blistered/Serum response factor


MADS-box proteins and invertebrate SRF homologs

There is a striking homology between human SRF and Drosophila SERF. Within the conserved 55 amino acid 'MADS-box' motif (derived from MCM1-ARG80-Agamous-Deficiens-SRF), only two conserved amino acid replacements are found. C-terminal to the MADS-box, only three amino acid replacements are found between human SRF, MCM1, ARG80 and Drosophila SERF. In addition to this domain of 93% amino acid identity, there are additional smaller regions of similarity with human SRF (Affolter, 1994).

The serum response factor (SRF) and myocyte enhancer factor 2A (MEF2A) (see Drosophila Mef2) represent two human members of the MADS-box transcription factor family. Each protein has a distinct biological function that is reflected by the distinct specificities of the proteins for their coregulatory protein partners and DNA-binding sites. The mechanism of DNA binding utilized by these two related transcription factors was examined. Although SRF and MEF2A belong to the same family and contain related DNA-binding domains, their DNA-binding mechanisms differ in several key aspects. In contrast to the dramatic DNA bending induced by SRF, MEF2A induces minimal DNA distortion. A combination of loss- and gain-of-function mutagenesis identifies a single amino acid residue located at the N terminus of the recognition helices as the critical mediator of this differential DNA bending. This residue is also involved in determining DNA-binding specificity, thus indicating a link between DNA bending and DNA-binding specificity determination. Different basic residues within the putative recognition alpha-helices are critical for DNA binding, and the role of the C-terminal extensions to the MADS box in dimerization between SRF and MEF2A also differs. These important differences in the molecular interactions of SRF and MEF2A are likely to contribute to their differing roles in the regulation of specific gene transcription (West, 1997).

The ExPASy World Wide Web (WWW) molecular biology server of the Geneva University Hospital and the University of Geneva provides extensive documentation for the SRF-type transcription factors DNA-binding and dimerization domain.

The CURLEY LEAF gene of Arabidopsis is necessary for stable repression of a floral homeotic gene (AGAMOUS) required to specify stamen and carpel identity in whorls 3 and 4 respectively. AGAMOUS is a MADS box protein. The CURLEY LEAF protein shows extensive homology to Enhancer of zeste, with three regions conserved between the two proteins. First, the C-terminus contains a 115 amino-acid region, the SET domain, previously recognized as a conserved region in the products of E(z), Trithorax and Suppressor of variegation 3-9. Second, residues 655-720 of CLF show 46% identity with the region of E(Z) (residues 538-603) that are rich in cysteine residues, but with an arrangement unlike that of zinc fingers. Third, residues 270-317 of CLF contain seven cysteines with a similar spacing to a region of E(Z) (residues 321-367) containing five cysteines, and there is a small region of sequence similarity at the N termini of these regions (Goodrich, 1997).

In the yeast Saccharomyces cerevisiae, the MADS-box protein Mcm1, which is highly related to mammalian SRF (serum response factor), forms a ternary complex with SFF (Swi five factor) to regulate the cell cycle expression of genes such as SWI5, CLB2 and ACE2. The forkhead protein Fkh2 is a component of SFF and is essential for ternary complex formation on the SWI5 and ACE2 promoters. Fkh2 is essential for the correct cell cycle periodicity of SWI5 and CLB2 gene expression and is phosphorylated with a timing that is consistent with a role in this expression. Furthermore, investigation of the relationship between Fkh2 and a related forkhead protein, Fkh1, demonstrates that these proteins act in overlapping pathways to regulate cell morphology and cell separation. This is the first example of a eukaryotic transcription factor complex containing both a MADS-box and a forkhead protein, and it has important implications for the regulation of mammalian gene expression (Pic, 2000).

How is the Mcm1-SFF transcription factor complex regulated during the cell cycle? The Mcm1-SFF complex may be detectable in extracts prepared from G1 cells. This is consistent with a model where Mcm1-SFF binds to the SWI5 promoter throughout the cell cycle and that the Mcm1-SFF complex undergoes cell cycle regulation to activate transcription. In agreement with this, the Fkh2 protein can be detected in the nucleus of cells throughout the cell cycle. What other mechanism(s) could be responsible for the regulation of Mcm1-SFF? Previous studies have shown that the activity of Cdc28-Clb2 is required for the periodic activation of expression of the SWI5 and CLB2 genes, leading to the suggestion that a positive feedback loop acts to regulate G2 periodic gene expression. It is possible that the Mcm1-SFF transcription factor complex is phosphorylated directly by Cdc28-Clb2. However, although Mcm1 is known to be a phosphoprotein, it also regulates the expression of several genes in a non-cell cycle-dependent manner. Hence Fkh2 is a more likely target for specific cell cycle phosphorylation. Indeed, examination of the predicted sequence of the Fkh2 protein reveals several S/TP motifs, which may be potential targets for Cdc28-Clb2. Furthermore, Fkh2 becomes phosphorylated with a cell cycle timing that is consistent with the role of this protein in the activation of gene expression at G2/M. The molecular basis of this phosphorylation of Fkh2 is under investigation, but data suggest that the activity of the Mcm1-SFF transcription factor complex may be regulated by a cell cycle-regulated protein kinase(s) (Pic, 2000 and references therein).

The yeast Mcm1-SFF complex has been proposed to represent a functionally analogous complex to the mammalian SRF-TCF complex, which plays a pivotal role in regulating the induction of immediate-early genes such as the proto-oncogene c-fos in response to mitogenic stimuli. Thus the SRF-TCF complex is thought to function to promote cell cycle entry. Similarly, the Mcm1-SFF complex plays a role in regulating cell cycle progression, albeit at a later time point. Molecularly, their mechanisms of complex assembly and function appear to be similar, with both TCF (in mammals) and SFF (in yeast) being recruited to promoters by the highly related MADS-box proteins SRF and Mcm1. Both SFF and TCF are critical for the transcriptional activity of these complexes. The identification of Fkh2 as a component of SFF has uncovered a number of other intriguing parallels between these mammalian and yeast complexes. (1) While the TCFs and Fkh2 are members of different transcription factor families, ETS-domain and forkhead, respectively, their DNA-binding domains are related and form part of the larger winged helix-turn-helix family. Indeed, ETS-domain proteins appear unique to the metazoan lineage. (2) Both Fkh2 and TCF are modified by phosphorylation. In the case of Fkh2, there are numerous S/TP motifs in its C-terminal region, which is highly reminiscent of TCF's structure. Hence these similarities in structure and regulation suggest that studies of the mechanisms of regulation of activity and interaction between these highly conserved proteins will shed new light on the roles and activities of these proteins in eukaryotes (Pic, 2000 and references therein).

A homolog of the serum response factor (SRF) has been isolated from Dictyostelium discoideum and its function studied by analyzing the consequences of its gene disruption. The MADS-box region of Dictyostelium SRF (DdSRF) is highly conserved with those of the human, Drosophila and yeast homologs. srfA is a developmentally regulated gene expressed in prespore and spore cells. This gene plays an essential role in sporulation, since its disruption leads to abnormal spore morphology and loss of viability. The mutant spores are round and cellulose deposition seems to be partially affected. Initial prestalk and prespore cell differentiation does not seem to be compromised in the mutant since the expression of several cell-type-specific markers were found to be unaffected. However, the mRNA level of the spore marker spiA is greatly reduced. Activation of the cAMP-dependent protein kinase (PKA) by 8-Br-cAMP is not able to fully bypass the morphological defects of srfA- mutant spores, although this treatment induces spiA mRNA expression. These results suggest that DdSRF is required for full maturation of spores and participates in the regulation of the expression of the spore-coat marker spiA and probably other maturation genes necessary for proper spore cell differentiation (Escalante, 1998).

Myogenic regulatory factors (MRFs) are required for mammalian skeletal myogenesis. In contrast, bodywall muscle is readily detectable in C. elegans embryos lacking activity of the lone MRF ortholog HLH-1, indicating that additional myogenic factors must function in the nematode. Two additional C. elegans proteins, UNC-120/SRF and HND-1/HAND, can convert naive blastomeres to muscle when overproduced ectopically in the embryo. In addition, genetic null mutants were used to demonstrate that both of these factors act in concert with HLH-1 to regulate myogenesis. Loss of all three factors results in embryos that lack detectable bodywall muscle differentiation, identifying this trio as a set that is both necessary and sufficient for bodywall myogenesis in C. elegans. In mammals, SRF and HAND play prominent roles in regulating smooth and cardiac muscle development. That C. elegans bodywall muscle development is dependent on transcription factors that are associated with all three types of mammalian muscle supports a theory that all animal muscle types are derived from a common ancestral contractile cell type (Fukushige, 2006).

The morphological and functional evolution of appendages has played a critical role in animal evolution, but the developmental genetic mechanisms underlying appendage diversity are not understood. Given that homologous appendage development is controlled by the same Hox gene in different organisms, and that Hox genes are transcription factors, diversity may evolve from changes in the regulation of Hox target genes. Two impediments to understanding the role of Hox genes in morphological evolution have been the limited number of organisms in which Hox gene function can be studied and the paucity of known Hox-regulated target genes. An analysis was carried out of Hindsight, a butterfly homeotic mutant in which portions of the ventral hindwing pattern are transformed to ventral forewing identity, and the regulation of target genes by the Ultrabithorax (Ubx) gene product was compared in Lepidopteran and Dipteran hindwings. Ubx gene expression is lost from patches of cells in developing Hindsight hindwings, which correlates with changes in wing pigmentation, color pattern elements, and scale morphology. This mutant was used to study how regulation of target genes by Ubx protein differs between species. Drosophila Serum response factor (blistered), Achaete-Scute Complex, and wingless are repressed in Drosophila halteres. Portions of the expression pattern of Lepidopteran homologs of these genes are not repressed in butterfly hindwings. Unlike the expression patterns of the homologous genes in halteres, butterfly wg is not repressed along the posterior margin in the hindwing, nor is butterfly SRF repressed in intervein regions, and the AS-C homologs are not repressed in cells flanking the dorsal-ventral boundary. These differences in the regulation of wg, SRF and AS-C between Drosophila halteres and butterfly hindwings suggest that these genes became repressed by Ubx when an ancestral hindwing evolved into a haltere in the dipteran lineage, with a concomitant reduction of appendage size, loss of margin bristles, and changes in shape. Two additional exampes of Ubx-regulated differences in gene expression between fly and butterfly flight appendages were found. (1) wg is expressed in two stripes in butterfly forewings that roughly correspond to the future location of the proximal band elements. This protein of the wg pattern is absent from butterfly hindwings and has not counterpart in flies and represents a novel feature regulated by Ubx in butterflies. (2) Dll is expressed along the margin of both butterfly wings and the Drosophila forewing, but this expression is modified in halteres and may be regulated by Ubx. Changes in Hox-regulated target gene sets are, in general, likely to underlie the morphological divergence of homologous structures between animals (Weatherbee, 1999).

Zebrafish serum response factor

Serum response factor (SRF) was identified as an activity that binds, upon serum stimulation of HeLa cells, to a motif known as the serum response element in the c-fos promoter. This element is also found in the regulatory regions of many muscle-specific genes. srf expression has been characterized during early zebrafish embryogenesis. In addition to low-level expression in many or even all cells, elevated levels of srf RNA and protein are transiently expressed in skeletal muscle lineages during their differentiation (Vogel, 1999).

Serum response factor recruits Elk-1 and other Pointed homologs to the Fos promoter

The ternary complex factor (TCF) subfamily of ETS-domain transcription factors form ternary complexes with the serum response factor (SRF) and the c-fos (See Drosophila Fos-related antigen) SRE. Extracellular signals are relayed via MAP kinase signal transduction pathways through the TCF component of the ternary complex. Protein-protein interactions between TCFs and SRF play an essential role in formation of this ternary complex. A 30 amino acid sequence encompassing the TCF B-box is sufficient to mediate interactions with SRF. The amino acids that are critical for this interaction have been identified and a molecular model has been derived of the SRF binding interface. Alanine scanning of the Elk-1 B-box reveals five predominantly hydrophobic residues that are essential for binding to SRF and for ternary complex formation in vitro and in vivo. These amino acids are predicted to lie on one face of an alpha-helix. Peptides encompassing the B-box retain biological activity and exhibit a helix-forming propensity. alpha-Helix and ternary complex formation is disrupted by the introduction of helix-breaking proline residues. These results are consistent with a model in which the Elk-1 B-box forms an inducible alpha-helix, which presents a hydrophobic face for interaction with SRF (Ling, 1997).

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET (Homologs of Drosophila Pointed and Yan), have been reported to have the properties of TCF. Elk-1 and SAP-1a were compared with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation (Price, 1995).

The serum response element (SRE) of the mammalian c-fos gene forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA (see Drosophila Rho1), Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK (Drosophila homolog:Rolled Rolled), SAPK/JNK (Drosophila homolog: Basket/JNK), or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway (Hill, 1995).

Signal transduction pathways that mediate activation of serum response factor (SRF) by heterotrimeric G protein alpha subunits were characterized in transfection systems. Galphaq, Galpha12, and Galpha13 (but not Galphai) activate SRF through RhoA. When Galphaq, alpha12, or alpha13 are coexpressed with a Rho-specific guanine nucleotide exchange factor GEF115, Galpha13 (but not Galphaq or Galpha12) shows synergistic activation of SRF with GEF115. The synergy between Galpha13 and GEF115 depends on the N-terminal part of GEF115, and there is no synergistic effect between Galpha13 and another Rho-specific exchange factor: Lbc. In addition, the Dbl-homology (DH)-domain-deletion mutant of GEF115 inhibits Galpha13- and Galpha12-induced SRF activation, but not GEF115- or Galphaq-induced SRF activation. The DH-domain-deletion mutant also suppresses thrombin- and lysophosphatidic acid-induced SRF activation in NIH 3T3 cells, probably by inhibition of Galpha12/13. The N-terminal part of GEF115 contains a sequence motif that is homologous to the regulator of G protein signaling (RGS) domain of RGS12. RGS12 can inhibit both Galpha12 and Galpha13. Thus, the inhibition of Galpha12/13 by the DH-deletion mutant may be due to the RGS activity of the mutant. The synergism between Galpha13 and GEF115 indicates that GEF115 mediates Galpha13-induced activation of Rho and SRF (Mao, 1998b).

The rapid and transient induction of the human proto-oncogene c-fos in response to a variety of stimuli depends on the serum response element (SRE). In vivo footprinting experiments show that this promoter element is bound by a multicomponent complex that includes the serum response factor (SRF) and a ternary complex factor such as Elk-1. SRF is thought to recruit a ternary complex factor monomer into an asymmetric complex. A quaternary complex that binds the SRE is described that, in addition to an SRF dimer, contains two Elk-1 molecules. Its formation at the SRE is strictly dependent on phosphorylation of S-383 in the Elk-1 regulatory domain and appears to involve a weak intermolecular association between the two Elk-1 molecules. The influence of mutations in Elk-1 on quaternary complex formation in vitro correlates with their effect on the induction of c-fos reporter expression in response to mitogenic stimuli in vivo (Gille, 1998).

Mitogenic and stress signals result in the activation of extracellular signal-regulated kinases (ERKs) and stress-activated protein kinase/c-Jun N-terminal kinases (SAPK/JNKs), respectively, which are two subgroups of the mitogen-activated protein kinases. A nuclear target of mitogen-activated protein (MAP) kinases is the ternary complex factor Elk-1, which underlies its involvement in the regulation of c-fos gene expression by mitogenic and stress signals. A second ternary complex factor, Sap1a, is coexpressed with Elk-1 in several cell types and shares attributes of Elk-1, the significance of which has not been clear. Sap1a is phosphorylated efficiently by ERKs but not by SAPK/JNKs. Serum response factor-dependent ternary complex formation by Sap1a is stimulated by ERK phosphorylation but not by SAPK/JNKs. Sap1a-mediated transcription is activated by mitogenic signals but not by cell stress. These results suggest that Sap1a and Elk-1 have distinct physiological functions (Strahl, 1996).

Cytoplasmic proteins such as small GTPases and Tec non-receptor tyrosine kinases activate SRF

Serum response factor: Evolutionary homologs part 2/2

blistered/Serum response factor: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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