ABSTRACT
Serum response factor (SRF), a key transcription factor, plays an important role in regulating cell functions such as proliferation and differentiation. Most proteins are unstable, and protein stability is regulated through the ubiquitin-proteasome system (UPS) or the autophagy lysosome pathway (ALP). Whether SRF is degraded and what mechanisms control SRF protein stability remain unexplored. Western blot analyses of cells treated with cycloheximide (CHX), a protein synthesis inhibitor, showed that SRF was degraded in a time-dependent manner. Moreover, we observed that SRF undergoes autophagy-dependent destruction, which is accelerated by serum deprivation. Through bioinformatics screening, we found that SRF contains the GSK3ß phosphorylation motif (T/SPPXS): SPDSPPRSDPT, which is conserved from zebrafish to humans. Serum deprivation stimulated GSK3ß activation that then potentiates SRF degradation through the autophagy lysosome pathway. Since SRF is important for numerous cellular activities, our results suggest that the autophagy-dependent SRF degradation pathway may provide a new avenue to modulate SRF-mediated cell functions.
Subject(s)
Autophagy , Serum Response Factor/chemistry , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Culture Media, Serum-Free/pharmacology , Cycloheximide/pharmacology , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Lysosomes/metabolism , Protein Stability , Rats , Serum Response Factor/metabolismABSTRACT
BACKGROUND/AIMS: Increasing evidence links microRNAs to the pathogenesis of peripheral vascular disease. We recently found microRNA-125b (miR-125b) to be one of the most significantly downregulated microRNAs in human arteries with arteriosclerosis obliterans (ASO) of the lower extremities. However, its function in the process of ASO remains unclear. This study aimed to investigate the expression, regulatory mechanisms, and functions of miR-125b in the process of ASO. METHODS: Using the tissue explants adherent method, vascular smooth muscle cells (VSMCs) were prepared for this study. A rat carotid artery balloon injury model was constructed to simulate the development of vascular neointima, and a lentiviral transduction system was used to overexpress serum response factor (SRF) or miR-125b. Quantitative realtime PCR (qRTPCR) was used to detect the expression levels of miR125b and SRF mRNA. Western blotting was performed to determine the expression levels of SRF and Ki67. In situ hybridization analysis was used to analyze the location and expression levels of miR-125b. CCK-8 and EdU assays were used to assess cell proliferation, and transwell and wound closure assays were performed to measure cell migration. Flow cytometry was used to evaluate cell apoptosis, and a dual-luciferase reporter assay was conducted to examine the effects of miR125b on SRF. Immunohistochemistry and immunofluorescence analyses were performed to analyze the location and expression levels of SRF and Ki67. RESULTS: miR-125b expression was decreased in ASO arteries and platelet-derived growth factor (PDGF)-BB-stimulated VSMCs. miR-125b suppressed VSMC proliferation and migration but promoted VSMC apoptosis. SRF was determined to be a direct target of miR-125b. Exogenous miR-125b expression modulated SRF expression and inhibited vascular neointimal formation in balloon-injured rat carotid arteries. CONCLUSIONS: These findings demonstrate a specific role of the miR-125b/SRF pathway in regulating VSMC function and suggest that modulating miR-125b levels might be a novel approach for treating ASO.
Subject(s)
MicroRNAs/metabolism , Serum Response Factor/metabolism , 3' Untranslated Regions , Adult , Aged , Animals , Antagomirs/metabolism , Apoptosis/drug effects , Arteriosclerosis Obliterans/genetics , Arteriosclerosis Obliterans/metabolism , Arteriosclerosis Obliterans/pathology , Base Sequence , Becaplermin , Carotid Artery Injuries/metabolism , Carotid Artery Injuries/pathology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Down-Regulation/drug effects , Female , Humans , In Situ Hybridization, Fluorescence , Male , MicroRNAs/antagonists & inhibitors , MicroRNAs/genetics , Middle Aged , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Proto-Oncogene Proteins c-sis/pharmacology , Rats , Rats, Sprague-Dawley , Sequence Alignment , Serum Response Factor/chemistry , Serum Response Factor/geneticsABSTRACT
GPR55 is a newly deorphanized class A G-protein-coupled receptor that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Few potent GPR55 ligands have been identified to date. This is largely due to an absence of information about salient features of GPR55, such as residues important for signaling and residues implicated in the GPR55 signaling cascade. The goal of this work was to identify residues that are key for the signaling of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol (LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433, 3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-N,N-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}. Serum response element (SRE) and serum response factor (SRF) luciferase assays were used as readouts for studying LPI and ML184 signaling at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid receptor (DOR) crystal structure was used to interpret the resultant mutation data. Two residues were found to be crucial for agonist signaling at GPR55, K2.60 and E3.29, suggesting that these residues form the primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F, and F6.55A/L mutation results suggested that these residues are part of the orthosteric binding site for ML184, while Y3.32F and H(170)F mutation results suggest that these two residues are part of the LPI binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling cascade. C(10)A and C(260)A mutations suggest that these residues form a second disulfide bridge in the extracellular domain of GPR55, occluding ligand extracellular entry in the TMH1-TMH7 region of GPR55. Taken together, these results provide the first set of discrete information about GPR55 residues important for LPI and ML184 signaling and for GPR55 activation. This information should aid in the rational design of next-generation GPR55 ligands and the creation of the first high-affinity GPR55 radioligand, a tool that is sorely needed in the field.
Subject(s)
Lysophospholipids/chemistry , Piperazines/chemistry , Pyrrolidines/chemistry , Receptors, G-Protein-Coupled/chemistry , Recombinant Fusion Proteins/chemistry , Serum Response Element , Amino Acid Motifs , Binding Sites , Crystallography, X-Ray , Gene Expression , HEK293 Cells , Humans , Kinetics , Ligands , Lysophospholipids/pharmacology , Molecular Docking Simulation , Mutation , Piperazines/pharmacology , Protein Binding , Pyrrolidines/pharmacology , Receptors, Cannabinoid , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Opioid, delta/chemistry , Receptors, Opioid, delta/genetics , Receptors, Opioid, delta/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serum Response Factor/chemistry , Serum Response Factor/genetics , Serum Response Factor/metabolism , Signal Transduction , Glycine max , Structural Homology, Protein , ThermodynamicsABSTRACT
Selectively regulating genes is an important goal in Chemical Biology. We report the development of a peptide-based synthetic transcription factor which binds the targeted DNA sequence with high affinity and single base-pair discrimination capability. When delivered inside a tumor cell, it regulated targeted genes selectively and inhibited cell proliferation.
Subject(s)
Down-Regulation/drug effects , Peptides/chemical synthesis , Peptides/pharmacology , Proto-Oncogene Proteins c-fos/genetics , Serum Response Factor/metabolism , ets-Domain Protein Elk-1/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , Proto-Oncogene Mas , Proto-Oncogene Proteins c-fos/chemistry , Proto-Oncogene Proteins c-fos/metabolism , Serum Response Factor/chemistry , ets-Domain Protein Elk-1/chemistryABSTRACT
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.
Subject(s)
Cell Differentiation/genetics , MEF2 Transcription Factors/chemistry , Mitochondria/physiology , Muscle Fibers, Skeletal/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Smooth Muscle/metabolism , Serum Response Factor/chemistry , Amino Acid Motifs , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Diabetes, Gestational , Female , Gene Expression Regulation , Humans , MEF2 Transcription Factors/metabolism , MEF2 Transcription Factors/physiology , Membrane Potential, Mitochondrial/genetics , MicroRNAs/metabolism , Mitochondria/genetics , Muscle Fibers, Skeletal/cytology , Mutagenesis, Site-Directed , Myocytes, Cardiac/cytology , Myocytes, Smooth Muscle/cytology , Phosphorylation , Pregnancy , Prenatal Exposure Delayed Effects , Rats , Serum Response Factor/metabolism , Serum Response Factor/physiology , Tandem Mass SpectrometryABSTRACT
The transcriptional activity of the serum response factor (SRF) protein is triggered by its binding to a 10-base-pair DNA consensus sequence designated the CArG box, which is the core sequence of the serum response element (SRE). Sequence-specific recognition of the CArG box by a core domain of 100 amino acid residues of SRF (core-SRF) was asserted to depend almost exclusively on the intrinsic SRE conformation and on the degree of protein-induced SRE bending. Nevertheless, this paradigm was invalidated by a temperature-dependent Raman spectroscopy study of 20-mer oligonucleotides involved in bonding interactions with core-SRF that reproduced both wild type and mutated c-fos SREs. Indeed, the SRE moieties that are complexed with core-SRF exhibit permanent interconversion dynamics between bent and linear conformers. Thus, sequence-specific recognition of the CArG box by core-SRF cannot be explained only in terms of the three-dimensional structure of the SRE. A particular dynamic pairing process discriminates between the wild type and mutated complexes. Specific oscillations of the phosphate charge network of the SRE govern the recognition between both partners rather than an intrinsic set of conformations of the SRE.
Subject(s)
DNA/chemistry , Oligonucleotides/chemistry , Phosphates/chemistry , Serum Response Element/genetics , Serum Response Factor/chemistry , Binding Sites , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Nucleic Acid Conformation , Nucleotide Motifs , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Serum Response Factor/genetics , Spectrum Analysis, Raman , Static Electricity , Thermodynamics , Transcription, GeneticABSTRACT
MADS box family transcription factors are involved in signal transduction and development control through DNA specific sequence recognition. The DNA binding domain of these proteins contains a conservative 55-60 amino acid sequence which defines the membership of this large family. Here we present a thorough study of the MADS segment of serum response factor (MADS(SRF)). Fluorescence, UV-absorption, and Raman spectroscopy studies were performed in order to disclose its behavior and basic functional properties in an aqueous environment. The secondary structure of MADS(SRF) estimated by analysis of Raman spectra and supported by CD has revealed only the C-terminal part as homologous with those of free core-SRF, while the N-terminal part has lost the stable α-helical structure found in both the free core-SRF and its specific complex with DNA. The three tyrosine residues of the MADS(SRF) were used as spectroscopic inner probes. The effect of environmental conditions, especially pH variations and addition of variously charged quenchers, on their spectra was examined. Two-component fluorescence quenching was revealed using factor analysis and corresponding Stern-Volmer constants determined. Factor analysis of absorbance and fluorescence pH titration led to determination of three dissociation constants pKa1 = 6.4 ± 0.2, pKa2 = 7.3 ± 0.2, and pKa3 = 9.6 ± 0.6. Critical comparison of all experiments identified the deprotonation of His193 hydrogen bonded to Tyr195 as a candidate for pKa1 (and that of Tyr158 as a candidate for pKa2). Within MADS(SRF), His193 is a key intermediary between the N-terminal primary DNA binding element and the hydrophobic C-terminal protein dimerization element.
Subject(s)
Serum Response Factor/chemistry , Tyrosine/chemistry , Amino Acid Sequence , Humans , Hydrogen-Ion Concentration , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Protein Structure, Secondary , Serum Response Factor/metabolism , Spectrophotometry, Ultraviolet , Spectrum Analysis, Raman , Tyrosine/metabolismABSTRACT
The megakaryoblastic leukemia 1 (MKL1) protein functions as a transcriptional coactivator of the serum response factor. MKL1 has three RPEL motifs (RPEL1, RPEL2, and RPEL3) in its N-terminal region. MKL1 binds to monomeric G-actin through RPEL motifs, and the dissociation of MKL1 from G-actin promotes the translocation of MKL1 to the nucleus. Although structural data are available for RPEL motifs of MKL1 in complex with G-actin, the structural characteristics of RPEL motifs in the free state have been poorly defined. Here we characterized the structures of free RPEL motifs using NMR and CD spectroscopy. NMR and CD measurements showed that free RPEL motifs are largely unstructured in solution. However, NMR analysis identified transient α-helices in the regions where helices α1 and α2 are induced upon binding to G-actin. Proline mutagenesis showed that the transient α-helices are locally formed without helix-helix interactions. The helix content is higher in the order of RPEL1, RPEL2, and RPEL3. The amount of preformed structure may correlate with the binding affinity between the intrinsically disordered protein and its target molecule.
Subject(s)
Protein Structure, Secondary/physiology , Serum Response Factor/chemistry , Trans-Activators/chemistry , Actins/chemistry , Actins/metabolism , Amino Acid Motifs/physiology , Cell Nucleus/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Magnetic Resonance Spectroscopy/methods , Protein Binding/physiology , Serum Response Factor/metabolism , Trans-Activators/metabolismABSTRACT
The high sensitivity to pH of a short segment (an octamer) of serum response factor (SRF), an important member of the MADS box family of transcription factors, was investigated by Raman scattering, infrared and circular dichroism spectroscopies. Molecular dynamics (MD) and density functional theory (DFT) calculations enabled interpretation of spectral changes in close detail. Although there was a negligible difference between spectra in acidic and neutral environments, the spectrum in basic pH was substantially different. The major changes were attributed to the deprotonation of tyrosine. The secondary structure of the SRF octamer fragment was estimated experimentally as well as predicted theoretically by MD. All techniques proved that it exists in a dynamical equilibrium among several conformations mostly close to ß turn, unordered conformations, and extended structure, in contrast to the stable secondary structure it possesses as a part of SRF. Generally, this approach represents a useful tool for the study of various short oligopeptides.
Subject(s)
Molecular Dynamics Simulation , Protons , Serum Response Factor/chemistry , Tyrosine/chemistryABSTRACT
The epicardium and coronary vessels originate from progenitor cells in the proepicardium. Here we show that Tbx18, a T-box family member highly expressed in the proepicardium, controls critical early steps in coronary development. In Tbx18(-/-) mouse embryos, both the epicardium and coronary vessels exhibit structural and functional defects. At E12.5, the Tbx18-deficient epicardium contains protrusions and cyst-like structures overlying a disorganized coronary vascular plexus that contains ectopic structures resembling blood islands. At E13.5, the left and right coronary stems form correctly in mutant hearts. However, analysis of PECAM-1 whole mount immunostaining, distribution of SM22α(lacZ/+) activity, and analysis of coronary vascular casts suggest that defective vascular plexus remodeling produces a compromised arterial network at birth consisting of fewer distributing conduit arteries with smaller lumens and a reduced capacity to conduct blood flow. Gene expression profiles of Tbx18(-/-) hearts at E12.5 reveal altered expression of 79 genes that are associated with development of the vascular system including sonic hedgehog signaling components patched and smoothened, VEGF-A, angiopoietin-1, endoglin, and Wnt factors compared to wild type hearts. Thus, formation of coronary vasculature is responsive to Tbx18-dependent gene targets in the epicardium, and a poorly structured network of coronary conduit vessels is formed in Tbx18 null hearts due to defects in epicardial cell signaling and fate during heart development. Lastly, we demonstrate that Tbx18 possesses a SRF/CArG box dependent repressor activity capable of inhibiting progenitor cell differentiation into smooth muscle cells, suggesting a potential function of Tbx18 in maintaining the progenitor status of epicardial-derived cells.
Subject(s)
Coronary Vessels/embryology , Coronary Vessels/metabolism , Pericardium/embryology , Pericardium/metabolism , T-Box Domain Proteins/metabolism , Animals , Animals, Newborn , Cell Differentiation , Coronary Circulation , Coronary Vessels/pathology , Coronary Vessels/ultrastructure , Embryo, Mammalian/metabolism , Embryo, Mammalian/pathology , Gene Expression Profiling , Gene Expression Regulation, Developmental , Mice , Myocytes, Smooth Muscle/metabolism , Pericardium/pathology , Pericardium/ultrastructure , Repressor Proteins/metabolism , Serum Response Factor/chemistry , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , Transcription, Genetic , beta-Galactosidase/metabolismABSTRACT
Full genome annotation requires gene expression analysis and elucidation of promoter activity. Here, we analyzed the expression and promoter of a highly restricted integrin gene, Itga8. RNase protection and quantitative RT-PCR showed Itga8 to be expressed most abundantly in vascular smooth muscle cells (SMC). Transcription start site mapping of Itga8 revealed the immediate 5' promoter region to be poorly conserved with orthologous sequences in the human genome. Further comparative sequence analysis showed a number of conserved non-coding sequence modules around the Itga8 gene. The immediate promoter region and an upstream conserved sequence module were each found to contain a CArG box, which is a binding site for serum response factor (SRF). Luciferase reporter assays revealed activity of several Itga8 promoter constructs with no apparent restricted activity to SMC types. Further, neither SRF nor its coactivator, Myocardin (MYOCD), was able to induce several distinct Itga8 promoter constructs. Transgenic mouse studies failed to reveal Itga8 promoter activity, indicating distal regulatory elements likely control this gene's in vivo expression profile. Interestingly, although the promoter was unresponsive to SRF/MYOCD, the endogenous Itga8 gene showed increases in expression upon ectopic MYOCD expression even though knockdown of SRF both in vitro and in vivo failed to demonstrate a corresponding change in Itga8. Collectively, these data demonstrate that Itga8 expression is CArG-SRF independent, but MYOCD dependent through an as yet unknown sequence module that is distal from the promoter region.
Subject(s)
Gene Expression Regulation , Integrin alpha Chains/genetics , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Conserved Sequence/genetics , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/metabolism , Protein Binding/genetics , Rats , Serum Response Factor/chemistry , Serum Response Factor/metabolism , Trans-Activators/metabolism , TranscriptomeABSTRACT
Enteroviral infection can lead to dilated cardiomyopathy (DCM), which is a major cause of cardiovascular mortality worldwide. However, the pathogenetic mechanisms have not been fully elucidated. Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes, including those involved in the contractile apparatus and immediate early response, as well as microRNAs that silence the expression of cardiac regulatory factors. Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM. The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM. Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes. This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes. Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A. Moreover, we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain, resulting in the disruption of SRF-mediated gene transactivation. In addition to loss of functional SRF, finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor, which likely competes with the native SRF for DNA binding. Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.
Subject(s)
Myocytes, Cardiac/metabolism , Peptide Hydrolases/metabolism , Serum Response Factor/metabolism , Viral Proteins/metabolism , Amino Acid Sequence , Animals , Cardiomyopathy, Dilated/etiology , Cardiomyopathy, Dilated/metabolism , Caspase Inhibitors , Caspases/metabolism , Cell Line , Enterovirus/enzymology , Gene Expression , HeLa Cells , Heart/physiopathology , Humans , Male , Mice , MicroRNAs/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Serum Response Factor/chemistry , Serum Response Factor/genetics , Virus ReplicationABSTRACT
Despite extensive studies on GnRH regulation of the gonadotropin subunit genes, very little is known about mechanism of induction of intermediary immediate early genes, such as c-Fos, that are direct targets of GnRH signaling and that upon induction, activate transcription of gonadotropin genes. Although c-Fos is induced by a variety of stimuli in other cell types, in the gonadotropes, only GnRH induces c-Fos and through it FSHß. Thus, understanding the specificity of c-Fos induction by GnRH will provide insight into GnRH regulation of FSHß gene expression. GnRH induction of c-Fos in LßT2 cells requires the serum response factor (SRF)-binding site, but not the Ets/ELK1 site. This is in contrast to c-Fos induction by growth factors in other cells, which activate c-Fos transcription via phosphorylation of ELK1 and require the ELK1-binding site. The SRF site alone is sufficient for induction by GnRH, whereas induction by 12-tetradecanoylphorbol-13-acetate (TPA) requires both the ELK1 and SRF sites. Although ELK1 site is not required, upon GnRH stimulation, ELK1 interacts with SRF and is recruited to the SRF site. GnRH phosphorylates ELK1 through ERK1/2 and p38 MAPK, which correlates with the signaling pathways necessary for c-Fos and FSHß induction. GnRH also causes phosphorylation of SRF through calmodulin-dependent kinase II (CamKII), which leads to increased binding to its site. CamKII activation is sufficient for phosphorylation of SRF and for induction of the c-Fos gene through the SRF site. Thus, GnRH uses a combination of growth factor signaling and the CamKII pathway to induce c-Fos to regulate FSHß gene expression in gonadotrope cells.
Subject(s)
Follicle Stimulating Hormone, beta Subunit/metabolism , Genes, fos , Gonadotropin-Releasing Hormone/metabolism , Serum Response Factor/metabolism , Signal Transduction , Animals , Binding Sites , Blotting, Western , COS Cells , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Line , Chlorocebus aethiops , Gene Expression Regulation , Mice , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Reverse Transcriptase Polymerase Chain Reaction , Serum Response Factor/chemistry , Tetradecanoylphorbol Acetate/pharmacology , Transcription, Genetic , ets-Domain Protein Elk-1ABSTRACT
Serum response factor (SRF) is a transcription factor that regulates many genes involved in cellular activities such as proliferation, migration, differentiation, angiogenesis, and apoptosis. Although it has only been known for about two decades, SRF has been studied extensively. To date, over a thousand SRF studies have been published, but it still remains a hot topic. Due to its critical role in mesoderm-derived tissues, most of the SRF studies focused on muscle structure/function, cardiovascular development/maintenance, and smooth muscle generation/repair. Recently, SRF has received more attention in the digestive field and several important discoveries have been made. This review will summarize what we have learned about SRF in the gastrointestinal tract and provide insights into possible future directions in this area.
Subject(s)
Digestive System Physiological Phenomena , Serum Response Factor/physiology , Animals , Digestive System Diseases/etiology , Digestive System Diseases/physiopathology , Humans , Serum Response Factor/chemistry , Serum Response Factor/geneticsABSTRACT
This study identifies KLF3 as a transcriptional regulator of muscle genes and reveals a novel synergistic interaction between KLF3 and serum response factor (SRF). Using quantitative proteomics, KLF3 was identified as one of several candidate factors that recognize the MPEX control element in the Muscle creatine kinase (MCK) promoter. Chromatin immunoprecipitation analysis indicated that KLF3 is enriched at many muscle gene promoters (MCK, Myosin heavy chain IIa, Six4, Calcium channel receptor alpha-1, and Skeletal alpha-actin), and two KLF3 isoforms are upregulated during muscle differentiation. KLF3 and SRF physically associate and synergize in transactivating the MCK promoter independently of SRF binding to CArG motifs. The zinc finger and repression domains of KLF3 plus the MADS box and transcription activation domain of SRF are implicated in this synergy. Our results provide the first evidence of a role for KLF3 in muscle gene regulation and reveal an alternate mechanism for transcriptional regulation by SRF via its recruitment to KLF binding sites. Since both factors are expressed in all muscle lineages, SRF may regulate many striated- and smooth-muscle genes that lack known SRF control elements, thus further expanding the breadth of the emerging CArGome.
Subject(s)
Kruppel-Like Transcription Factors/metabolism , Myoblasts, Skeletal/metabolism , Serum Response Factor/metabolism , Amino Acid Sequence , Animals , Base Sequence , Binding Sites/genetics , Cell Differentiation , Cell Line , Creatine Kinase, MM Form/genetics , DNA Primers/genetics , Humans , In Vitro Techniques , Kruppel-Like Transcription Factors/chemistry , Kruppel-Like Transcription Factors/genetics , Mice , Molecular Sequence Data , Myoblasts, Skeletal/cytology , Promoter Regions, Genetic , Protein Interaction Domains and Motifs , Proteomics , Serum Response Factor/chemistry , Serum Response Factor/genetics , Transcriptional ActivationABSTRACT
Transcriptional activity of serum response factor (SRF) is dependent on its binding to the CC(A/T)(6)GG box (CArG box) of serum response element (SRE). By Raman spectroscopy, we carried out a comparative analysis, in solution, of the complexes obtained from the association of core-SRF with 20-mer SREs bearing wild-type and mutated c-fos CArG boxes. In case of association with the wild type c-fos CArG box, the complex does not bring out the expected Raman signature of a stable bending of the targeted SRE but keeps a bend-linear conformer oligonucleotide interconversion. The linear conformer population is larger than that of free oligonucleotide. In the core-SRF moiety of the wild-type complex a large spectral change associated with the CO-groups from Asp and/or Glu residues shows that their ionization states and the strength of their interactions decrease as compared to those of mutated non-specific complexes. Structural constraints evidenced on the free core-SRF are released in the wild-type complex and environmental heterogeneities appear in the vicinity of Tyr residues, due to higher water molecule access. The H-bonding configuration of one Tyr OH-group, in average, changes with a net transfer from H-bond acceptor character to a combined donor and acceptor character. A charge repartition distributed on both core-SRF and targeted SRE stabilizes the specific complex, allowing the two partners to experience a variety of conformations.
Subject(s)
Gene Expression Regulation , Serum Response Element , Serum Response Factor/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Tertiary , Serum Response Factor/chemistry , Serum Response Factor/genetics , Spectrum Analysis, Raman , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
OBJECTIVE: Our goal was to identify phosphorylation sites that regulate serum response factor (SRF) activity to gain a better understanding of the signaling mechanisms that regulate SRF's involvement in smooth muscle cell (SMC)-specific and early response gene expression. METHODS AND RESULTS: By screening phosphorylation-deficient and mimetic mutations in SRF(-/-) embryonic stem cells, we identified T159 as a phosphorylation site that significantly inhibits SMC-specific gene expression in an embryonic stem cell model of SMC differentiation. This residue conforms to a highly conserved consensus cAMP-dependent protein kinase (PKA) site, and in vitro and in vivo labeling studies demonstrated that it was phosphorylated by PKA. Results from gel shift and chromatin immunoprecipitation assays demonstrated that T159 phosphorylation inhibited SRF binding to SMC-specific CArG elements. Interestingly, the myocardin factors could at least partially rescue the effects of the T159D mutation under some conditions, but this response was promoter specific. Finally, PKA signaling had much less of an effect on c-fos promoter activity and SRF binding to the c-fos CArG. CONCLUSIONS: Our results indicate that phosphorylation of SRF by PKA inhibits SMC-specific transcription suggesting a novel signaling mechanism for the control of SMC phenotype.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , Myocytes, Smooth Muscle/metabolism , Serum Response Factor/metabolism , Amino Acid Sequence , Amino Acid Substitution , Animals , Binding Sites/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Gene Expression , Molecular Sequence Data , Mutagenesis, Site-Directed , Myocytes, Smooth Muscle/cytology , Nuclear Proteins/genetics , Phosphorylation , Promoter Regions, Genetic , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serum Response Factor/chemistry , Serum Response Factor/deficiency , Serum Response Factor/genetics , Signal Transduction , Trans-Activators/genetics , Transcriptional ActivationABSTRACT
NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.
Subject(s)
Genes, Tumor Suppressor , Homeodomain Proteins/metabolism , Serum Response Factor/metabolism , Transcription Factors/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Line, Tumor , Circular Dichroism , Homeodomain Proteins/chemistry , Humans , Male , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Phosphorylation , Protein Binding , Protein Folding , Serum Response Factor/chemistry , Transcription Factors/chemistryABSTRACT
Megakaryoblastic leukemia 1 (MAL) is a transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding one twenty-two (OTT). Herein, we show that MAL expression increases during the late differentiation steps of neonate and adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin. MAL knockdown in megakaryocyte progenitors reduced the percentage of cells forming filopodia, lamellipodia, and stress fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic megakaryocytes with disorganized demarcation membranes and alpha granules heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression after MAL inhibition. Luciferase assays in HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro. Megakaryocyte migration in response to stromal cell-derived factor 1, through Matrigel was considerably decreased after MAL knockdown, implicating MMP9 in migration. Finally, the use of a shRNA to decrease MYL9 expression showed that MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved in platelet formation and megakaryocyte migration by regulating MYL9 and MMP9.
Subject(s)
Blood Platelets/cytology , Blood Platelets/metabolism , Cardiac Myosins/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Matrix Metalloproteinase 9/genetics , Megakaryocytes/physiology , Myosin Light Chains/genetics , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Serum Response Factor/genetics , Serum Response Factor/metabolism , Adult , Cell Differentiation , Cell Line , Cell Movement/genetics , Cell Movement/physiology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , Fetal Blood/cytology , Focal Adhesions , Gene Expression Profiling , Humans , In Vitro Techniques , Infant, Newborn , Megakaryocytes/cytology , Multiprotein Complexes , Oligonucleotide Array Sequence Analysis , Oncogene Proteins, Fusion/antagonists & inhibitors , Oncogene Proteins, Fusion/chemistry , RNA, Small Interfering/genetics , Serum Response Factor/chemistry , Thrombopoiesis , Trans-Activators , rho GTP-Binding Proteins/metabolismABSTRACT
By binding to the CArG box sequence, the serum response factor (SRF) activates several muscle-specific genes, as well as genes that respond to mitogens. The core domain of the SRF (core-SRF) binds as a dimer to the CArG box C-5C-4A-3T-2A-1T+1T+2A+3G+4G+5 of the c-fos serum response element (SREfos). However, previous studies using 20-mer DNAs have shown that the binding stoichiometry of core-SRF is significantly altered by mutations C-5-->G (SREGfos) and C-5C-4-->GG (SREGGfos) of the CArG box [A Huet, A Parlakian, M-C Arnaud, J-M Glandières, P Valat, S Fermandjian, D Paulin, B Alpert & C Zentz (2005) FEBS J272, 3105-3119]. To understand these effects, we carried out a comparative analysis of the three 20-mer DNAs SREfos, SREGfos and SREGGfos in aqueous solution. Their CD spectra were of the B-DNA type with small differences generated by variations in the mutual arrangement of the base pairs. Analysis by singular value decomposition of a set of Raman spectra recorded as a function of temperature, revealed a premelting transition associated with a conformational shift in the DNA double helices from a bent to a linear form. Time-resolved fluorescence anisotropy shows that the fluorescein reporter linked to the oligonucleotide 5'-ends experiences twisting motions of the double helices related to the interconversion between bent and linear conformers. The three SREs present various bent populations submitted, however, to particular internal dynamics, decisive for the mutual adjustment of binding partners and therefore specific complex formation.