scholarly journals Rac1 and a GTPase-activating protein, MgcRacGAP, are required for nuclear translocation of STAT transcription factors

2006 ◽  
Vol 175 (6) ◽  
pp. 937-946 ◽  
Author(s):  
Toshiyuki Kawashima ◽  
Ying Chun Bao ◽  
Yasushi Nomura ◽  
Yuseok Moon ◽  
Yukio Tonozuka ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Target Genes ◽  
Nuclear Translocation ◽  
Nuclear Transport ◽  
Gtpase Activating Protein ◽  
Permeabilized Cells ◽  
Importin Α ◽  
Cytokine Stimulation

STAT transcription factors are tyrosine phosphorylated upon cytokine stimulation and enter the nucleus to activate target genes. We show that Rac1 and a GTPase-activating protein, MgcRacGAP, bind directly to p-STAT5A and are required to promote its nuclear translocation. Using permeabilized cells, we find that nuclear translocation of purified p-STAT5A is dependent on the addition of GTP-bound Rac1, MgcRacGAP, importin α, and importin β. p-STAT3 also enters the nucleus via this transport machinery, and mutant STATs lacking the MgcRacGAP binding site do not enter the nucleus even after phosphorylation. We conclude that GTP-bound Rac1 and MgcRacGAP function as a nuclear transport chaperone for activated STATs.

scholarly journals A Rac GTPase-Activating Protein, MgcRacGAP, Is a Nuclear Localizing Signal-Containing Nuclear Chaperone in the Activation of STAT Transcription Factors

2009 ◽  
Vol 29 (7) ◽  
pp. 1796-1813 ◽  
Author(s):  
Toshiyuki Kawashima ◽  
Ying Chun Bao ◽  
Yukinori Minoshima ◽  
Yasushi Nomura ◽  
Tomonori Hatori ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Tertiary Structure ◽  
Nuclear Translocation ◽  
Computer Assisted ◽  
Rac Gtpase ◽  
Loop Region ◽  
Importin Α ◽  
Cytokine Stimulation

ABSTRACT In addition to their pleiotropic functions under physiological conditions, transcription factors STAT3 and STAT5 also have oncogenic activities, but how activated STATs are transported to the nucleus has not been fully understood. Here we show that an MgcRacGAP mutant lacking its nuclear localizing signal (NLS) blocks nuclear translocation of p-STATs both in vitro and in vivo. Unlike wild-type MgcRacGAP, this mutant did not promote complex formation of phosphorylated STATs (p-STATs) with importin α in the presence of GTP-bound Rac1, suggesting that MgcRacGAP functions as an NLS-containing nuclear chaperone. We also demonstrate that mutants of STATs lacking the MgcRacGAP binding site (the strand βb) are hardly tyrosine phosphorylated after cytokine stimulation. Intriguingly, mutants harboring small deletions in the C′-adjacent region (βb-βc loop region) of the strand βb became constitutively active with the enhanced binding to MgcRacGAP. The molecular basis of this phenomenon will be discussed, based on the computer-assisted tertiary structure models of STAT3. Thus, MgcRacGAP functions as both a critical mediator of STAT's tyrosine phosphorylation and an NLS-containing nuclear chaperone of p-STATs.

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α

2017 ◽  
Vol 474 (24) ◽  
pp. 4091-4104 ◽  
Author(s):  
Natália E. Bernardes ◽  
Agnes A.S. Takeda ◽  
Thiago R. Dreyer ◽  
Fernanda B. Cupertino ◽  
Stela Virgilio ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Neurospora Crassa ◽  
Binding Site ◽  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Protein Translocation ◽  
Nuclear Transport ◽  
Nuclear Localization Sequence ◽  
Primary Role ◽  
Importin Α

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP–NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Mechanisms regulating target gene selection by the homeodomain-containing protein Fushi tarazu

Development ◽  
2000 ◽  
Vol 127 (13) ◽  
pp. 2965-2976 ◽  
Author(s):  
A. Nasiadka ◽  
A. Grill ◽  
H.M. Krause
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Binding Site ◽  
Target Gene ◽  
Target Genes ◽  
Gene Selection ◽  
Activity Regulation ◽  
Fushi Tarazu ◽  
Activation Domain ◽  
Regulation Model

Homeodomain proteins are DNA-binding transcription factors that control major developmental patterning events. Although DNA binding is mediated by the homeodomain, interactions with other transcription factors play an unusually important role in the selection and regulation of target genes. A major question in the field is whether these cofactor interactions select target genes by modulating DNA binding site specificity (selective binding model), transcriptional activity (activity regulation model) or both. A related issue is whether the number of target genes bound and regulated is a small or large percentage of genes in the genome. In this study, we have addressed these issues using a chimeric protein that contains the strong activation domain of the viral VP16 protein fused to the Drosophila homeodomain-containing protein Fushi tarazu (Ftz). We find that genes previously thought not to be direct targets of Ftz remain unaffected by FtzVP16. Addition of the VP16 activation domain to Ftz does, however, allow it to regulate previously identified target genes at times and in regions that Ftz alone cannot. It also changes Ftz into an activator of two genes that it normally represses. Taken together, the results suggest that Ftz binds and regulates a relatively limited number of target genes, and that cofactors affect target gene specificity primarily by controlling binding site selection. Activity regulation then fine-tunes the temporal and spatial domains of promoter responses, the magnitude of these responses, and whether they are positive or negative.

scholarly journals miR-1322 Binding Sites in Paralogous and Orthologous Genes

2015 ◽  
Vol 2015 ◽  
pp. 1-7 ◽  
Author(s):  
Raigul Niyazova ◽  
Olga Berillo ◽  
Shara Atambayeva ◽  
Anna Pyrkova ◽  
Aigul Alybayeva ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transcription Factors ◽  
Amino Acid ◽  
Binding Site ◽  
Binding Sites ◽  
Target Genes ◽  
Amino Acid Sequences ◽  
Orthologous Genes ◽  
Start Position ◽  
Human Genes

We searched for 2,563 microRNA (miRNA) binding sites in 17,494 mRNA sequences of human genes. miR-1322 has more than 2,000 binding sites in 1,058 genes withΔG/ΔGmratio of 85% and more. miR-1322 has 1,889 binding sites in CDSs, 215 binding sites in 5′ UTRs, and 160 binding sites in 3′ UTRs. From two to 28 binding sites have arranged localization with the start position through three nucleotides of each following binding site. The nucleotide sequences of these sites in CDSs encode oligopeptides with the same and/or different amino acid sequences. We found that 33% of the target genes encoded transcription factors. miR-1322 has arranged binding sites in the CDSs of orthologousMAMLD1,MAML2, andMAML3genes. These sites encode a polyglutamine oligopeptide ranging from six to 47 amino acids in length. The properties of miR-1322 binding sites in orthologous and paralogous target genes are discussed.

scholarly journals LncRNAOIP5-AS1is overexpressed in undifferentiated oral tumors and integrated analysis identifies as a downstream effector of stemness-associated transcription factors

2018 ◽  
Author(s):  
Ganesan Arunkumar ◽  
Shankar Anand ◽  
Partha Raksha ◽  
Shankar Dhamodharan ◽  
Harikrishnan Prasanna Srinivasa Rao ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Target Genes ◽  
Integrated Analysis ◽  
Downstream Target ◽  
Cellular Processes ◽  
Downstream Effector ◽  
Undifferentiated Tumors ◽  
Oral Tumors ◽  
High Level

AbstractLong non-coding RNAs (lncRNAs) play an important role in the regulation of key cellular processes in early development and in cancer. LncRNAOip5-as1facilitates stem cell self-renewal in mouse by sponging mmu-miR-7 and modulating NANOG level, yet its role in cancer is less understood. We analyzedOIP5-AS1expression in oral tumors and in TCGA datasets. We observed overexpression ofOIP5-AS1in oral tumors (P<0.001) and in tumors of epithelial origin from TCGA.OIP5-AS1expression was strongly associated with undifferentiated tumors (P=0.0038).In silicoanalysis showed miR-7 binding site is conserved in mouse and humanOIP5-AS1. However, humanNANOG3’-UTR lost the binding site for hsa-miR-7a-3. Therefore, we screened for other miRNAs that can be sponged byOIP5-AS1and identified six potential miRNAs and their downstream target genes. Expression analysis showed downregulation of miRNAs and upregulation of downstream target genes, particularly in undifferentiated tumors with high-level ofOIP5-AS1suggesting thatOIP5-AS1could post-transcriptionally modulate the downstream target genes. Further, systematic epigenomic analysis ofOIP5-AS1promoter revealed binding motifs for MYC, NANOG and KLF4 suggesting thatOIP5-AS1could be transactivated by stemness-associated transcription factors in cancer. Overexpression of OIP5-AS1 in undifferentiated oral tumors may confer poor prognosis through maintenance of cancer stemness.

scholarly journals Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

2015 ◽  
Vol 112 (8) ◽  
pp. E852-E861 ◽  
Author(s):  
Shengzhan D. Luo ◽  
Bruce S. Baker
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Target Genes ◽  
Fat Body ◽  
Regulatory Sequence ◽  
Sexually Dimorphic ◽  
Regulatory Modules ◽  
Sexually Dimorphic Expression ◽  
Specific Tissue ◽  
Female Specific

“Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.

scholarly journals Molecular Dynamics of Cobalt Protoporphyrin Antagonism of the Cancer Suppressor REV-ERBβ

Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3251
Author(s):  
Taufik Muhammad Fakih ◽  
Fransiska Kurniawan ◽  
Muhammad Yusuf ◽  
Mudasir Mudasir ◽  
Daryono Hadi Tjahjono
Keyword(s):  
Binding Site ◽  
Nuclear Receptor ◽  
Target Genes ◽  
Protoporphyrin Ix ◽  
Binding Energies ◽  
Metal Center ◽  
Complex Nature ◽  
Dynamic Simulations ◽  
Subtle Change ◽  
Cobalt Protoporphyrin

Nuclear receptor REV-ERBβ is an overexpressed oncoprotein that has been used as a target for cancer treatment. The metal-complex nature of its ligand, iron protoporphyrin IX (Heme), enables the REV-ERBβ to be used for multiple therapeutic modalities as a photonuclease, a photosensitizer, or a fluorescence imaging agent. The replacement of iron with cobalt as the metal center of protoporphyrin IX changes the ligand from an agonist to an antagonist of REV-ERBβ. The mechanism behind that phenomenon is still unclear, despite the availability of crystal structures of REV-ERBβ in complex with Heme and cobalt protoporphyrin IX (CoPP). This study used molecular dynamic simulations to compare the effects of REV-ERBβ binding to Heme and CoPP, respectively. The initial poses of Heme and CoPP in complex with agonist and antagonist forms of REV-ERBβ were predicted using molecular docking. The binding energies of each ligand were calculated using the MM/PBSA method. The computed binding affinity of Heme to REV-ERBβ was stronger than that of CoPP, in agreement with experimental results. CoPP altered the conformation of the ligand-binding site of REV-ERBβ, disrupting the binding site for nuclear receptor corepressor, which is required for REV-ERBβ to regulate the transcription of downstream target genes. Those results suggest that a subtle change in the metal center of porphyrin can change the behavior of porphyrin in cancer cell signaling. Therefore, modification of porphyrin-based agents for cancer therapy should be conducted carefully to avoid triggering unfavorable effects.

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