗
PubMed1990

Casein kinase II enhances the DNA binding activity of serum response factor.

Serum response factor (SRF) is a mammalian transcription factor that binds to the serum response element in the enhancer of the c-fos proto-oncogene and thus may mediate serum-induction of c-fos transcription. We report here that the DNA binding activity of recombinant SRF made in Escherichia coli can be greatly enhanced by incubation of the protein with HeLa cell nuclear extract. The enhancing activity is ATP or GTP dependent and cofractionates with a protein kinase that phosphorylates SRF on a specific tryptic peptide. Coincubation with phosphatase blocks the enhancing activity, further suggesting that the enhanced binding activity is due to phosphorylation. The specific tryptic phosphopeptide phosphorylated in vitro is also phosphorylated in vivo, demonstrating that this phosphorylation is physiologically important. We have localized the phosphorylation site by a small deletion mutant. Finally, we show that the kinase activity is provided by casein kinase II (CKII) or a close variant. The potential role of CKII as either a regulatory or constitutive modifier of SRF in vivo will be discussed.

Adenosine Triphosphate↗
PubMed1994

Suppression of Ras transformation by serum response factor.

Serum response factor (SRF) is a nuclear transcription factor that binds to the serum response element (SRE) found in the promoter regions of a number of growth factor-inducible genes, as well as muscle-specific genes. The smooth muscle alpha-actin promoter contains two SRE sequences that can bind to SRF. Its expression is repressed in Ras-transformed fibroblast cells and derepressed in revertant cells. In this study, we demonstrate that SRF can activate alpha-actin expression in Ras-transformed cells and that overexpression of SRF in Ras-transformed cells can revert their transformed phenotype. The ability of SRF to bind to the SRE was required for this effect, since mutations that inhibit DNA binding abolish SRF's ability to activate alpha-actin expression and suppress transformation by the ras oncogene. These results show that SRF, thought to be involved in stimulation of cell growth through activation of growth factor-inducible genes, can actually have the opposite effect and suggest a novel mechanism for suppression of transformation by Ras.

Actins↗
PubMed2001

Early postnatal cardiac changes and premature death in transgenic mice overexpressing a mutant form of serum response factor.

Serum response factor (SRF) is a key regulator of a number of extracellular signal-regulated genes important for cell growth and differentiation. A form of the SRF gene with a double mutation (dmSRF) was generated. This mutation reduced the binding activity of SRF protein to the serum response element and reduced the capability of SRF to activate the atrial natriuretic factor promoter that contains the serum response element. Cardiac-specific overexpression of dmSRF attenuated the total SRF binding activity and resulted in remarkable morphologic changes in the heart of the transgenic mice. These mice had dilated atrial and ventricular chambers, and their ventricular wall thicknesses were only 1/2 to 1/3 the thickness of that of nontransgenic mice. Also these mice had smaller cardiac myocytes and had less myofibrils in their myocytes relative to nontransgenic mice. Altered gene expression and slight interstitial fibrosis were observed in the myocardium of the transgenic mice. All the transgenic mice died within the first 12 days after birth, because of the early onset of severe, dilated cardiomyopathy. These results indicate that dmSRF overexpression in the heart apparently alters cardiac gene expression and blocks normal postnatal cardiac growth and development.

Age of Onset↗
PubMed2001

Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor.

Serum response factor (SRF) regulates transcription of numerous muscle and growth factor-inducible genes. Because SRF is not muscle specific, it has been postulated to activate muscle genes by recruiting myogenic accessory factors. Using a bioinformatics-based screen for unknown cardiac-specific genes, we identified a novel and highly potent transcription factor, named myocardin, that is expressed in cardiac and smooth muscle cells. Myocardin belongs to the SAP domain family of nuclear proteins and activates cardiac muscle promoters by associating with SRF. Expression of a dominant negative mutant of myocardin in Xenopus embryos interferes with myocardial cell differentiation. Myocardin is the founding member of a class of muscle transcription factors and provides a mechanism whereby SRF can convey myogenic activity to cardiac muscle genes.

Amino Acid Sequence↗
PubMed2001

Cardiomyopathy in transgenic mice with cardiac-specific overexpression of serum response factor.

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

Actins↗
PubMed2003

Model of functional cardiac aging: young adult mice with mild overexpression of serum response factor.

Serum response factor (SRF) is an important transcription factor that may have a role in the maintenance of cardiac structure and function. The level of SRF mRNA expression increases approximately 16% in the hearts of mice during adult aging. To model the effect of mild SRF elevation in the aging heart, transgenic mice with low levels of SRF overexpression were generated. By 6 mo of age, the transgenic mice had a 19% increase of heart-to-body weight ratio compared with nontransgenic mice. In addition, they had a 12% increase in myocyte size, a 6.7% increase in collagen deposition, and altered gene expression of a number of muscle-specific and cardiac genes. Doppler echocardiography revealed that these transgenic mice had increased left ventricular wall thickness and decreased left ventricular (LV) volumes, increased LV stiffness with 20% reduction in early diastolic LV filling (peak E), and 35% decline in peak E-to-peak A (late diastolic filling) ratio. The observed changes, especially those in the E/A ratio, are similar to those seen clinically in late life as a part of human adult myocardial aging.

Aging↗
PubMed2003

Inhibition of the cardiac alpha-actin gene in embryonic cardiac myocytes by dominant-negative serum response factor.

Serum response factor (SRF), a transcription factor ubiquitously involved in the processes of cellular proliferation and differentiation, has been implicated in cardiac and skeletal muscle development because of its strong expression in embryonic muscle lineages, and its necessity for the transcription of transiently transfected muscle genes that contain SRF binding sites. This study was designed to ascertain whether SRF is required for the expression of an endogenous SRF-dependent gene during differentiation of early embryonic cardiac myocytes by introducing a dominant-negative SRF construct via retroviral delivery. Although no effect on overt cellular differentiation was detected, semi-quantitative RT-PCR revealed that expression of the SRF-dependent gene cardiac alpha-actin was inhibited, whereas expression of the non-SRF-dependent genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cardiac troponin-C was unaffected. No effect on myocyte proliferation was detected. Curiously, immunohistochemical localization of SRF protein suggested that whereas endogenous SRF was homogeneously dispersed throughout the cytoplasm and nucleus, the dominant-negative SRF protein was concentrated in the nucleus. These results extend previous findings using transiently transfected genes to the endogenous level, indicating that SRF is required for the full expression of muscle genes that contain SRF binding sites during cardiac myocyte differentiation.

Actins↗
PubMed2000

Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators.

Combinatorial interaction among cardiac tissue-restricted enriched transcription factors may facilitate the expression of cardiac tissue-restricted genes. Here we show that the MADS box factor serum response factor (SRF) cooperates with the zinc finger protein GATA-4 to synergistically activate numerous myogenic and nonmyogenic serum response element (SRE)-dependent promoters in CV1 fibroblasts. In the absence of GATA binding sites, synergistic activation depends on binding of SRF to the proximal CArG box sequence in the cardiac and skeletal alpha-actin promoter. GATA-4's C-terminal activation domain is obligatory for synergistic coactivation with SRF, and its N-terminal domain and first zinc finger are inhibitory. SRF and GATA-4 physically associate both in vivo and in vitro through their MADS box and the second zinc finger domains as determined by protein A pullout assays and by in vivo one-hybrid transfection assays using Gal4 fusion proteins. Other cardiovascular tissue-restricted GATA factors, such as GATA-5 and GATA-6, were equivalent to GATA-4 in coactivating SRE-dependent targets. Thus, interaction between the MADS box and C4 zinc finger proteins, a novel regulatory paradigm, mediates activation of SRF-dependent gene expression.

Actins↗
PubMed2007

Transcription factor serum response factor is selectively involved in the mechanisms of long-term synapse-specific plasticity.

Our previous studies demonstrated that acquisition of nociceptive sensitization in common snails is accompanied by long-term facilitation of the responses of defensive behavior command neurons LPl1 and RPl1 to sensory stimuli, this being dependent on the processes of translation and transcription. The mechanism of induction of long-term synaptic facilitation at the sensory inputs of neurons from chemoreceptors on the head involves cAMP and the immediate early gene transcription factor C/EBP (CCAAT-enhancer-binding protein), while regulation of the other sensory input of neurons LPl1 and RPl1 - from mechanoreceptors on the head - depends on protein kinase C. The present report describes studies of the involvement of the transcription factor serum response factor (SRF) in the processes of the synapse-specific plasticity of neuron LPl1 during the acquisition of sensitization in snails. The acquisition of sensitization during intracellular administration of oligonucleotides specifically inhibiting SRF led to the selective suppression of synaptic facilitation in the responses of neuron LPl1 to tactile stimulation of the snail's head. Synaptic facilitation of responses to chemical stimulation of the head and tactile stimulation of the foot developed just as in neurons in control sensitized animals. The results were assessed in relation to a hypothesis postulating that synapse-specific plasticity on learning may occur because of selective neurochemical "projection" of synaptic connections to various genes within neurons.

Analgesics, Non-Narcotic↗
PubMed2004

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Alleles↗
PubMed2005

Mechanism of binding of serum response factor to serum response element.

Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle-specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core-SRF (the 124-245 fragment of serum response factor) with specific oligonucleotides from c-fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core-SRF labeled with bis-ANS showed that the protein is monomeric at low concentration (10(-7) m). The titration curves for the fluorescence anisotropy of fluorescein-labeled oligonucleotide revealed that under equilibrium conditions, the core-SRF monomers were bound sequentially to SRE at very low concentration (10(-9) m). Curve-fitting data showed also major differences between the wild-type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core-SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core-SRF was bound as a dimer to the wild-type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far-UV CD spectra indicated that the flexibility of core-SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein-labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA-protein interaction.

Amino Acid Sequence↗
PubMed1999

Transformation blocks differentiation-induced inhibition of serum response factor interactions with serum response elements.

The differentiation of nontransformed 3T3T mesenchymal stem cells is a multistep process that is associated with the progressive repression of mitogenic responsiveness to serum growth factors that ultimately results in expression of the terminally differentiated adipocyte phenotype. The repression of serum-induced mitogenesis by differentiation correlates with repression of the serum-inducible transcription of junB and c-fos. In contrast, the differentiation of neoplastically transformed cells does not repress mitogenic responsiveness or junB or c-fos inducibility. Because the junB and c-fos promoters both contain serum response elements (SREs), the current studies tested the possibility that differentiation might repress the ability of serum response factor (SRF) to bind to the SRE in normal cells but not in transformed cells. We now report that differentiation represses SRE serum inducibility using nontransformed cells transiently transfected with pjunB SRE thymidine kinase/chloroamphenicol acetyltransferase (SREtk/CAT) or pc-fos SREtk/CAT containing an intact SRF-binding domain. Adipocyte differentiation of nontransformed cells also markedly represses the ability of SRF to bind to the junB SRE, the c-fos SRE, and other SREs, as determined by mobility shift and gel supershift assays, without affecting the DNA binding characteristics of the nuclear protein SP-1. By comparison, the ability of SRF to bind SRE is not repressed by the differentiation of SV40 large T antigen-transformed 3T3T cells. The results further establish that adipocyte differentiation blocks the nuclear localization of SRF, thus preventing its interaction with SREs in nontransformed cells but not in transformed cells.

Adipocytes↗
PubMed1998

Steroid receptor coactivator-1 interacts with serum response factor and coactivates serum response element-mediated transactivations.

Steroid receptor coactivator-1 (SRC-1) specifically bound to serum response factor (SRF), as demonstrated by glutathione S-transferase pull down assays, and the yeast and mammalian two-hybrid tests. In mammalian cells, SRC-1 potentiated serum response element (SRE)-mediated transactivations in a dose-dependent manner. Coexpression of p300 synergistically enhanced this SRC-1-potentiated level of transactivations, consistent with the recent finding (Ramirez, S., Ali, S. A. S., Robin, P., Trouche, D., and Harel-Bellan, A. (1997) J. Biol. Chem. 272, 31016-31021) in which the p300 homologue CREB-binding protein was shown to be a transcription coactivator of SRF. Thus, we concluded that at least two distinct classes of coactivator molecules may cooperate to regulate SRF-dependent transactivations in vivo.

DNA-Binding Proteins↗
PubMed1996

Expression of the serum response factor gene is regulated by serum response factor binding sites.

The serum response factor (SRF) is a ubiquitous transcription factor that plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. Notably, SRF has been found to be a key regulator of members of a class of cellular response genes termed immediate-early genes (IEGs), many of which are believed to be involved in regulating cell growth and differentiation. The mechanism by which SRF activates transcription of IEGs in response to mitogenic agents has been extensively studied. Significantly less is known about how expression of the SRF gene itself is mediated. We and others have previously shown that the SRF gene is itself transiently induced by a variety of mitogenic agents and belongs to a class of "delayed" early response genes. We have cloned the SRF promoter and in the present study have analyzed the upstream regulatory sequences involved in mediating serum responsiveness of the SRF gene. Our analysis indicates that inducible SRF expression requires both SRF binding sites located within the first 63 nucleotides upstream from the start site of transcriptional initiation and an Sp1 site located 83 nucleotides upstream from the start site. Maximal transcriptional activity of the promoter also requires two CCAATT box sites located 90 and 123 nucleotides upstream of the start site.

3T3 Cells↗
PubMed1997

Growth hormone regulates ternary complex factors and serum response factor associated with the c-fos serum response element.

For insight into the mechanisms of gene regulation by growth hormone (GH), the regulation of transcription factors associated with the serum response element (SRE) located upstream of c-fos was examined. The SRE can mediate induction of reporter expression in response to GH. For insight into the mechanism by which GH regulates transcription factors, regulation of SRE-associated proteins by GH was examined. In nuclear extracts from 3T3-F442A fibroblasts, several SRE-binding complexes were identified by electrophoretic mobility shift assay. GH treatment for 2-10 min transiently increased binding of two complexes; binding returned to control values within 30 min. The two GH-stimulated complexes were supershifted by antibodies against the serum response factor (SRF), indicating that they contained SRF or an antigenically related protein. One of the GH-stimulated complexes was supershifted by antibody against Elk-1, suggesting that it contains a ternary complex factor (TCF) such as Elk-1 in addition to SRF. Induction of binding by GH was lost when the SRF binding site in the SRE was mutated, and mutation of either the SRF or TCF binding site altered the pattern of protein binding to the SRE. Mutation of the SRF or TCF binding site in SRE-luciferase plasmids inhibited the ability of GH to stimulate reporter expression, supporting a role for both SRF and TCF in GH-induced transcription of c-fos via the SRE. The TCF family member Elk-1 is capable of mediating GH-stimulated transcription, since GH-stimulated reporter expression was mediated by the transcriptional activation domain of Elk-1. Consistent with this stimulation, GH rapidly and transiently stimulated the serine phosphorylation of Elk-1. The increase was evident within 10 min and subsided after 30 min. Taken together, these data indicate that SRF and TCF contribute to GH-promoted transcription of c-fos via the SRE and are consistent with GH-promoted phosphorylation of Elk-1 contributing to GH-promoted transcriptional activation via the SRE.

3T3 Cells↗
PubMed1988

Phosphorylation of serum response factor, a factor that binds to the serum response element of the c-FOS enhancer.

Serum and growth factor regulation of c-FOS protooncogene transcription is mediated by the serum response element. A factor, serum response factor, binding to this element has already been identified. We demonstrate that serum response factor is phosphorylated in vivo on serine residues and that phosphatase treatment of this factor in vitro abolishes its DNA-binding activity. These results show phosphorylation of serum response factor to be required for its DNA-binding activity. The importance of serum response factor phosphorylation for the regulation of c-FOS expression is discussed.

Animals↗
PubMed1999

Basic fibroblast growth factor activates serum response factor gene expression by multiple distinct signaling mechanisms.

Serum response factor (SRF) plays a central role in the transcriptional response of mammalian cells to a variety of extracellular signals. It is a key regulator of many cellular early response genes which are believed to be involved in cell growth and differentiation. The mechanism by which SRF activates transcription in response to mitogenic agents has been extensively studied; however, significantly less is known about regulation of the SRF gene itself. Previously, we identified distinct regulatory elements in the SRF promoter that play a role in activation, including a consensus ETS domain binding site, a consensus overlapping Sp/Egr-1 binding site, and two SRF binding sites. We further showed that serum induces SRF by a mechanism that requires an intact SRF binding site, also termed a CArG box. In the present study we demonstrate that in response to stimulation of cells by a purified growth factor, basic fibroblast growth factor (bFGF), the SRF promoter is upregulated by a complex pathway that involves at least two independent mechanisms: a CArG box-independent mechanism that is mediated by an ETS binding site, and a novel CArG box-dependent mechanism that requires both an Sp factor binding site and the CArG motifs for maximal stimulation. Our analysis indicates that the CArG/Sp element activation mechanism is mediated by distinct signaling pathways. The CArG box-dependent component is targeted by a Rho-mediated pathway, and the Sp binding site-dependent component is targeted by a Ras-mediated pathway. Both SRF and bFGF have been implicated in playing an important role in mediating cardiogenesis during development. The implications of our findings for SRF expression during development are discussed.

3T3 Cells↗