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Results for “Multiprotein Complexes”

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At least 343 records · Page 19Linked to original sources

PubMed2004

Cooperative activation of atrial naturetic peptide promoter by dHAND and MEF2C.

An intricate array of cell-specific multiprotein complexes participate in programs of cell-specific gene expression through combinatorial interaction with different transcription factors and cofactors. The dHAND basic helix-loop-helix (bHLH) transcription factor, which is essential for heart development and extra embryonic structures, is thought to regulate cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted transcription factors such as GATA4 and NKX2.5. Here, we determine that dHAND also interacts with the myocyte enhancer binding factor-2c (MEF2C) protein, which belongs to MADS-box transcription factors and is essential for heart development. dHAND and MEF2C synergistically activated expression of the atrial naturetic peptide gene (ANP) in transfected HeLa cells. GST-pulldown and immunoprecipitation assay demonstrate that full-length MEF2C protein is able to interact with dHAND in vitro and in vivo, just like MEF2A and bHLH transcription factors MyoD in skeletal muscle cells. In addition, electrophoretic mobility shift assays (EMSAs) demonstrate that MEF2C and dHAND do not influence each other's DNA binding activity. Using chromatin immunoprecipitation (ChIP) analysis in H9c2 cells we show that dHAND interact with MEF2C to form protein complex and bind A/T sequence in promoter of ANP. Taken together with previous observations, these results suggest the existence of large multiprotein transcriptional complex with core DNA binding proteins that physically interact with other transcriptional factors to form favorable conformation to potentiate transcription.

Atrial Natriuretic Factor↗

PubMed2006

Transcriptional regulation of PEN-2, a key component of the gamma-secretase complex, by CREB.

Gamma-secretase, which is responsible for the intramembranous cleavage of Alzheimer's beta-amyloid precursor protein (APP), the signaling receptor Notch, and many other substrates, is a multiprotein complex consisting of at least four components: presenilin (PS), nicastrin, APH-1, and PEN-2. Despite the fact that PEN-2 is known to mediate endoproteolytic cleavage of full-length PS and APH-1 and nicastrin are required for maintaining the stability of the complex, the detailed physiological function of each component remain elusive. Unlike that of PS, the transcriptional regulation of PEN-2, APH-1, and nicastrin has not been investigated. Here, we characterized the upstream regions of the human PEN-2 gene and identified a 238-bp fragment located 353 bp upstream of the translational start codon as the key region necessary for the promoter activity. Further analysis revealed a CREB binding site located in the 238-bp region that is essential for the transcriptional activity of the PEN-2 promoter. Mutation of the CREB site abolished the transcriptional activity of the PEN-2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed the binding of CREB to the PEN-2 promoter region both in vitro and in vivo. Activation of the CREB transcriptional factor by forskolin dramatically promoted the expression of PEN-2 mRNA and protein, whereas the other components of the gamma-secretase complex remained unaffected. Forskolin treatment slightly increases the secretion of soluble APPalpha and Abeta without affecting Notch cleavage. These results demonstrate that expression of PEN-2 is regulated by CREB and suggest that the specific control of PEN-2 expression may imply additional physiological functions uniquely assigned to PEN-2.

Amyloid Precursor Protein Secretases↗

PubMed2003

A nucleoprotein complex containing Sp1, C/EBP beta, and HMGI-Y controls human insulin receptor gene transcription.

HMGI-Y is an architectural transcription factor that regulates gene expression in vivo by controlling the formation of stereospecific multiprotein complexes on the AT-rich regions of certain gene promoters. Recently, we demonstrated that HMGI-Y is required for proper transcription of the insulin receptor (IR) gene. Here we provide evidence that transcriptional activation of the human IR promoter requires the assembly of a transcriptionally active multiprotein-DNA complex which includes, in addition to HMGI-Y, the ubiquitously expressed transcription factor Sp1 and the CCAAT-enhancer binding protein beta (C/EBP beta). Functional integrity of this nucleoprotein complex is required for full transactivation of the IR gene by Sp1 and C/EBP beta in cells readily expressing IRs. We show that HMGI-Y physically interacts with Sp1 and C/EBP beta and facilitates the binding of both factors to the IR promoter in vitro. Furthermore, HMGI-Y is needed for transcriptional synergism between these factors in vivo. Repression of HMGI-Y function adversely affects both Sp1- and C/EBP beta-induced transactivation of the IR promoter. Together, these findings demonstrate that HMGI-Y plays significant molecular roles in the transcriptional activities of these factors in the context of the IR gene and provide concordant support for the hypothesis that, in affected individuals, a putative defect in these nuclear proteins may cause decreased IR expression with subsequent impairment of insulin signaling and action.

3T3 Cells↗

PubMed2001

Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIRI in mediating auxin response.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Arabidopsis↗

PubMed1999

A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex.

Nuclear receptors play critical roles in the regulation of eukaryotic gene expression. We report the isolation and functional characterization of a novel transcriptional coactivator, termed steroid receptor RNA activator (SRA). SRA is selective for steroid hormone receptors and mediates transactivation via their amino-terminal activation function. We provide functional and mechanistic evidence that SRA acts as an RNA transcript; transfected SRA, unlike other steroid receptor coregulators, functions in the presence of cycloheximide, and SRA mutants containing multiple translational stop signals retain their ability to activate steroid receptor-dependent gene expression. Biochemical fractionation shows that SRA exists in distinct ribonucleoprotein complexes, one of which contains the nuclear receptor coactivator steroid receptor coactivator 1. We suggest that SRA may act to confer functional specificity upon multiprotein complexes recruited by liganded receptors during transcriptional activation.

Animals↗

PubMed2006

Tiam1 takes PARt in cell polarity.

Cell polarity is an essential requirement for the proper tissue development of complex organisms. This is underscored by in vivo studies showing that loss of cell polarity contributes to the formation and progression of tumours. Evolutionary conserved multiprotein complexes, such as the Par3-Par6-aPKC or, in short, the Par polarity complex, regulate the establishment of cell polarity. The small Rho GTPases CDC42 and Rac control the activation of the Par polarity complex. Evidence now implicates the Rac activator Tiam1 as a crucial component of the Par complex in regulating neuronal (axonal) and epithelial (apical-basal) polarity. Our current knowledge places Tiam1 at the centre of a pivotal biological process, the establishment and maintenance of cell polarity, and suggests that deregulation of the Tiam1-Par complex contributes to tumourigenicity.

Adaptor Proteins, Signal Transducing↗

PubMed1997

The N-terminus of Nef from HIV-1/SIV associates with a protein complex containing Lck and a serine kinase.

The Nef protein of human and primate lentiviruses is a key factor in HIV/SIV pathogenesis. Here we report that Nef associates with two different kinases, forming a multiprotein complex at the far N-terminus of the viral protein. One of the kinases was identified as Lck, whereas the second protein was found to be a serine kinase that phosphorylated Nef and Lck in vitro and could be discriminated from the serine kinase identified previously. The Nef-associated kinase complex (NAKC) was demonstrated in COS cells, in HIV-infected cells, and in vitro using recombinant Lck and Nef proteins. Deletion of a short amphipathic alpha-helix in the N-terminus, which was found to be conserved in all Nef proteins, inhibited association of the NAKC and significantly reduced virion infectivity.

Amino Acid Sequence↗

PubMed1997

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation.

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

Adaptor Proteins, Signal Transducing↗

PubMed1998

cDNA of eight nuclear encoded subunits of NADH:ubiquinone oxidoreductase: human complex I cDNA characterization completed.

NADH:ubiquinone oxidoreductase (complex I) is an extremely complicated multiprotein complex located in the inner mitochondrial membrane. Its main function is the transport of electrons from NADH to ubiquinone, which is accompanied by translocation of protons from the mitochondrial matrix to the intermembrane space. Human complex I appears to consist of 41 subunits of which 34 are encoded by nDNA. Here we report the cDNA sequences of the hitherto uncharacterized 8 nuclear encoded subunits, all located within the hydrophobic protein (HP) fraction of complex I. Now all currently known 41 proteins of human NADH:ubiquinone oxidoreductase have been characterized and reported in literature, which enables more complete mutational analysis studies of isolated complex I-deficient patients.

Adult↗

PubMed1998

Mapping of the human genes encoding cyclin H (CCNH) and the CDK-activating kinase (CAK) assembly factor MAT1 (MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively.

Cyclin-dependent kinases (CDKs), which play a key role in cell cycle control, are activated by the CDK-activating kinase (CAK), which activates cyclin-bound CDKs by phosphorylation at the specific threonine residue. Mammalian CAK contains three components: CDK7, cyclin H, and an assembly factor called MAT1. The CDK7-cyclin H-MAT1 complex is tightly associated with a multiprotein complex TFIIH, which plays a dual role in transcription and DNA repair. Here, we have determined chromosomal localizations of the human genes encoding cyclin H (CCNH) and MAT1 (HGMW-approved symbol MNAT1) to chromosome bands 5q13.3-q14 and 14q23, respectively, by using fluorescence in situ hybridization, somatic cell hybrid analyses, and mapping to the human YAC contigs.

Animals↗

PubMed2000

The COP/DET/FUS proteins-regulators of eukaryotic growth and development.

Eleven recessive mutant loci define the class of cop / det / fus mutants of Arabidopsis. The cop / det / fus mutants mimic the phenotype of light-grown seedlings when grown in the dark. At least four cop / det / fus mutants carry mutations in subunits of the COP9 signalosome, a multiprotein complex paralogous to the 'lid' subcomplex of the 26S proteasome. COP1, another COP/DET/FUS protein, is itself not a subunit of the COP9 signalosome. In the dark, COP1 accumulates in the nucleus where it is required for the degradation of the HY5 protein, a positive regulator of photomorphogenesis. In the light, COP1 is excluded from the nucleus and the constitutively nuclear HY5 protein can accumulate. Nuclear accumulation of COP1 and degradation of HY5 are impaired in the cop / det / fus mutants that carry mutations in subunits of the COP9 signalosome. Although the cellular function of the COP/DET/FUS proteins is not yet well understood, taken together the current findings suggest that the COP/DET/FUS proteins repress photomorphogenesis in the dark by mediating specific protein degradation.

Arabidopsis↗

PubMed2002

The proteasome regulatory particle subunit Rpn6 is required for Drosophila development and interacts physically with signalosome subunit Alien/CSN2.

The eukaryotic 26S proteasome plays a central role in ubiquitin-dependent intracellular protein metabolism. The multimeric holoenzyme is composed of two major subcomplexes, known as the 20S proteolytic core particle and the 19S regulatory particle (RP). The RP can be further dissected into two multisubunit complexes, the lid and the base complex. The lid complex shares striking similarities with another multiprotein complex, the COP9 signalosome. Several subunits of both complexes contain the characteristic PCI domain, a structural motif important for complex assembly. The COP9 signalosome was shown to act as a versatile regulator in numerous pathways. To help define the molecular interactions of the signalosome during Drosophila development, we performed a yeast two-hybrid screen to identify proteins that physically interact with subunit 2 of the complex, namely Alien/CSN2. Here, we report that Drosophila Rpn6, a non-ATPase subunit of the RP lid complex, interacts with Alien/CSN2 via its PCI domain. The temporal and spatial expression patterns of Rpn6 and alien/CSN2 overlap on a large scale during development providing additional evidence for their interaction in vivo. Analyses of an Rpn6 P element insertion mutant and newly generated Rpn6 alleles reveal that Rpn6 is essential for Drosophila development.

Amino Acid Sequence↗

PubMed2003

A single malaria merozoite serine protease mediates shedding of multiple surface proteins by juxtamembrane cleavage.

Erythrocyte invasion by the malaria merozoite is accompanied by the regulated discharge of apically located secretory organelles called micronemes. Plasmodium falciparum apical membrane antigen-1 (PfAMA-1), which plays an indispensable role in invasion, translocates from micronemes onto the parasite surface and is proteolytically shed in a soluble form during invasion. We have previously proposed, on the basis of incomplete mass spectrometric mapping data, that PfAMA-1 shedding results from cleavage at two alternative positions. We now show conclusively that the PfAMA-1 ectodomain is shed from the merozoite solely as a result of cleavage at a single site, just 29 residues away from the predicted transmembrane-spanning sequence. Remarkably, this cleavage is mediated by the same membrane-bound parasite serine protease as that responsible for shedding of the merozoite surface protein-1 (MSP-1) complex, an abundant, glycosylphosphatidylinositol-anchored multiprotein complex. Processing of MSP-1 is essential for invasion. Our results indicate the presence on the merozoite surface of a multifunctional serine sheddase with a broad substrate specificity. We further demonstrate that translocation and shedding of PfAMA-1 is an actin-independent process.

Amino Acid Sequence↗

PubMed1998

The human polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain.

The Polycomb group (PcG) complex is a chromatin-associated multiprotein complex, involved in the stable repression of homeotic gene activity in Drosophila. Recently, a mammalian PcG complex has been identified with several PcG proteins implicated in the regulation of Hox gene expression. Although the mammalian PcG complex appears analogous to the complex in Drosophila, the molecular mechanisms and functions for the mammalian PcG complex remain unknown. Here we describe a detailed characterization of the human PcG complex in terms of cellular localization and chromosomal association. By using antibodies that specifically recognize three human PcG proteins- RING1, BMI1, and hPc2-we demonstrate in a number of human cell lines that the PcG complex forms a unique discrete nuclear structure that we term PcG bodies. PcG bodies are prominent novel nuclear structures with the larger PcG foci generally localized near the centromeres, as visualized with a kinetochore antibody marker. In both normal fetal and adult fibroblasts, PcG bodies are not randomly dispersed, but appear clustered into defined areas within the nucleus. We show in three different human cell lines that the PcG complex can tightly associate with large pericentromeric heterochromatin regions (1q12) on chromosome 1, and with related pericentromeric sequences on different chromosomes, providing evidence for a mammalian PcG-heterochromatin association. Furthermore, these heterochromatin-bound PcG complexes remain stably associated throughout mitosis, thereby allowing the potential inheritance of the PcG complex through successive cell divisions. We discuss these results in terms of the known function of the PcG complex as a transcriptional repression complex.

Cell Line↗

PubMed2000

Histone H1 is a specific repressor of core histone acetylation in chromatin.

Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

Acetylation↗

PubMed2003

Recognition of a bicoid mRNA localization signal by a protein complex containing Swallow, Nod, and RNA binding proteins.

Localization of mRNAs, a process essential for embryonic body patterning in Drosophila, requires recognition of cis-acting signals by cellular components responsible for movement and anchoring. We have purified a large multiprotein complex that binds a minimal form of the bicoid mRNA localization signal in a manner both specific and sensitive to inactivating mutations. Identified complex components include the RNA binding proteins Modulo, PABP, and Smooth, the known localization factor Swallow, and the kinesin family member Nod. We demonstrate that localization of bcd mRNA is defective in modulo mutants. The presence of three required localization components (Swallow, Modulo, and specific RNA binding activity) within the recognition complex strongly implicates it in mRNA localization.

Animals↗

PubMed2004

Rad62 protein functionally and physically associates with the smc5/smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast.

Smc5 and Smc6 proteins form a heterodimeric SMC (structural maintenance of chromosome) protein complex like SMC1-SMC3 cohesin and SMC2-SMC4 condensin, and they associate with non-SMC proteins Nse1 and Nse2 stably and Rad60 transiently. This multiprotein complex plays an essential role in maintaining chromosome integrity and repairing DNA double strand breaks (DSBs). This study characterizes a Schizosaccharomyces pombe mutant rad62-1, which is hypersensitive to methyl methanesulfonate (MMS) and synthetically lethal with rad2 (a feature of recombination mutants). rad62-1 is hypersensitive to UV and gamma rays, epistatic with rhp51, and defective in repair of DSBs. rad62 is essential for viability and genetically interacts with rad60, smc6, and brc1. Rad62 protein physically associates with the Smc5-6 complex. rad62-1 is synthetically lethal with mutations in the genes promoting recovery from stalled replication, such as rqh1, srs2, and mus81, and those involved in nucleotide excision repair like rad13 and rad16. These results suggest that Rad62, like Rad60, in conjunction with the Smc5-6 complex, plays an essential role in maintaining chromosome integrity and recovery from stalled replication by recombination.

Base Sequence↗

PubMed1999

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome.

Alleles↗