scholarly journals Separation of v-Src-induced mitogenesis and morphological transformation by inhibition of AP-1.

1994 ◽  
Vol 5 (11) ◽  
pp. 1177-1184 ◽  
Author(s):  
M C Frame ◽  
K Simpson ◽  
V J Fincham ◽  
D H Crouch
Keyword(s):  
Cell Cycle ◽  
Leucine Zipper ◽  
Binding Activity ◽  
Temperature Sensitive ◽  
Morphological Transformation ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cell Cycle Reentry ◽  
Gel Mobility Shift ◽  
Basic Region

v-Src activity results in both morphological transformation and reentry of quiescent chick embryo fibroblasts (CEF) into cell cycle. We have previously used temperature-sensitive v-Src mutants to show that enhanced activity of cellular AP-1 in the first few hours after activation of v-Src invariably precedes the biological consequences. Here we have investigated whether the early activation of AP-1 is essential for any or all of the v-Src responses by using a mutant c-Fos that comprises the leucine zipper and a disrupted basic region. Expression of the c-Fos mutant partially reduced cellular AP-1 activity in exponentially growing cells. However, in CEF that had been made quiescent by serum deprivation, v-Src-induced stimulation of AP-1 DNA binding activity was substantially reduced. In addition, quiescent CEF stably transfected with this mutant show an impaired mitogenic response to v-Src, indicating that the AP-1 stimulation is a necessary prerequisite for cell-cycle reentry. The ability of v-Src to morphologically transform quiescent CEF was not impaired by the inhibition of AP-1 stimulation, indicating that the mitogenic and morphological consequences of v-Src have distinguishable biochemical mediators. Focal adhesion kinase, a recently identified determinant of cell morphology, undergoes a gel mobility shift, characteristic of its hyperphosphorylated state, in response to v-Src activation in cells expressing the inhibitory AP-1 protein. This provides further evidence that the pathways that regulate morphological transformation are independent of AP-1.

scholarly journals The CDK7-cycH-p36 complex of transcription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro.

1997 ◽  
Vol 17 (10) ◽  
pp. 5923-5934 ◽  
Author(s):  
H Lu ◽  
R P Fisher ◽  
P Bailey ◽  
A J Levine
Keyword(s):  
Transcription Factor ◽  
Excision Repair ◽  
Binding Activity ◽  
Mobility Shift ◽  
Trimeric Complex ◽  
Dna Binding Activity ◽  
Terminal Amino ◽  
Gel Mobility Shift ◽  
Kinase Phosphorylation

Phosphorylation is believed to be one of the mechanisms by which p53 becomes activated or stabilized in response to cellular stress. Previously, p53 was shown to interact with three components of transcription factor IIH (TFIIH): excision repair cross-complementing types 2 and 3 (ERCC2 and ERCC3) and p62. This communication demonstrates that p53 is phosphorylated by the TFIIH-associated kinase in vitro. The phosphorylation was found to be catalyzed by the highly purified kinase components of TFIIH, the CDK7-cycH-p36 trimeric complex. The phosphorylation sites were mapped to the C-terminal amino acids located between residues 311 and 393. Serines 371, 376, 378, and 392 may be the potential sites for this kinase. Phosphorylation of p53 by this kinase complex enhanced the ability of p53 to bind to the sequence-specific p53-responsive DNA element as shown by gel mobility shift assays. These results suggest that the CDK7-cycH-p36 trimeric complex of TFIIH may play a role in regulating p53 functions in cells.

IL-17 stimulates inflammatory responses via NF-κB and MAP kinase pathways in human colonic myofibroblasts

2002 ◽  
Vol 282 (6) ◽  
pp. G1035-G1044 ◽  
Author(s):  
Kazunori Hata ◽  
Akira Andoh ◽  
Mitsue Shimada ◽  
Sanae Fujino ◽  
Shigeki Bamba ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Northern Blotting ◽  
Binding Activity ◽  
Mitogen Activated Protein Kinase ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Tnf Α ◽  
Mobility Shift Assay ◽  
Mapk Inhibitors ◽  
Gel Mobility Shift

Colonic subepithelial myofibroblasts (SEMFs) may play a role in the modulation of mucosal inflammatory responses. We investigated the effects of interleukin (IL)-17 on IL-6 and chemokine [IL-8 and monocyte chemoattractant protein (MCP)-1] secretion in colonic SEMFs. Cytokine expression was determined by ELISA and Northern blotting. Nuclear factor kappa B (NF-κB) DNA-binding activity was evaluated by electrophortetic gel mobility shift assay (EMSA). The activation of mitogen-activated protein kinase (MAPK) was assessed by immunoblotting. IL-6, IL-8, and MCP-1 secretions were rapidly induced by IL-17. IL-17 induced NF-κB activation within 45 min after stimulation. A blockade of NF-κB activation markedly reduced these responses. MAPK inhibitors (SB-203580, PD-98059, and U-0126) significantly reduced the IL-17-induced IL-6 and chemokine secretion. The combination of either IL-17 + IL-1β or IL-17 + tumor necrosis factor (TNF)-α enhanced cytokine secretion; in particular, the effects of IL-17 + TNF-α on IL-6 secretion were much stronger than the other responses. This was dependent on the enhancement of IL-6 mRNA stability. In conclusion, human SEMFs secreted IL-6, IL-8, and MCP-1 in response to IL-17. These responses might play an important role in the pathogenesis of gut inflammation.

Activation of the NF-κB Pathway by Inflammatory Stimuli in Human Neutrophils

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3421-3433 ◽  
Author(s):  
Patrick P. McDonald ◽  
Anette Bald ◽  
Marco A. Cassatella
Keyword(s):  
Cell Activation ◽  
Protein Complexes ◽  
Binding Activity ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cytokines And Chemokines ◽  
Inflammatory Stimuli ◽  
Gel Mobility Shift

Abstract Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-κB, for inducible expression. We therefore investigated whether NF-κB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFκB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IκB-α in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-α [TNF-α], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-κB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-κB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IκB-α degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IκB-α mRNA transcripts, resulting in the reexpression of the IκB-α protein. To our knowledge, this constitutes the first indication that NF-κB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology.

scholarly journals Redox-mediated Mechanisms Regulate DNA Binding Activity of the G-group of Basic Region Leucine Zipper (bZIP) Transcription Factors inArabidopsis

2012 ◽  
Vol 287 (33) ◽  
pp. 27510-27525 ◽  
Author(s):  
Jehad Shaikhali ◽  
Louise Norén ◽  
Juan de Dios Barajas-López ◽  
Vaibhav Srivastava ◽  
Janine König ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Bzip Transcription Factors ◽  
Basic Region

scholarly journals Inhibition of the expression of ornithine decarboxylase and c-Myc by cell-permeant ceramide in difluoromethylornithine-resistant leukaemia cells

1997 ◽  
Vol 324 (3) ◽  
pp. 783-789 ◽  
Author(s):  
Flavio FLAMIGNI ◽  
Irene FAENZA ◽  
Sandra MARMIROLI ◽  
Ivana STANIC' ◽  
Antonella GIACCARI ◽  
...  
Keyword(s):  
Cell Growth ◽  
Ornithine Decarboxylase ◽  
Thymidine Incorporation ◽  
Binding Activity ◽  
Lipid Mediator ◽  
Mobility Shift ◽  
Experimental Conditions ◽  
Dna Binding Activity ◽  
C6 Ceramide ◽  
Gel Mobility Shift

Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 μM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.

scholarly journals E2F-4 switches from p130 to p107 and pRB in response to cell cycle reentry.

1996 ◽  
Vol 16 (4) ◽  
pp. 1436-1449 ◽  
Author(s):  
K Moberg ◽  
M A Starz ◽  
J A Lees
Keyword(s):  
Cell Cycle ◽  
Binding Activity ◽  
Cell Cycle Regulators ◽  
Dna Binding Activity ◽  
Cell Cycle Reentry ◽  
E2f Transcription Factor ◽  
Transition Points ◽  
The Individual ◽  
G1 Cells ◽  
Almost All

The E2F transcription factor couples the coordinate expression of cell cycle proteins to their appropriate transition points. Its activity is controlled by the cell cycle regulators pRB, p107, and p130. These bind to E2F at defined but distinct stages of the cell cycle. Using specific antisera, we have identified the DP and E2F components of each of these species. Although present at very different levels, DP-1 and DP-2 are evenly distributed among each of these complexes. In contrast, the individual E2Fs have distinctly different binding profiles. Consistent with previous studies, E2F-1, E2F-2, and E2F-3 bind specifically to the retinoblastoma protein. In each case, their expression and DNA binding activity are restricted to post-G1/S fractions. Surprisingly, E2F-1 and E2F-3 make unequal contributions to the pRB-associated and free E2F activity, suggesting that these proteins perform different cell cycle functions. Most significantly, this study showed E2F-4 accounts for the vast majority of the endogenous E2F activity. In arrested cells, E2F-4 is sequestered by the p130 protein. However, as the cells pass the G1-to-S transition, the levels of pRB and p107 increase and E2F-4 now associates with both of these regulators. Despite this, a considerable amount of E2F-4 exists as free E2F. In G1 cells, this accounts for almost all of the free activity. Once the cells enter S phase, free E2F is composed of an equal mixture of E2F-4 and E2F-1.

scholarly journals The DNA Binding Domains of P1 ParB and the Architecture of the P1 Plasmid Partition Complex

2001 ◽  
Vol 276 (15) ◽  
pp. 12385-12394 ◽  
Author(s):  
Jennifer A. Surtees ◽  
Barbara E. Funnell
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Integration Host Factor ◽  
Mobility Shift ◽  
Dna Binding Domains ◽  
High Affinity ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
C Terminus ◽  
Gel Mobility Shift

Stable maintenance of P1 plasmids inEscherichia coliis mediated by a high affinity nucleoprotein complex called the partition complex, which consists of ParB and theE. coliintegration host factor (IHF) bound specifically to the P1parSsite. IHF strongly stimulates ParB binding toparS, and the minimal partition complex contains a single dimer of ParB. To examine the architecture of the partition complex, we have investigated the DNA binding activity of various ParB fragments. Gel mobility shift and DNase I protection assays showed that the first 141 residues of ParB are dispensable for the formation of the minimal, high affinity partition complex. A fragment missing only the last 16 amino acids of ParB bound specifically toparS, but binding was weak and was no longer stimulated by IHF. The ability of IHF to stimulate ParB binding toparScorrelated with the ability of ParB to dimerize via its C terminus. Using full and partialparSsites, we show that two regions of ParB, one in the center and the other near the C terminus of the protein, interact with distinct sequences withinparS. Based on these data, we have proposed a model of how the ParB dimer bindsparSto form the minimal partition complex.

DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 281-290 ◽  
Author(s):  
Yu. Rebets ◽  
B. Ostash ◽  
A. Luzhetskyy ◽  
S. Kushnir ◽  
M. Fukuhara ◽  
...  
Keyword(s):  
Dna Binding ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Gene Promoter ◽  
Binding Activity ◽  
Mobility Shift ◽  
Chitin Binding Domain ◽  
Dna Binding Activity ◽  
Gel Mobility Shift

The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912. LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure. Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE. Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription. Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass. In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.

scholarly journals Transcriptional Regulation of theStreptococcus mutans gal Operon by the GalR Repressor

1998 ◽  
Vol 180 (21) ◽  
pp. 5727-5732 ◽  
Author(s):  
Dragana Ajdić ◽  
Joseph J. Ferretti
Keyword(s):  
Transcription Initiation ◽  
Binding Activity ◽  
Regulatory Function ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Dnase I Footprinting ◽  
Vitro Binding ◽  
Gel Mobility Shift ◽  
Transcriptional Start Sites

ABSTRACT The galactose operon of Streptococcus mutans is transcriptionally regulated by a repressor protein (GalR) encoded by the galR gene, which is divergently oriented from the structural genes of the gal operon. To study the regulatory function of GalR, we partially purified the protein and examined its DNA binding activity by gel mobility shift and DNase I footprinting experiments. The protein specifically bound to thegalR-galK intergenic region at an operator sequence, the position of which would suggest that GalR plays a role in the regulation of the gal operon as well as autoregulation. To further examine this hypothesis, transcriptional start sites of the gal operon and thegalR gene were determined. Primer extension analysis showed that both promoters overlap the operator, indicating that GalR most likely represses transcription initiation of both promoters. Finally, the results from in vitro binding experiments with potential effector molecules suggest that galactose is a true intracellular inducer of the galactose operon.

scholarly journals Curdlan sulphate modulates protein synthesis and enhances NF-κB and C/EBP binding activity in HepG2 cells

1997 ◽  
Vol 6 (1) ◽  
pp. 58-63 ◽  
Author(s):  
A. Guzdek ◽  
H. Rokita
Keyword(s):  
Protein Synthesis ◽  
Hepg2 Cells ◽  
Manganese Superoxide Dismutase ◽  
Mrna Level ◽  
Binding Activity ◽  
Mobility Shift ◽  
Intracellular Enzyme ◽  
Dna Binding Activity ◽  
Regulatory Effects ◽  
Gel Mobility Shift

In human hepatoma HepG2 cell line curdlan sulphate enhances basal and interleukin-6-stimulated fibrinogen and antichymotrypsin (ACT) synthesis, slightly increases basal ceruloplasmin production and exerts only minor effects on alpha-1-proteinase inhibitor and transferrin. Curdlan sulphate may, at least in part, affect protein synthesis at a pretranslational level, as the expression of ACT mRNA was found to be increased, whereas intracellular enzyme, manganese superoxide dismutase mRNA level was decreased in the cell culture treated with curdlan sulphate. Gel mobility shift analysis revealed that curdlan sulphate increases the DNA binding activity of NF-κB and C/EBP, suggesting that these transcription factors may participate in the regulatory effects of curdlan sulphate in HepG2 cells.

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