Cross-regulatory protein-protein interactions between Hox and Pax transcription factors

Protein–protein interactions of core pluripotency transcription factors play an important role during cell reprogramming. Cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. Thus, methods that help to quantify protein–protein interactions may be useful for understanding the mechanisms of pluripotency at the molecular level. Here, a detailed protocol for the detection and quantitative analysis of in vivo protein–protein proximity of Sox2 and Oct4 using the proximity-utilizing biotinylation (PUB) method is described. The method is based on the coexpression of two proteins of interest fused to a biotin acceptor peptide (BAP)in one case and a biotin ligase enzyme (BirA) in the other. The proximity between the two proteins leads to more efficient biotinylation of the BAP, which can be either detected by Western blotting or quantified using proteomics approaches, such as a multiple reaction monitoring (MRM) analysis. Coexpression of the fusion proteins BAP-X and BirA-Y revealed strong biotinylation of the target proteins when X and Y were, alternatively, the pluripotency transcription factors Sox2 and Oct4, compared with the negative control where X or Y was green fluorescent protein (GFP), which strongly suggests that Sox2 and Oct4 come in close proximity to each other and interact.

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Dependence of KCC2 K-Cl cotransporter activity on a conserved carboxy terminus tyrosine residue

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.3.c860 ◽

2000 ◽

Vol 279 (3) ◽

pp. C860-C867 ◽

Cited By ~ 73

Author(s):

Kevin Strange ◽

Thomas D. Singer ◽

Rebecca Morrison ◽

Eric Delpire

Keyword(s):

Protein Interactions ◽

Regulatory Protein ◽

Phosphorylation Site ◽

Fold Increase ◽

Tyrosine Residue ◽

Carboxy Terminus ◽

Protein Protein Interactions ◽

Pharmacological Studies ◽

Osmotic Homeostasis ◽

Tyrosine Phosphorylation Site

K-Cl cotransporters (KCC) play fundamental roles in ionic and osmotic homeostasis. To date, four mammalian KCC genes have been identified. KCC2 is expressed exclusively in neurons. Injection of Xenopus oocytes with KCC2 cRNA induced a 20-fold increase in Cl−-dependent, furosemide-sensitive K+ uptake. Oocyte swelling increased KCC2 activity 2–3 fold. A canonical tyrosine phosphorylation site is located in the carboxy termini of KCC2 (R1081–Y1087) and KCC4, but not in other KCC isoforms. Pharmacological studies, however, revealed no regulatory role for phosphorylation of KCC2 tyrosine residues. Replacement of Y1087 with aspartate or arginine dramatically reduced K+ uptake under isotonic and hypotonic conditions. Normal or near-normal cotransporter activity was observed when Y1087 was mutated to phenylalanine, alanine, or isoleucine. A tyrosine residue equivalent to Y1087 is conserved in all identified KCCs from nematodes to humans. Mutation of the Y1087 congener in KCC1 to aspartate also dramatically inhibited cotransporter activity. Taken together, these results suggest that replacement of Y1087 and its congeners with charged residues disrupts the conformational state of the carboxy terminus. We postulate that the carboxy terminus plays an essential role in maintaining the functional conformation of KCC cotransporters and/or is involved in essential regulatory protein-protein interactions.

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A yeast library-hybrid assay to screen maize- Rhizoctonia transcription factors and protein-protein interactions in one experimental pipeline

Agri Gene ◽

10.1016/j.aggene.2016.05.002 ◽

2016 ◽

Vol 1 ◽

pp. 15-22 ◽

Cited By ~ 1

Author(s):

Xuan Tang ◽

Junwei Shi ◽

Wubei Dong

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Protein Protein Interactions

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Dof DNA-Binding Domains of Plant Transcription Factors Contribute to Multiple Protein-Protein Interactions

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1997.0403a.x ◽

1997 ◽

Vol 250 (2) ◽

pp. 403-410 ◽

Cited By ~ 58

Author(s):

Shuichi Yanagisawa

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Protein Interactions ◽

Protein Protein Interactions ◽

Dna Binding Domains ◽

Binding Domains ◽

Multiple Protein

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Targeting of the Visna Virus Tat Protein to AP-1 Sites: Interactions with the bZIP Domains of Fos and Jun In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.73.1.37-45.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 37-45 ◽

Cited By ~ 15

Author(s):

B. A. Morse ◽

L. M. Carruth ◽

J. E. Clements

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Tat Protein ◽

Protein Protein Interactions ◽

Viral Promoter ◽

Visna Virus ◽

Cellular Transcription ◽

Virus Promoter

ABSTRACT The visna virus Tat protein is required for efficient viral transcription from the visna virus long terminal repeat (LTR). AP-1 sites within the visna virus LTR, which can be bound by the cellular transcription factors Fos and Jun, are also necessary for Tat-mediated transcriptional activation. A potential mechanism by which the visna virus Tat protein could target the viral promoter is by protein-protein interactions with Fos and/or Jun bound to AP-1 sites in the visna virus LTR. Once targeted to the visna virus promoter, the Tat protein could then interact with basal transcription factors to activate transcription. To examine protein-protein interactions with cellular proteins at the visna virus promoter, we used an in vitro protein affinity chromatography assay and electrophoretic mobility shift assay, in addition to an in vivo two-hybrid assay, to show that the visna virus Tat protein specifically interacts with the cellular transcription factors Fos and Jun and the basal transcription factor TBP (TATA binding protein). The Tat domain responsible for interactions with Fos and Jun was localized to an alpha-helical domain within amino acids 34 to 69 of the protein. The TBP binding domain was localized to amino acids 1 to 38 of Tat, a region previously described by our laboratory as the visna virus Tat activation domain. The bZIP domains of Fos and Jun were found to be important for the interactions with Tat. Mutations within the basic domains of Fos and Jun abrogated binding to Tat in the in vitro assays. The visna virus Tat protein was also able to interact with covalently cross-linked Fos and Jun dimers. Thus, the visna virus Tat protein appears to target AP-1 sites in the viral promoter in a mechanism similar to the interaction of human T-cell leukemia virus type 1 Tax with the cellular transcription factor CREB, by binding the basic domains of an intact bZIP dimer. The association between Tat, Fos, and Jun would position Tat proximal to the viral TATA box, where the visna virus Tat activation domain could contact TBP to activate viral transcription.

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Sequence conservation, domain architectures, and phylogenetic distribution of the HD-GYP type c-di-GMP phosphodiesterases

10.1101/2021.11.05.467447 ◽

2021 ◽

Author(s):

Michael Y. Galperin ◽

Shan-Ho Chou

Keyword(s):

Protein Interactions ◽

Regulatory Protein ◽

Second Messenger ◽

Distribution Patterns ◽

Distribution Functions ◽

Amino Acid Residues ◽

Sequence Motifs ◽

Protein Protein Interactions ◽

Conserved Sequence ◽

Common Domain

The HD-GYP domain, named after two of its conserved sequence motifs, was first described in 1999 as a specialized version of the widespread HD phosphohydrolase domain that had additional highly conserved amino acid residues. Domain associations of HD-GYP indicated its involvement in bacterial signal transduction and distribution patterns of this domain suggested that it could serve as a hydrolase of the bacterial second messenger c-di-GMP, in addition to or instead of the EAL domain. Subsequent studies confirmed the ability of various HD-GYP domains to hydrolyze c-di-GMP to linear pGpG and/or GMP. Certain HD-GYP-containing proteins hydrolyze another second messenger, cGAMP, and some HD-GYP domains participate in regulatory protein-protein interactions. The recently solved structures of HD-GYP domains from four distinct organisms clarified the mechanisms of c-di-GMP binding and metal-assisted hydrolysis. However, the HD-GYP domain is poorly represented in public domain databases, which causes certain confusion about its phylogenic distribution, functions, and domain architectures. Here, we present a refined sequence model for the HD-GYP domain and describe the roles of its most conserved residues in metal and/or substrate binding. We also calculate the numbers of HD-GYPs encoded in various genomes and list the most common domain combinations involving HD-GYP, such as the RpfG (REC-HD-GYP), Bd1817 (DUF3391-HD-GYP), and PmGH (GAF-HD-GYP) protein families. We also provide the descriptions of six HD-GYP-associated domains, including four novel integral membrane sensor domains. This work is expected to stimulate studies of diverse HD-GYP-containing proteins, their N-terminal sensor domains, and the signals to which they respond.

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ApicoTFdb: the comprehensive web repository of apicomplexan transcription factors and transcription-associated co-factors

Database ◽

10.1093/database/baz094 ◽

2019 ◽

Vol 2019 ◽

Author(s):

Rahila Sardar ◽

Abhinav Kaushik ◽

Rajan Pandey ◽

Asif Mohmmed ◽

Shakir Ali ◽

...

Keyword(s):

Transcription Factors ◽

Protein Interactions ◽

Drug Targets ◽

Complex Life Cycle ◽

Protein Protein Interactions ◽

Apicomplexan Parasites ◽

Reference Protein ◽

Ap2 Family ◽

Novel Drug ◽

Novel Drug Targets

Abstract Despite significant progress in apicomplexan genome sequencing and genomics, the current list of experimentally validated transcription factors (TFs) in these genomes is incomplete and mainly consists of AP2 family of proteins, with only a limited number of non-AP2 family TFs and transcription-associated co-factors (TcoFs). We have performed a systematic bioinformatics-aided prediction of TFs and TcoFs in apicomplexan genomes and developed the ApicoTFdb database which consists of experimentally validated as well as computationally predicted TFs and TcoFs in 14 apicomplexan species. The predicted TFs are manually curated to complement the existing annotations. The current version of the database includes 1292 TFs which includes experimentally validated and computationally predicted TFs, representing 20 distinct families across 14 apicomplexan species. The predictions include TFs of TUB, NAC, BSD, HTH, Cupin/Jumonji, winged helix and FHA family proteins, not reported earlier as TFs in the genomes. Apart from TFs, ApicoTFdb also classifies TcoFs into three main subclasses: TRs, CRRs and RNARs, representing 2491 TcoFs in 14 apicomplexan species, are analyzed in this study. The database is designed to integrate different tools for comparative analysis. All entries in the database are dynamically linked with other databases, literature reference, protein–protein interactions, pathways and annotations associated with each protein. ApicoTFdb will be useful to the researchers interested in less-studied gene regulatory mechanisms mediating the complex life cycle of the apicomplexan parasites. The database will aid in the discovery of novel drug targets to much needed combat the growing drug resistance in the parasites.

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The transcription factors Elk-1 and serum response factor interact by direct protein-protein contacts mediated by a short region of Elk-1.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3283 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3283-3291 ◽

Cited By ~ 103

Author(s):

P Shore ◽

A D Sharrocks

Keyword(s):

Transcription Factor ◽

Transcription Factors ◽

Protein Interactions ◽

Serum Response Factor ◽

Specific Protein ◽

Response Factor ◽

Protein Protein Interactions ◽

Amino Acid Peptide ◽

Necessary And Sufficient ◽

Serum Response

Transcriptional induction of the c-fos gene in response to epidermal growth factor stimulation is mediated in part by a ternary nucleoprotein complex within the promoter consisting of serum response factor (SRF), p62TCF/Elk-1 and the serum response element (SRE). Both SRF and p62TCF/Elk-1 contact the DNA and bind in a cooperative manner to the SRE. In this study, we demonstrate that SRF and Elk-1 interact directly in the absence of the SRE. A 30-amino-acid peptide from Elk-1 (B-box) is both necessary and sufficient to mediate protein-protein contacts with SRF. Moreover, the Elk-1 B-box is necessary to enable SRF-dependent binding of an alternative ETS domain (from the transcription factor PU.1) to the c-fos SRE. Mutations in either the Elk-1 B-box or the C-terminal half of the SRF DNA-binding domain (coreSRF) which show reduced ability to form ternary complexes also show greatly reduced protein-protein interactions in the absence of the SRE. Our results clearly demonstrate that direct protein-protein interactions between the transcription factors Elk-1 and SRF, in addition to DNA contacts, contribute to the formation of a ternary complex on the c-fos SRE. We discuss the wider applicability of our results in describing specific protein-protein interactions between short well-defined transcription factor domains.

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