scholarly journals The Alpha Chain of the Nascent Polypeptide-Associated Complex Functions as a Transcriptional Coactivator

1998 ◽  
Vol 18 (3) ◽  
pp. 1303-1311 ◽  
Author(s):  
Wagner V. Yotov ◽  
Alain Moreau ◽  
René St-Arnaud
Keyword(s):  
Affinity Chromatography ◽  
Protein Interactions ◽  
Transcriptional Control ◽  
Specific Protein ◽  
Transcriptional Coactivator ◽  
Protein Blotting ◽  
Nascent Polypeptide ◽  
Alpha Chain ◽  
Tata Box Binding Protein

ABSTRACT We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (α-NAC). Based on sequence similarities between α-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of α-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434–440, 1994), we investigated a putative role for α-NAC/1.9.2 in transcriptional control. The α-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because α-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of α-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between α-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the α-NAC/1.9.2 gene product functions as a transcriptional coactivator.

scholarly journals Bone-Specific Expression of the Alpha Chain of the Nascent Polypeptide-Associated Complex, a Coactivator Potentiating c-Jun-Mediated Transcription

1998 ◽  
Vol 18 (3) ◽  
pp. 1312-1321 ◽  
Author(s):  
Alain Moreau ◽  
Wagner V. Yotov ◽  
Francis H. Glorieux ◽  
René St-Arnaud
Keyword(s):  
Leucine Zipper ◽  
Transcriptional Coactivator ◽  
Recognition Sequence ◽  
Developmentally Regulated ◽  
Specific Expression ◽  
Nascent Polypeptide ◽  
Alpha Chain ◽  
Two Hybrid

ABSTRACT The alpha chain of the nascent polypeptide-associated complex (α-NAC) coactivator was shown to potentiate the activity of the homodimeric c-Jun activator, while transcription mediated by the c-Fos/c-Jun heterodimer was unaffected. The use of deletion mutants in pull-down assays revealed that α-NAC interacted with amino acids 1 to 89 of the c-Jun protein and that the coactivator could interact with both the unphosphorylated and the serine 73-phosphorylated form of c-Jun. N-terminal-deleted c-Jun protein failed to interact with α-NAC in mammalian two-hybrid assays, while mutant c-Jun proteins lacking the leucine zipper or the basic domain retained interaction with α-NAC in vivo. Kinetics studies with purified c-Jun homodimer and recombinant α-NAC proteins allowed determination of the mechanism of coactivation by α-NAC: the coactivator stabilized the AP-1 complex formed by the c-Jun homodimer on its DNA recognition sequence through an eightfold reduction in the dissociation constant (kd ) of the complex. This effect of α-NAC was specific, because α-NAC could not stabilize the interactions of JunB or Sp1 with their cognate binding sites. Interestingly, the expression of α-NAC was first detected at 14.5 to 15 days postconception, concomitantly with the onset of ossification during embryogenesis. The α-NAC protein was specifically expressed in differentiated osteoblasts at the centers of ossification. Thus, the α-NAC gene product exhibits the properties of a developmentally regulated, bone-specific transcriptional coactivator.

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.

The study of protein–protein interactions in bacteria

2012 ◽  
Vol 58 (11) ◽  
pp. 1241-1257 ◽  
Author(s):  
Roberto Velasco-García ◽  
Rocío Vargas-Martínez
Keyword(s):  
Hybrid System ◽  
High Throughput ◽  
Protein Interactions ◽  
Specific Protein ◽  
False Positives ◽  
Interaction Networks ◽  
Protein Protein Interactions ◽  
Extensive Data ◽  
False Positives And Negatives

Many of the functions fulfilled by proteins in the cell require specific protein–protein interactions (PPI). During the last decade, the use of high-throughput experimental technologies, primarily based on the yeast 2-hybrid system, generated extensive data currently located in public databases. This information has been used to build interaction networks for different species. Unfortunately, due to the nature of the yeast 2-hybrid system, these databases contain many false positives and negatives, thus they require purging. A method for confirming these PPI is to test them using a technique that operates in vivo and detects binary PPI. This article comprises an overview of the study of PPI and describes the main techniques that have been used to identify bacterial PPI, prioritizing those that can be used for their verification, and it also mentions a number of PPI that have been identified or confirmed using these methods.

scholarly journals A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

1995 ◽  
Vol 15 (10) ◽  
pp. 5214-5225 ◽  
Author(s):  
A D Catling ◽  
H J Schaeffer ◽  
C W Reuter ◽  
G R Reddy ◽  
M J Weber
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Phosphorylation Site ◽  
Specific Protein ◽  
Protein Protein Interactions ◽  
Serum Stimulation ◽  
And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

scholarly journals Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C Branon ◽  
Alice Y Ting ◽  
Dominique C Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

scholarly journals The Transcriptional Coactivator Cbp Interacts with β-Catenin to Activate Gene Expression

2000 ◽  
Vol 149 (2) ◽  
pp. 249-254 ◽  
Author(s):  
Ken-Ichi Takemaru ◽  
Randall T. Moon
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Target Genes ◽  
Terminal Region ◽  
Creb Binding Protein ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid System ◽  
Recruitment System

β-Catenin plays a pivotal role in the transcriptional activation of Wnt-responsive genes by binding to TCF/LEF transcription factors. Although it has been suggested that the COOH-terminal region of β-catenin functions as an activation domain, the mechanisms of activation remain unclear. To screen for potential transcriptional coactivators that bind to the COOH-terminal region of β-catenin, we used a novel yeast two-hybrid system, the Ras recruitment system (RRS) that detects protein–protein interactions at the inner surface of the plasma membrane. Using this system, we isolated the CREB-binding protein (CBP). Armadillo (Arm) repeat 10 to the COOH terminus of β-catenin is involved in binding to CBP, whereas β-catenin interacts directly with the CREB-binding domain of CBP. β-Catenin synergizes with CBP to stimulate the activity of a synthetic reporter in vivo. Conversely, β-catenin–dependent transcriptional activation is repressed by E1A, an antagonist of CBP function, but not by an E1A mutant that does not bind to CBP. The activation of Wnt target genes such as siamois and Xnr3 in Xenopus embryos is also sensitive to E1A. These findings suggest that CBP provides a link between β-catenin and the transcriptional machinery, and possibly mediates the oncogenic function of β-catenin.

scholarly journals The SH2 domain of P210BCR/ABL is not required for the transformation of hematopoietic factor-dependent cells

Blood ◽  
1995 ◽  
Vol 86 (10) ◽  
pp. 3897-3904 ◽  
Author(s):  
RL Jr Ilaria ◽  
RA Van Etten
Keyword(s):  
Cell Lines ◽  
Protein Interactions ◽  
Specific Protein ◽  
Sh2 Domain ◽  
Myelogenous Leukemia ◽  
Mutant Proteins ◽  
Abl Tyrosine Kinase ◽  
Negative Effect ◽  
Dependent Cell

Src-homology region 2 (SH2) domains, by binding to tyrosine- phosphorylated sequences, mediate specific protein-protein interactions important in diverse signal transduction pathways. Previous studies have shown that activated forms of the Abl tyrosine kinase, including P210BCR/ABL of human chronic myelogenous leukemia, require the SH2 domain for the transformation of fibroblasts. To determine whether SH2 is also required for Bcr/Abl to transform hematopoietic cells, we have studied two SH2 domain mutations in P210BCR/ABL: a point mutation in the conserved FLVRES motif (P210/R1033K), which interferes with phosphotyrosine-binding by SH2, and a complete deletion of SH2 (P210/delta SH2). Despite a negative effect on intrinsic Abl kinase activity, both P210 SH2 mutants were still able to transform the hematopoietic factor-dependent cell lines Ba/F3 and FDC-P1 to growth factor independence. Unexpectedly, both mutants showed greater transforming activity than wild-type P210 in a quantitative transformation assay, probably as a consequence of increased stability of the SH2 mutant proteins in vivo. Cells transformed by both P210 SH2 mutants were leukemogenic in synaptic mice and P210/r1053K mice exhibited a distinct disease phenotype, reminiscent of that induced by v-Abl. These results demonstrate that while the Abl SH2 domain is essential for BCR/ABL transformation of fibroblasts, it is dispensable for the transformation of hematopoietic factor-dependent cell lines.

scholarly journals In vivo FRET–FLIM reveals cell-type-specific protein interactions in Arabidopsis roots

Nature ◽  
2017 ◽  
Vol 548 (7665) ◽  
pp. 97-102 ◽  
Author(s):  
Yuchen Long ◽  
Yvonne Stahl ◽  
Stefanie Weidtkamp-Peters ◽  
Marten Postma ◽  
Wenkun Zhou ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Specific Protein ◽  
Cell Type ◽  
Cell Type Specific

scholarly journals CIITA is a transcriptional coactivator that is recruited to MHC class II promoters by multiple synergistic interactions with an enhanceosome complex

2000 ◽  
Vol 14 (9) ◽  
pp. 1156-1166
Author(s):  
Krzysztof Masternak ◽  
Annick Muhlethaler-Mottet ◽  
Jean Villard ◽  
Madeleine Zufferey ◽  
Viktor Steimle ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Protein Interactions ◽  
Class Ii ◽  
Hereditary Disease ◽  
Transcriptional Coactivator ◽  
Protein Protein Interactions ◽  
Mhc I ◽  
Mhc Ii ◽  
Class Ii Mhc

By virtue of its control over major histocompatibility complex class II (MHC-II) gene expression, CIITA represents a key molecule in the regulation of adaptive immune responses. It was first identified as a factor that is defective in MHC-II deficiency, a hereditary disease characterized by the absence of MHC-II expression. CIITA is a highly regulated transactivator that governs all spatial, temporal, and quantitative aspects of MHC-II expression. It has been proposed to act as a non-DNA-binding transcriptional coactivator, but evidence that it actually functions at the level of MHC-II promoters was lacking. By means of chromatin immunoprecipitation assays, we show here for the first time that CIITA is physically associated with MHC-II, as well asHLA–DM, Ii, MHC-I, and β2mpromoters in vivo. To dissect the mechanism by which CIITA is recruited to the promoter, we have developed a DNA-dependent coimmunoprecipitation assay and a pull-down assay using immobilized promoter templates. We demonstrate that CIITA recruitment depends on multiple, synergistic protein–protein interactions with DNA-bound factors constituting the MHC-II enhanceosome. CIITA therefore represents a paradigm for a novel type of regulatory and gene-specific transcriptional cofactor.

scholarly journals Proximity labeling of protein complexes and cell type-specific organellar proteomes in Arabidopsis enabled by TurboID

2019 ◽  
Author(s):  
Andrea Mair ◽  
Shou-Ling Xu ◽  
Tess C. Branon ◽  
Alice Y. Ting ◽  
Dominique C. Bergmann
Keyword(s):  
Transcription Factor ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Cell Types ◽  
Specific Protein ◽  
Cell Type ◽  
Subcellular Compartment ◽  
Cellular Processes ◽  
Highly Active

AbstractDefining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurboID), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurboID in Arabidopsis and N. benthamiana and versatile vectors enable customization by plant researchers.

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