c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein.

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029 ◽  
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

Intergenic region between TATA-box binding protein and proteasome subunit C3 genes of Medaka function as the bidirectional promoter in vitro and in vivo

Gene ◽  
2012 ◽  
Vol 511 (2) ◽  
pp. 177-186 ◽  
Author(s):  
Kazuhiro E. Fujimori ◽  
Kumiko Hazama ◽  
Takashi Kawasaki ◽  
Tomonori Deguchi ◽  
Syunsuke Yuba
Keyword(s):  
Intergenic Region ◽  
Binding Protein ◽  
Bidirectional Promoter ◽  
Tata Box ◽  
Proteasome Subunit ◽  
Tata Box Binding Protein

scholarly journals Evidence that TAF-TATA Box-Binding Protein Interactions Are Required for Activated Transcription in Mammalian Cells

2002 ◽  
Vol 22 (8) ◽  
pp. 2788-2798 ◽  
Author(s):  
Lisa S. Martel ◽  
Helen J. Brown ◽  
Arnold J. Berk
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Convex Surface ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Surface ◽  
Substitution Mutations ◽  
Residue Substitution ◽  
Tata Box Binding Protein

ABSTRACT Surfaces of human TATA box-binding protein (hsTBP) required for activated transcription in vivo were defined by constructing a library of surface residue substitution mutations and assaying them for their ability to support activated transcription in transient-transfection assays. In earlier work, three regions were identified where mutations inhibited activated transcription without interfering with TATA box DNA binding. One region is on the upstream surface of the N-terminal TBP repeat with respect to the direction of transcription and corresponds to the TBP surface that interacts with TFIIA. A second region on the stirrup of the C-terminal TBP repeat corresponds to the TFIIB-binding surface. Here we report that the third region where mutations inhibit activated transcription in mammalian cells, the convex surface of the N-terminal repeat, corresponds to a surface on TBP that interacts with hsTAF1, the major scaffold subunit of TFIID. Since mutations at the center of the hsTAF1-interacting region inhibit the ability of the protein to support activated transcription in vivo, these results are consistent with the conclusion that an interaction between hsTBP and TAFIIs is required for activated transcription in mammalian cells.

scholarly journals In-vivo crystals reveal protein interactions

2019 ◽  
Author(s):  
Eleanor R. Martin ◽  
Alessandro Barbieri ◽  
Robert C. Ford ◽  
Robert C. Robinson
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Pdz Domain ◽  
Cell Types ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
Reporter System ◽  
X Ray Crystallography

AbstractCrystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at −1 and −3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

scholarly journals Differentiation-Dependent Interactions between RUNX-1 and FLI-1 during Megakaryocyte Development

2009 ◽  
Vol 29 (15) ◽  
pp. 4103-4115 ◽  
Author(s):  
Hui Huang ◽  
Ming Yu ◽  
Thomas E. Akie ◽  
Tyler B. Moran ◽  
Andrew J. Woo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Transcriptional Activation ◽  
Fetal Liver ◽  
Gel Filtration ◽  
Cell Types ◽  
Protein Protein Interactions ◽  
Megakaryocyte Differentiation

ABSTRACT The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

scholarly journals Ways into Understanding HIF Inhibition

Cancers ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 159
Author(s):  
Tina Schönberger ◽  
Joachim Fandrey ◽  
Katrin Prost-Fingerle
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Protein Protein Interactions ◽  
Low Oxygen ◽  
Drug Candidates ◽  
Open Questions ◽  
Wide Range ◽  
Hif Pathway

Hypoxia is a key characteristic of tumor tissue. Cancer cells adapt to low oxygen by activating hypoxia-inducible factors (HIFs), ensuring their survival and continued growth despite this hostile environment. Therefore, the inhibition of HIFs and their target genes is a promising and emerging field of cancer research. Several drug candidates target protein–protein interactions or transcription mechanisms of the HIF pathway in order to interfere with activation of this pathway, which is deregulated in a wide range of solid and liquid cancers. Although some inhibitors are already in clinical trials, open questions remain with respect to their modes of action. New imaging technologies using luminescent and fluorescent methods or nanobodies to complement widely used approaches such as chromatin immunoprecipitation may help to answer some of these questions. In this review, we aim to summarize current inhibitor classes targeting the HIF pathway and to provide an overview of in vitro and in vivo techniques that could improve the understanding of inhibitor mechanisms. Unravelling the distinct principles regarding how inhibitors work is an indispensable step for efficient clinical applications and safety of anticancer compounds.

scholarly journals EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling

2021 ◽  
Author(s):  
Liqing Jia ◽  
Xiaolu Ge ◽  
Chao Du ◽  
Linna Chen ◽  
Yanhong Zhou ◽  
...  
Keyword(s):  
Lung Adenocarcinoma ◽  
Protein Interactions ◽  
Elongation Factor ◽  
Protein Translation ◽  
Protein Protein Interactions ◽  
Promising Target ◽  
Tgfβ Receptor ◽  
Mesenchymal Transformation

Abstract Background Eukaryotic protein translation elongation factor 1α2 (EEF1A2) is an oncogene that promotes the progression of breast and pancreatic cancer. In this study, we aimed to elucidate the oncogenic function of EEF1A2 in the metastasis of lung adenocarcinoma (LUAD). Methods Immunohistochemistry and western blot were used to study EEF1A2 expression levels in LUAD tissues and cells, respectively. The role of EEF1A2 in LUAD progression were investigated in vitro and in vivo. We identified potential EEF1A2-binding proteins by liquid chromatography-electrospray mass spectrometry (LC-MS)/MS. Protein–protein interactions were determined by immunofluorescence and co-immunoprecipitation (Co-IP). Results In this study, we report that EEF1A2 mediates the epithelial–mesenchymal transformation (EMT), to promote the metastasis of LUAD cells in vitro and in vivo. Moreover, EEF1A2 interacts with HSP90AB1 to increase TGFβ Receptor (TβR)-I, and TβRII expression, followed by enhanced SMAD3 and pSMAD3 expression and nuclear localisation, which promotes the EMT of LUAD cells. Overexpression of EEF1A2 in cancer tissues is associated with poor prognosis and short survival of patients with LUAD. Conclusions These findings underscore the molecular functions of EEF1A2 in LUAD metastasis and indicate that EEF1A2 represents a promising target in the treatment of aggressive LUAD.

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