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At least 19 recordsLinked to original sources

PubMed2004

Conversion of [Pt(SRf)2(PPh2 -n(C6F5)n+ 1)2]( n = 0 or 1, Rf=C6HF4-4) through carbon-fluorine bond activation to [Pt(SRf)2(1,2-C6F4(SRf)-(PPh2))] and chiral [Pt(SRf)2(1,2-C6F4(SRf)(PPh(C6F5)))].

Treatment of trans-[PtCl(2)(PPh(2 - n)(C(6)F(5))(n + 1))(2)](n = 0 or 1) with Pb(SC(6)HF(4)-4)(2) yields a mixture of monometallic cis/trans [Pt(SC(6)HF(4)-4)(2)(PPh(2 - n)(C(6)F(5))(n + 1))(2)], thiolate-bridged bimetallic cis/trans [Pt(2)(mu-SC(6)HF(4)-4)(2)(SC(6)HF(4)-4)(2)(PPh(2 - n)(C(6)F(5))(n + 1))(2)] and [Pt(SC(6)HF(4)-4)(2)(1,2-C(6)F(4)(SC(6)HF(4)-4)(PPh(2 - n)(C(6)F(5))(n))].

Journal Article↗

PubMed1993

Expression and purification of the DNA-binding domain of SRF: SRF-DB, a part of a DNA-binding protein which can act as a dominant negative mutant in vivo.

We have developed an approach which allows functional in vivo examination of DNA-binding proteins through microinjection of polypeptides containing the DNA-binding domain into living fibroblasts. The present analysis utilizes serum response factor (SRF), a transcription factor that binds to the serum response element. We have expressed in bacteria a 30-kDa portion of this protein (amino acids 113 to 265) containing the DNA-binding domain of SRF (SRF-DB) and purified it to homogeneity by a single DNA affinity chromatography step using the high-affinity SRF-binding site (ACT.L). We have tested the efficiency of SRF-DB to prevent endogenous SRF function through analysis of c-fos expression and DNA synthesis stimulated by fetal calf serum, two events known to require SRF. Injection of purified SRF-DB into rat embryo fibroblasts inhibits c-fos induction by growth factors. Moreover, DNA synthesis, induced after serum addition, is also suppressed by SRF-DB injection. This implies that overproduction of SRF-DB makes the cell deficient in the function of wild-type SRF and that SRF-DB acts as a dominant negative mutant. These data show that, for the study of DNA-binding proteins, expressing and using portions of the protein that corresponds to the DNA-binding domain present a useful method for generating dominant negative mutants and illustrate the potential application of the DNA-binding region to facilitate the study of events at the DNA/protein level.

Animals↗

PubMed1993

Identification of transcriptional activation and inhibitory domains in serum response factor (SRF) by using GAL4-SRF constructs.

The binding of serum response factor (SRF) to the c-fos serum response element has been shown to be essential for serum and growth factor activation of c-Fos. Since SRF is ubiquitously expressed, it has been difficult to measure the activity of SRF introduced into cells. To assay for functions of SRF in cells, we have changed its DNA binding specificity by fusing it to the DNA binding domain of GAL4. Transfection of GAL4-SRF constructs into cells has allowed us to identify SRF's transcriptional activation domain as well as domains which inhibit this activity. First, we found that the transcriptional activation domain maps to between amino acids 339 and 508 in HeLa cells and to between amino acids 414 and 508 in NIH 3T3 cells. Second, we show that in the context of GAL4-SRF constructs, there are two separate domains of SRF that can inhibit its activation domain. Although these domains overlap the DNA binding and dimerization domains of SRF, these functions were not required for inhibition. Finally, we show that one of the inhibitory domains is modular in that it can also inhibit activation when it is moved amino terminal to GAL4's DNA binding domain in an SRF-GAL4-SRF construct. The implications of these inhibitory domains for SRF regulation are discussed.

Base Sequence↗

PubMed2002

Interaction of serum response factor (SRF) with the Elk-1 B box inhibits RhoA-actin signaling to SRF and potentiates transcriptional activation by Elk-1.

Serum response factor (SRF) is a transcription factor which regulates many immediate-early genes. Rho GTPases regulate SRF activity through changes in actin dynamics, but some SRF target genes, such as c-fos, are insensitive to this pathway. At the c-fos promoter, SRF recruits members of the ternary complex factor (TCF) family of Ets domain proteins through interactions with the TCF B-box region. Analysis of c-fos promoter mutations demonstrates that the TCF and ATF/AP1 sites adjoining the SRF binding site inhibit activation of the promoter by RhoA-actin signaling. The presence of the TCF binding site is sufficient for inhibition, and experiments with an altered-specificity Elk-1 derivative demonstrate that inhibition can be mediated by the Elk-1 TCF. Using Elk-1 fusion proteins that can bind DNA autonomously, we show that inhibition of RhoA-actin signaling requires physical interaction between the Elk-1 B box and SRF. These results account for the insensitivity of c-fos to RhoA-actin signaling. Interaction of the B box with SRF also potentiates transcriptional activation by the Elk-1 C-terminal activation domain. Combinatorial interactions between SRF and TCF proteins are thus likely to play an important role in determining the relative sensitivity of SRF target genes to Ras- and Rho-controlled signal transduction pathways.

3T3 Cells↗

PubMed2004

The SRF target gene Fhl2 antagonizes RhoA/MAL-dependent activation of SRF.

RhoA signaling regulates the activity of the transcription factor SRF (serum response factor) during muscle differentiation. How RhoA signaling is integrated at SRF target promoters to achieve muscle-lineage-specific expression is largely unknown. Using large-scale expression profiling combined with bioinformatic and biochemical approaches, we identified several SRF target genes, including Fhl2, encoding a transcriptional cofactor that is highly expressed in the heart. SRF binds the Fhl2 promoter in vivo and regulates Fhl2 expression in response to RhoA activation. FHL2 protein and SRF interact physically, and FHL2 binds the promoters of SRF-responsive smooth muscle (SM) genes, but not the promoters of immediate-early genes (IEGs), in response to RhoA. FHL2 antagonizes induction of SM genes, but not IEGs or cardiac genes, by competing with the coactivator MAL/MRTF-A for SRF binding. Our findings identify an autoregulatory mechanism to selectively regulate subsets of RhoA-activated SRF target genes.

Animals↗

PubMedDate not supplied

Cloning and preliminary characterization of srf-2020 and srf-801, the recF partial suppressor mutations which map in recA of Escherichia coli K-12.

We have located the single nucleotide changes suffered in recA sequence of 2 recF partial suppressor mutations: srf-2020 at codon 121 and srf-801 at codon 257. srf-2020 changes codon 121 from threonine (ACC) to isoleucine (ATC). srf-801 changes codon 257 from glutamine (CAG) to proline (CCG). Consequently these mutations were renamed recA2020 and recA801 respectively. Preliminary characterization of recA2020 revealed that it is transdominant to recA+, like recA803, another recF partial suppressor. Interactions of recA2020 with recA803 were examined using genetic studies. Heterozygotes containing recA2020 and recA803 failed to produce a synergistic suppression effect in suppressing the recF deficiency. Presence of both recA2020 and recA803 mutations in the same recA gene also failed to produce any greater amount of UV resistance to a uvrA6recF143del(recA) strain indicating no interactions between these suppressors.

Alleles↗

PubMed1991

Expression of genes encoding the transcription factor SRF during early development of Xenopus laevis: identification of a CArG box-binding activity as SRF.

cDNA clones encoding the sequence-specific DNA binding protein, serum response factor (SRF), have been isolated from a Xenopus laevis neurula library and their nucleotide sequence determined. The Xenopus SRF (SRFX) gene produces multiple-sized transcripts, present at 10(5) copies per unfertilized egg. A similar level is detected in the embryo during early cleavage, but SRFX transcripts accumulate rapidly following gastrulation. The protein they encode is similar in sequence to human SRF in its central and carboxy-terminal regions, but possesses a divergent amino-terminal portion. We have previously described a Xenopus embryo sequence-specific binding activity that recognized the CArG motif of the cardiac actin gene promoter. Here we show that the DNA-binding characteristics of synthetic SRFX are indistinguishable from those of the embryo factor. Moreover, antiserum raised against the synthetic SRFX recognizes this factor. Together, these results establish that the same factor binds to elements required for constitutive transcription in Xenopus oocytes, muscle-specific gene expression in Xenopus embryos and serum-responsive transcription in cultured amphibian cells.

Aging↗

PubMed2003

Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes.

Megakaryoblastic leukemia 1 (MKL1) is a myocardin-related transcription factor that we found strongly activated serum response element (SRE)-dependent reporter genes through its direct binding to serum response factor (SRF). The c-fos SRE is regulated by mitogen-activated protein kinase phosphorylation of ternary complex factor (TCF) but is also regulated by a RhoA-dependent pathway. The mechanism of this pathway is unclear. Since MKL1 (also known as MAL, BSAC, and MRTF-A) is broadly expressed, we assessed its role in serum induction of c-fos and other SRE-regulated genes with a dominant negative MKL1 mutant (DN-MKL1) and RNA interference (RNAi). We found that DN-MKL1 and RNAi specifically blocked SRE-dependent reporter gene activation by serum and RhoA. Complete inhibition by RNAi required the additional inhibition of the related factor MKL2 (MRTF-B), showing the redundancy of these factors. DN-MKL1 reduced the late stage of serum induction of endogenous c-fos expression, suggesting that the TCF- and RhoA-dependent pathways contribute to temporally distinct phases of c-fos expression. Furthermore, serum induction of two TCF-independent SRE target genes, SRF and vinculin, was nearly completely blocked by DN-MKL1. Finally, the RBM15-MKL1 fusion protein formed by the t(1;22) translocation of acute megakaryoblastic leukemia had a markedly increased ability to activate SRE reporter genes, suggesting that its activation of SRF target genes may contribute to leukemogenesis.

Animals↗

PubMed2005

SRF-dependent gene expression in isolated cardiomyocytes: regulation of genes involved in cardiac hypertrophy.

Serum response factor (SRF) is a transcription factor required for the regulation of genes important for cardiac structure and function. Notably, the "fetal gene expression profile" that is characteristic of cardiac hypertrophy consists of genes known to be regulated by SRF. Transgenic animal studies suggest that cardiac-specific overexpression of SRF induces this pattern of hypertrophic genes and subsequently causes the progression of pathologic adaptations. Furthermore, studies examining cardiac tissues from patients with severe heart failure indicate significant alterations in SRF expression that correspond with alterations in expression of SRF-dependent genes. Based on these observations, it has been postulated that SRF may be critical for stimulating pathologic gene expression at the onset of hypertrophic adaptation. To address the role of SRF in cardiac hypertrophy we investigated whether SRF is necessary and sufficient for the expression of genes associated with the hypertrophic response. We used isolated cardiomyocytes from both neonatal rats, and transgenic mice containing floxed SRF alleles, to examine cardiac gene expression in response to overexpression and absence of SRF. Using this approach, we demonstrate that SRF is required for the induction of atrial naturetic factor (ANF), c-fos, NCX1, BNP, alpha-actins, alpha-myosin heavy chain, and beta-myosin heavy chain genes. However, overexpression of exogenous SRF in isolated cardiomyocytes is only sufficient to induce NCX1 and alpha-myosin heavy chain. These results indicate that SRF is critical for the regulation and induction of genes associated with the progression of pathologic cardiac hypertrophy, however, the pattern of genes induced by overexpression of SRF in isolated cardiomyocytes is different from those genes expressed in hypertrophic transgenic hearts. This suggests that SRF-dependent gene expression is modulated in a complex manner by in vivo physiologic systems prior to and during heart failure as the organism adapts to cardiac stress.

Actins↗

PubMed1998

Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control.

The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression required spo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducing srf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of srf-lacZ at neutral pH. The results suggest that srf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.

Bacillus subtilis↗

PubMed2002

Increased expression of alternatively spliced dominant-negative isoform of SRF in human failing hearts.

Serum response factor (SRF) has been shown to play a key role in cardiac cell growth and muscle gene regulation. To understand the role of SRF in heart failure, we compared its expression pattern between control and failing human heart samples. Western blot analysis of control samples showed expression of four different isoforms of SRF, with ~67-kDa full-length SRF being the predominant isoform. Interestingly, in failing hearts we found robust expression of a low-molecular-mass (~52 kDa) SRF isoform, accompanied by decreased expression of full-length SRF. By RT-PCR and Southern blot analyses, we characterized this ~52-kDa SRF isoform as being encoded by an alternatively spliced form of SRF lacking exons 4 and 5 of the SRF primary RNA transcript (SRF-Delta4,5 isoform). We cloned SRF-Delta4,5 cDNA and showed that overexpression of this isoform into cells inhibits SRF-dependent activation of cardiac muscle genes. These results suggest that expression of SRF-Delta4,5 in failing hearts may in part contribute to impaired cardiac gene expression and consequently to the pathogenesis of heart failure.

Adult↗

PubMed2000

Srf(-/-) ES cells display non-cell-autonomous impairment in mesodermal differentiation.

The serum response factor (SRF) transcription factor is essential for murine embryogenesis. SRF+(-/-) embryos stop developing at the onset of gastrulation, lacking detectable mesoderm. This developmental defect may reflect cell-autonomous impairment of SRF(-/-) embryonic cells in mesoderm formation. Alternatively, it may be caused by a non-cell-autonomous defect superimposed upon inappropriate provision of mesoderm-inducing signals to primitive ectodermal cells. We demonstrate that the ability of SRF(-/-) embryonic stem (ES) cells to differentiate in vitro into mesodermal cells is indeed impaired. However, this impairment can be modulated by external, cell-independent factors. Retinoic acid, but not dimethylsulfoxide, permitted activation of the mesodermal marker gene T(Bra), which was also activated when SRF was expressed in SRF(-/-) ES cells. Embryoid bodies from SRF(-/-) ES cell aggregates also activated mesodermal marker genes, but displayed unusual morphologies and impairment in cavitation. Finally, in nude mice, Srf(-/-) ES cells readily differentiated into mesodermal cells of SRF(-/-) genotype, including cartilage, bone or muscle cells. We demonstrate that SRF contributes to mesodermal gene expression of ES cells and that SRF(-/-) ES cells display a non-cell-autonomous defect in differentiation towards mesoderm.

Animals↗

PubMed2003

Inhibitory cardiac transcription factor, SRF-N, is generated by caspase 3 cleavage in human heart failure and attenuated by ventricular unloading.

BACKGROUND: Knowledge about molecular mechanisms leading to heart failure is still limited, but reduced gene activities and modest activation of caspase 3 are hallmarks of end-stage heart failure. We postulated that serum response factor (SRF), a central cardiac transcription factor, might be a cleavage target for modest activated caspase 3, and this cleavage of SRF may play a dominant inhibitory role in propelling hearts toward failure. METHODS AND RESULTS: We examined SRF protein levels from cardiac samples taken at the time of transplantation in 13 patients with end-stage heart failure and 7 normal hearts. Full-length SRF was markedly reduced and processed into 55- and 32-kDa subfragments in all failing hearts. SRF was intact in normal samples. In contrast, the hearts of 10 patients with left ventricular assist devices showed minimal SRF fragmentation. Specific antibodies to N- and C-terminal SRF sequences and site-directed mutagenesis revealed 2 alternative caspase 3 cleavage sites, so that 2 fragments were detected of each containing either the N- or C-terminal SRF. Expression of SRF-N, the 32-kDa fragment, in myogenic cells inhibited the transcriptional activity of alpha-actin gene promoters by 50% to 60%, which suggests that truncated SRF functioned as a dominant-negative transcription factor. CONCLUSIONS: Caspase 3 activation in heart failure sequentially cleaved SRF and generated a dominant-negative transcription factor, which may explain the depression of cardiac-specific genes. Moreover, caspase 3 activation may be reversible in the failing heart with ventricular unloading.

Actins↗

PubMed2004

Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent.

BACKGROUND: Serum Response Factor (SRF) is a transcription factor that is required for the expression of many genes including immediate early genes, cytoskeletal genes, and muscle-specific genes. SRF is activated in response to extra-cellular signals by its association with a diverse set of co-activators in different cell types. In the case of the ubiquitously expressed immediate early genes, the two sets of SRF binding proteins that regulate its activity are the TCF family of proteins that include Elk1, SAP1 and SAP2 and the myocardin-related MKL family of proteins that include MKL1 and MKL2 (also known as MAL, MRTF-A and -B and BSAC). In response to serum or growth factors these two classes of co-activators are activated by different upstream signal transduction pathways. However, it is not clear how they differentially activate SRF target genes. RESULTS: In order to identify the serum-inducible SRF target genes that are specifically dependent on the MKL pathway, we have performed microarray experiments using a cell line that expresses dominant negative MKL1. This approach was used to identify SRF target genes whose activation is MKL-dependent. Twenty-eight of 150 serum-inducible genes were found to be MKL-dependent. The promoters of the serum-inducible genes were analyzed for SRF binding sites and other common regulatory elements. Putative SRF binding sites were found at a higher rate than in a mouse promoter database but were only identified in 12% of the serum-inducible promoters analyzed. Additional partial matches to the consensus SRF binding site were found at a higher than expected rate in the MKL-dependent gene promoters. The analysis for other common regulatory elements is discussed. CONCLUSIONS: These results suggest that a subset of immediate early and SRF target genes are activated by the Rho-MKL pathway. MKL may also contribute to the induction of other SRF target genes however its role is not essential, possibly due to other activation mechanisms such as MAPK phosphorylation of TCFs.

Animals↗

PubMed1998

SRF binding to SRE in the rat heart: influence of age.

One important promoter element at the 5' end of the c-fos gene is the serum response element (SRE). SRE is the site of attachment of the 67-kDa protein serum response factor (SRF) and several accessory proteins (Elk1, SAP1, SAP2/NET), termed the ternary complex factors. The binding of SRF to SRE plays an integral role in c-fos transcription and may occur independently of the association of the ternary complex factors. In the current study, we found that SRF protein expression was increased in the hearts of the old vs young adult rats in the basal condition. The hearts of old rats may have posttranslationally modified SRF proteins that are different compared to that of the young adults. The SRF increase was present both in the cytoplasm as well as in the nucleus in the old hearts. To test whether SRF protein levels in response to acute stress might be altered with age, we studied hearts of young adult and old rats during myocardial infarction. The young adult rat hearts responded to acute ischemic stress with an increase in both p62 and p67 SRF. The hearts of the old rats, however, did not exhibit a significant change in SRF protein expression. These findings demonstrate qualitative as well as quantitative age differences in SRF protein levels, both at baseline and following stimulation. The reduced SRF expression in response to acute cardiac ischemic stress in the old rats might contribute to the observed age-related decrease in the induction of immediate early genes such as c-fos in the heart.

Aging↗

PubMed2000

Skeletal muscle CaMKII enriches in nuclei and phosphorylates myogenic factor SRF at multiple sites.

We characterized the activity of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in homogenates and nuclear extracts of skeletal muscle and analyzed their capacity to phosphorylate the myogenic factor SRF. Isoforms of CaMKII enriched from skeletal muscle phosphorylated SRF in vitro to high stoichiometries and produced multiple forms on SDS-PAGE, suggesting that SRF was phosphorylated at multiple sites. Phosphopeptide-mapping experiments using truncated SRF proteins located the residues of SRF phosphorylated by recombinant CaMKII within amino acids 1-171, with at least one site residing in amino acids 142-171. Microsequencing of these phosphorylated peptides identified that both Ser-103 and a novel residue, Thr-160 in the MADS box of SRF, were sites of phosphorylation. CaMKII activity was enriched in nuclear extracts relative to crude homogenates from skeletal muscle and similarly phosphorylated the nuclear transcription factor SRF in vitro. The location of Thr-160 in the 3-D structure of SRF suggests that its phosphorylation by nuclear CaMKII may directly influence DNA binding of SRF and other MADS box factors.

Amino Acid Sequence↗

PubMed2004

SRF regulates Bcl-2 expression and promotes cell survival during murine embryonic development.

The transcription factor serum response factor (SRF) controls the expression of genes involved in cellular proliferation and differentiation. Interestingly, SRF also promotes cell survival by regulating the expression of antiapoptotic genes. In in vitro differentiating murine embryonic stem (ES) cells, SRF deficiency leads to increased apoptosis. Loss of SRF correlates with impaired expression of the antiapoptotic Bcl-2 and Bcl-xl genes. SRF binds the Bcl-2 promoter in vivo and activates Bcl-2 transcription. Reconstituting Bcl-2 in Srf-/- ES cells rescues these cells from apoptosis, demonstrating that SRF-dependent Bcl-2 expression is critical for ES cell survival. At the multicellular level, SRF deficiency leads to impaired cavitation and reduced Bcl-2 expression in embryoid bodies (EBs) and inappropriate apoptosis in both EBs and pregastrulation mouse embryos. Thus, our data from genetic and cellular studies uncover SRF-regulated Bcl-2 expression as a novel mechanism that is important for cell survival during early murine embryogenesis.

Animals↗

PubMed2003

Differential regulation of NFAT and SRF by the B cell receptor via a PLCgamma-Ca(2+)-dependent pathway.

NFAT and SRF are important in the regulation of proliferation and cytokine production in lymphocytes. NFAT activation by the B cell receptor (BCR) occurs via the PLCgamma-Ca(2+)-calcineurin pathway, however how the BCR activates SRF is unclear. We show here that like NFAT, BCR regulation of SRF occurs via an Src-Syk-Tec-PLCgamma-Ca(2+) (Lyn-Syk-Btk-PLCgamma-Ca(2+)) pathway. However, SRF responds to lower Ca(2+) and is less dependent on IP(3)R expression than NFAT. Ca(2+)-regulated calcineurin plays a partial role in SRF activation, in combination with diacylglycerol (DAG), while is fully required for NFAT activation. Signals from the DAG effectors protein kinase C, Ras and Rap1, and the downstream MEK-ERK pathway are required for both SRF and NFAT; however, NFAT but not SRF is dependent on JNK signals. Both SRF and NFAT were also dependent on Rac, Rho, CDC42 and actin. Finally, we show that Ca(2+) is not required for ERK activation, but instead for its association with nuclear areas of the cell. These data suggest that combinatorial assembly of signaling pathways emanating from the BCR differentially regulate NFAT and SRF, to activate gene expression.

Animals↗