scholarly journals Hypertonicity-induced phosphorylation and nuclear localization of the transcription factor TonEBP

2001 ◽  
Vol 280 (2) ◽  
pp. C248-C253 ◽  
Author(s):  
Stephen C. Dahl ◽  
Joseph S. Handler ◽  
H. Moo Kwon
Keyword(s):  
Transcription Factor ◽  
Nuclear Localization ◽  
Kinase Inhibitors ◽  
Dependent Manner ◽  
Binding Assays ◽  
P38 Inhibitor ◽  
Vitro Binding ◽  
Compatible Osmolytes

The accumulation of compatible osmolytes during osmotic stress is observed in virtually all organisms. In mammals, the hypertonicity-induced expression of osmolyte transporters and synthetic enzymes is conferred by the presence of upstream tonicity-responsive enhancer (TonE) sequences. Recently, we described the cloning and initial characterization of TonE-binding protein (TonEBP), a transcription factor that translocates to the nucleus and associates with TonE sequences in a tonicity-dependent manner. We now report that hypertonicity induces an increase in TonEBP phosphorylation that temporally correlates with increased nuclear localization of the molecule. TonEBP phosphorylation is not affected by a number of kinase inhibitors, including the p38 inhibitor SB-203580. In addition, in vitro binding assays show that the association of TonEBP with TonE sequences is not affected by phosphorylation. Thus TonEBP phosphorylation is an early step in the response of cells to hypertonicity and may be required for nuclear import or retention.

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

scholarly journals Endobrevin, a Novel Synaptobrevin/VAMP-Like Protein Preferentially Associated with the Early Endosome

1998 ◽  
Vol 9 (6) ◽  
pp. 1549-1563 ◽  
Author(s):  
Siew Heng Wong ◽  
Tao Zhang ◽  
Yue Xu ◽  
V. Nathan Subramaniam ◽  
Gareth Griffiths ◽  
...  
Keyword(s):  
Synaptic Vesicles ◽  
Early Endosome ◽  
Biological Characterization ◽  
Binding Assays ◽  
Indirect Immunofluorescence Microscopy ◽  
General Importance ◽  
Vitro Binding ◽  
Main Components

Synaptobrevins/vesicle-associated membrane proteins (VAMPs) together with syntaxins and a synaptosome-associated protein of 25 kDa (SNAP-25) are the main components of a protein complex involved in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, biochemical, and cell biological characterization of a novel member of the synaptobrevin/VAMP family. The amino acid sequence of endobrevin has 32, 33, and 31% identity to those of synaptobrevin/VAMP-1, synaptobrevin/VAMP-2, and cellubrevin, respectively. Membrane fractionation studies demonstrate that endobrevin is enriched in membrane fractions that are also enriched in the asialoglycoprotein receptor. Indirect immunofluorescence microscopy establishes that endobrevin is primarily associated with the perinuclear vesicular structures of the early endocytic compartment. The preferential association of endobrevin with the early endosome was further established by electron microscopy (EM) immunogold labeling. In vitro binding assays show that endobrevin interacts with immobilized recombinant α-SNAP fused to glutathioneS-transferase (GST). Our results highlight the general importance of members of the synaptobrevin/VAMP protein family in membrane traffic and provide new avenues for future functional and mechanistic studies of this protein as well as the endocytotic pathway.

scholarly journals Interactions of Pex7p and Pex18p/Pex21p with the Peroxisomal Docking Machinery: Implications for the First Steps in PTS2 Protein Import

2002 ◽  
Vol 22 (17) ◽  
pp. 6056-6069 ◽  
Author(s):  
Katharina Stein ◽  
Annette Schell-Steven ◽  
Ralf Erdmann ◽  
Hanspeter Rottensteiner
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Dependent Manner ◽  
Binding Assays ◽  
Interaction Domain ◽  
Vitro Binding ◽  
Targeting Signal ◽  
Two Hybrid ◽  
Import Receptor

ABSTRACT Peroxisomal PTS2-dependent matrix protein import starts with the recognition of the PTS2 targeting signal by the import receptor Pex7p. Subsequently, the formed Pex7p/cargo complex is transported from the cytosol to the peroxisomal docking complex, consisting of Pex13p and Pex14p. In Saccharomyces cerevisiae, the latter event is thought to require the redundant Pex18p and Pex21p. Here we mapped the Pex7p interaction domain of Pex13p to its N-terminal 100 amino acids. Pex18p and Pex21p also interacted with this region, albeit only in the presence of Pex7p. Expression of an N-terminally deleted version of Pex13p in a pex13Δ mutant failed to restore growth on fatty acids due to a specific defect in the import of PTS2-containing proteins. We further show by yeast two-hybrid analysis, coimmunoprecipitation, and in vitro binding assays that Pex7p can bind Pex13p and Pex14p in the absence of Pex18p/Pex21p. The PTS2 protein thiolase was shown to interact with Pex14p but not with Pex13p in a Pex7p- and Pex18p/Pex21p-dependent manner, suggesting that only Pex14p binds cargo-loaded PTS2 receptor. We also found that the cytosolic Pex7p/thiolase-containing complex includes Pex18p. This complex accumulated in docking mutants but was absent in cells lacking Pex18p/Pex21p, indicating that Pex18p/Pex21p are required already before the docking event.

scholarly journals CIF150, a Human Cofactor for Transcription Factor IID-Dependent Initiator Function

1998 ◽  
Vol 18 (1) ◽  
pp. 233-239 ◽  
Author(s):  
Jörg Kaufmann ◽  
Katharina Ahrens ◽  
Ronald Koop ◽  
Stephen T. Smale ◽  
Rolf Müller
Keyword(s):  
Transcription Factor ◽  
Core Promoter ◽  
Direct Interaction ◽  
Striking Similarity ◽  
Binding Assays ◽  
Vitro Binding ◽  
Transcription Factor Iid ◽  
Tata Box Binding Protein ◽  
Tfiid Complex

ABSTRACT The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAFII150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAFII150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAFII150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAFII135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals Identification of Potential Key lncRNAs in the Context of Mouse Myeloid Differentiation by Systematic Transcriptomics Analysis

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 630
Author(s):  
Yongqing Lan ◽  
Meng Li ◽  
Shuangli Mi
Keyword(s):  
Transcription Factor ◽  
Cell Model ◽  
Myeloid Differentiation ◽  
Rna Seq ◽  
Hematopoietic Differentiation ◽  
Granulocytic Differentiation ◽  
Expression Of Genes ◽  
Non Coding Rnas

Hematopoietic differentiation is a well-orchestrated process by many regulators such as transcription factor and long non-coding RNAs (lncRNAs). However, due to the large number of lncRNAs and the difficulty in determining their roles, the study of lncRNAs is a considerable challenge in hematopoietic differentiation. Here, through gene co-expression network analysis over RNA-seq data generated from representative types of mouse myeloid cells, we obtained a catalog of potential key lncRNAs in the context of mouse myeloid differentiation. Then, employing a widely used in vitro cell model, we screened a novel lncRNA, named Gdal1 (Granulocytic differentiation associated lncRNA 1), from this list and demonstrated that Gdal1 was required for granulocytic differentiation. Furthermore, knockdown of Cebpe, a principal transcription factor of granulocytic differentiation regulation, led to down-regulation of Gdal1, but not vice versa. In addition, expression of genes involved in myeloid differentiation and its regulation, such as Cebpa, were influenced in Gdal1 knockdown cells with differentiation blockage. We thus systematically identified myeloid differentiation associated lncRNAs and substantiated the identification by investigation of one of these lncRNAs on cellular phenotype and gene regulation levels. This study promotes our understanding of the regulation of myeloid differentiation and the characterization of roles of lncRNAs in hematopoietic system.

scholarly journals Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

1995 ◽  
Vol 15 (6) ◽  
pp. 3147-3153 ◽  
Author(s):  
G A Blobel ◽  
C A Sieff ◽  
S H Orkin
Keyword(s):  
Bone Marrow ◽  
Estrogen Receptor ◽  
Transcription Factor ◽  
Progenitor Cells ◽  
Erythroid Cell ◽  
High Dose ◽  
Dependent Manner ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

scholarly journals Two Isoforms of Npap60 (Nup50) Differentially Regulate Nuclear Protein Import

2010 ◽  
Vol 21 (4) ◽  
pp. 630-638 ◽  
Author(s):  
Yutaka Ogawa ◽  
Yoichi Miyamoto ◽  
Munehiro Asally ◽  
Masahiro Oka ◽  
Yoshinari Yasuda ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Protein ◽  
Time Lapse ◽  
Binding Assays ◽  
Vitro Binding ◽  
Importin Α ◽  
Nuclear Protein Import

Npap60 (Nup50) is a nucleoporin that binds directly to importin α. In humans, there are two Npap60 isoforms: the long (Npap60L) and short (Npap60S) forms. In this study, we provide both in vitro and in vivo evidence that Npap60L and Npap60S function differently in nuclear protein import. In vitro binding assays revealed that Npap60S stabilizes the binding of importin α to classical NLS-cargo, whereas Npap60L promotes the release of NLS-cargo from importin α. In vivo time-lapse experiments showed that when the Npap60 protein level is controlled, allowing CAS to efficiently promote the dissociation of the Npap60/importin α complex, Npap60S and Npap60L suppress and accelerate the nuclear import of NLS-cargo, respectively. These results demonstrate that Npap60L and Npap60S have opposing functions and suggest that Npap60L and Npap60S levels must be carefully controlled for efficient nuclear import of classical NLS-cargo in humans. This study provides novel evidence that nucleoporin expression levels regulate nuclear import efficiency.

scholarly journals Regulation of PDGFR-α in rat pulmonary myofibroblasts by staurosporine

2001 ◽  
Vol 280 (2) ◽  
pp. L354-L362 ◽  
Author(s):  
Pamela M. Lindroos ◽  
Yi-Zhe Wang ◽  
Annette B. Rice ◽  
James C. Bonner
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Protein A ◽  
Nuclear Factor Κb ◽  
Mitogen Activated Protein Kinase ◽  
Specific Gene ◽  
Autocrine Loop ◽  
P38 Inhibitor ◽  
Mitogen Activated Protein

Upregulation of the platelet-derived growth factor (PDGF) receptor-α (PDGFR-α) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1β as a major inducer of the PDGFR-α in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-α gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-α expression. Staurosporine did not act via an IL-1β autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-α expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1β, including nuclear factor-κB, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1β-induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-α by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-α expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-α as a growth arrest-specific gene.

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