scholarly journals The MEKK1 SWIM domain is a novel substrate receptor for c-Jun ubiquitylation

2012 ◽  
Vol 445 (3) ◽  
pp. 431-439 ◽  
Author(s):  
Michael A. Rieger ◽  
Tyler Duellman ◽  
Christopher Hooper ◽  
Magdalene Ameka ◽  
Joanna C. Bakowska ◽  
...  
Keyword(s):  
Dna Binding ◽  
Specific Interaction ◽  
Regulatory Region ◽  
Direct Interaction ◽  
Binding Domain ◽  
Ring Domain ◽  
Mitogen Activated Protein Kinase ◽  
Activator Protein 1 ◽  
Dna Binding Domains ◽  
Binding Domains

MEKK1 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase kinase 1] is a MAP3K (MAPK kinase kinase) that regulates MAPK activation, and is the only known mammalian kinase that is also a ubiquitin ligase. MEKK1 contains a RING domain within its N-terminal regulatory region, and MEKK1 has been shown to ubiquitylate the AP-1 (activator protein 1) transcription factor protein c-Jun, but the mechanism by which MEKK1 interacts with c-Jun to induce ubiquitylation has not been defined. Proximal to the RING domain is a SWIM (SWI2/SNF2 and MuDR) domain of undetermined function. In the present study, we demonstrate that the MEKK1 SWIM domain, but not the RING domain, directly associates with the c-Jun DNA-binding domain, and that the SWIM domain is required for MEKK1-dependent c-Jun ubiquitylation. We further show that this MEKK1 SWIM–Jun interaction is specific, as SWIM domains from other proteins failed to bind c-Jun. We reveal that, although the Jun and Fos DNA-binding domains are highly conserved, the MEKK1 SWIM domain does not bind Fos. Finally, we identify the sequence unique to Jun proteins required for specific interaction with the MEKK1 SWIM domain. Therefore we propose that the MEKK1 SWIM domain represents a novel substrate-binding domain necessary for direct interaction between c-Jun and MEKK1 that promotes MEKK1-dependent c-Jun ubiquitylation.

scholarly journals Structure of the XPA DNA binding domain and RPA high-affinity DNA binding domains on a model NER substrate

2019 ◽  
Vol 75 (a1) ◽  
pp. a203-a203
Author(s):  
Walter J. Chazin ◽  
Agnieszka M. Topolska-Woś ◽  
Norie Sugitani ◽  
John J. Cordoba ◽  
Hyun Suk Kim ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains

Biologic significance of GATA-1 activities in Ras-mediated megakaryocytic differentiation of hematopoietic cell lines

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2440-2450 ◽  
Author(s):  
Itaru Matsumura ◽  
Akira Kawasaki ◽  
Hirokazu Tanaka ◽  
Junko Sonoyama ◽  
Sachiko Ezoe ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Cell Lines ◽  
Direct Interaction ◽  
Myeloid Cell ◽  
Megakaryocytic Differentiation ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Endogenous Expression ◽  
Ras Activation

Abstract Lineage-specific transcription factors play crucial roles in the development of hematopoietic cells. In a previous study, it was demonstrated that Ras activation was involved in thrombopoietin-induced megakaryocytic differentiation. In this study, constitutive Ras activation by H-rasG12V evoked megakaryocytic maturation of erythroleukemia cell lines F-36P and K562, but not of myeloid cell line 32D cl3 that lacks GATA-1. However, the introduction of GATA-1 led to reprogramming of 32D cl3 toward erythrocytic/megakaryocytic lineage and enabled it to undergo megakaryocytic differentiation in response to H-rasG12V. In contrast, the overexpression of PU.1 and c-Myb changed the phenotype of K562 from erythroid to myeloid/monocytic lineage and rendered K562 to differentiate into granulocytes and macrophages in response to H-rasG12V, respectively. In GATA-1–transfected 32D cl3, the endogenous expression of PU.1 and c-Myb was easily detectable, but their activities were reduced severely. Endogenous GATA-1 activities were markedly suppressed in PU.1-transfected and c-myb–transfected K562. As for the mechanisms of these reciprocal inhibitions, GATA-1 and PU.1 were found to associate through their DNA-binding domains and to inhibit the respective DNA-binding activities of each other. In addition, c-Myb bound to GATA-1 and inhibited its DNA-binding activities. Mutant GATA-1 and PU.1 that retained their own transcriptional activities but could not inhibit the reciprocal partner were less effective in changing the lineage phenotype of 32D cl3 and K562. These results suggested that GATA-1 activities may be crucial for Ras-mediated megakaryocytic differentiation and that its activities may be regulated by the direct interaction with other lineage-specific transcription factors such as PU.1 and c-Myb.

scholarly journals The DNA Architectural Protein HMGB1 Displays Two Distinct Modes of Action That Promote Enhanceosome Assembly

2002 ◽  
Vol 22 (12) ◽  
pp. 4390-4401 ◽  
Author(s):  
Katherine Mitsouras ◽  
Ben Wong ◽  
Charina Arayata ◽  
Reid C. Johnson ◽  
Michael Carey
Keyword(s):  
Dna Binding ◽  
Mutational Analysis ◽  
Regulatory Protein ◽  
Specific Interaction ◽  
Regulatory Region ◽  
Direct Interaction ◽  
Single Amino Acid ◽  
Target Sequence ◽  
Mobility Shift ◽  
Epstein Barr

ABSTRACT HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites. Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA. Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1. HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer. DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex. Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence. The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU. These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence. The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA. We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts.

scholarly journals Specific Binding of Integrase to the Origin of Transfer (oriT) of the Conjugative Transposon Tn916

2001 ◽  
Vol 183 (9) ◽  
pp. 2947-2951 ◽  
Author(s):  
Douglas Hinerfeld ◽  
Gordon Churchward
Keyword(s):  
Dna Binding ◽  
Specific Binding ◽  
Binding Domain ◽  
Conjugal Transfer ◽  
Dna Binding Domain ◽  
Conjugative Transposon ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
A Site ◽  
Integrase Protein

ABSTRACT Purified integrase protein (Int) of the conjugative transposon Tn916 was shown, using nuclease protection experiments, to bind specifically to a site within the origin of conjugal transfer of the transposon, oriT. A sequence similar to the ends of the transposon that are bound by the C-terminal DNA-binding domain of Int was present in the protected region. However, Int binding tooriT required both the N- and C-terminal DNA-binding domains of Int, and the pattern of nuclease protection differed from that observed when Int binds to the transposon ends and flanking DNA. Binding of Int to oriT may be part of a mechanism to prevent premature conjugal transfer of Tn916 prior to excision from the donor DNA.

scholarly journals Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator

2004 ◽  
Vol 186 (11) ◽  
pp. 3499-3507 ◽  
Author(s):  
Hao Xu ◽  
Baohua Gu ◽  
B. Tracy Nixon ◽  
Timothy R. Hoover
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Sinorhizobium Meliloti ◽  
Atp Hydrolysis ◽  
Binding Domain ◽  
Stable Complex ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.

scholarly journals The Application of an Immobilized Molecular Beacon for the Analysis of the DNA Binding Domains from the Ecdysteroid Receptor Proteins Usp and EcR's Interaction with the hsp27 Response Element

2008 ◽  
Vol 13 (9) ◽  
pp. 899-905 ◽  
Author(s):  
Tomasz Krusiński ◽  
Anna Laskowska ◽  
Andrzej Ożyhar ◽  
Piotr Dobryszycki
Keyword(s):  
Dna Binding ◽  
Dissociation Constant ◽  
Molecular Beacon ◽  
Solid Phase ◽  
Specific Interaction ◽  
Resonance Energy ◽  
Response Element ◽  
Receptor Proteins ◽  
Dna Binding Domains ◽  
Binding Domains

The nonstandard molecular beacon described in this article consists of 2 fragments, each built of a short single-stranded oligonucleotide sequence and a double-stranded sequence. One of these hybridization probes, labeled with a fluorescence donor (fluorescein), is solid phase immobilized. The second nonimmobilized probe is labeled with a fluorescence quencher (dabcyl). Annealing of both probes via single-stranded sequences was possible only in the presence of a specific protein molecule that recognized the response element sequence initially separated between the immobilized and nonimmobilized fragments. The system was applied successfully to detect the sequence-specific interaction of a natural hsp27 response element from the promoter of the hsp27 gene with the DNA binding domains of 2 nuclear receptor proteins: ultraspiracle Usp (UspDBD) and the ecdysone receptor EcR (EcRDBD). Measured in the absence of EcRDBD, the dissociation constant, Kd of the UspDBD- hsp27 complex, was determined to be 3.26 nM, whereas for UspDBD devoid of the A-box (UspDBDΔA hsp27 ), the dissociation constant was 4.81 nM. The respective Kd values in the presence of EcRDBD were 2.43 nM and 10.80 nM. The results obtained with the immobilized molecular beacon technology were in agreement with those obtained by conventional fluorescence titrations and by fluorescence resonance energy transfer measurements with nonimmobilized beacons. ( Journal of Biomolecular Screening 2008:899-905)

scholarly journals Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.

1996 ◽  
Vol 16 (3) ◽  
pp. 792-799 ◽  
Author(s):  
S L Gregory ◽  
R D Kortschak ◽  
B Kalionis ◽  
R Saint
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Dna Binding Proteins ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
The Dead

We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.

scholarly journals B-Cell Coactivator OBF-1 Exhibits Unusual Transcriptional Properties and Functions in a DNA-Bound Oct-1-Dependent Fashion

1999 ◽  
Vol 19 (6) ◽  
pp. 4247-4254 ◽  
Author(s):  
Andrea Krapp ◽  
Michel Strubin
Keyword(s):  
Dna Binding ◽  
B Cell ◽  
Target Genes ◽  
Regulatory Elements ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT Eukaryotic transcriptional activators generally comprise both a DNA-binding domain that recognizes specific cis-regulatory elements in the target genes and an activation domain which is essential for transcriptional stimulation. Activation domains typically behave as structurally and functionally autonomous modules that retain their intrinsic activities when directed to a promoter by a variety of heterologous DNA-binding domains. Here we report that OBF-1, a B-cell-specific coactivator for transcription factor Oct-1, challenges this traditional view in that it contains an atypical activation domain that exhibits two unexpected functional properties when tested in the yeast Saccharomyces cerevisiae. First, OBF-1 by itself has essentially no intrinsic activation potential, yet it strongly synergizes with other activation domains such as VP16 and Gal4. Second, OBF-1 exerts its effect in association with DNA-bound Oct-1 but is inactive when attached to a heterologous DNA-binding domain. These findings suggest that activation by OBF-1 is not obtained by simple recruitment of the coactivator to the promoter but requires interaction with DNA-bound Oct-1 to stimulate a step distinct from those regulated by classical activation domains.

scholarly journals Expression and purification of 6xHis-tagged DNA binding domains of functional ecdysteroid receptor from drosophila melanogaster.

1996 ◽  
Vol 43 (4) ◽  
pp. 611-621 ◽  
Author(s):  
A Rusin ◽  
A Niedziela-Majka ◽  
G Rymarczyk ◽  
A Ozyhar
Keyword(s):  
Dna Binding ◽  
Dna Interaction ◽  
Binding Domain ◽  
Structural Basis ◽  
Dna Binding Domain ◽  
Affinity Tag ◽  
Mobility Shift ◽  
Ecdysteroid Receptor ◽  
Dna Binding Domains ◽  
Binding Domains

Two members of the nuclear receptor superfamily, EcR and Ultraspiracle (Usp) heterodimerize to form a functional receptor for 20-hydroxyecdysone-the key ecdysteroid controlling induction and modulation of morphogenetic events through Drosophila development. In order to study aspects of receptor function and ultimately the structural basis of the ecdysteroid receptor-DNA interaction, it is necessary to produce large quantities of purified EcR and Usp DNA-binding domains. Toward this end, we have expressed the EcR DNA-binding domain and the Usp DNA-binding domain as proteins with an affinity tag consisting of six histidine residues (6xHis-EcRDBD and 6xHis-UspDBD, respectively) using the expression vector pQE-30. Under optimal conditions, elaborated in this study, bacteria can express the recombinant 6xHis-EcRDBD to the levels of 11% of total soluble proteins and the 6xHis-UspDBD to the levels of 16%. Both proteins were purified to homogeneity from the soluble protein fraction using combination of ammonium sulphate fractionation and affinity chromatography on Ni-NTA agarose. The gel mobility shift experiments demonstrated that the purified 6xHis-EcRDBD and the 6xHis-UspDBD interact specifically with an 20-hydroxyecdysone response element from the promoter region of the hsp 27 Drosophila gene.

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