scholarly journals Multi-parameter analysis of the kinetics of NF-κB signalling and transcription in single living cells

2002 ◽  
Vol 115 (6) ◽  
pp. 1137-1148 ◽  
Author(s):  
Glyn Nelson ◽  
Luminita Paraoan ◽  
David G. Spiller ◽  
Geraint J. C. Wilde ◽  
Mark A. Browne ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Fluorescent Proteins ◽  
Parameter Analysis ◽  
Nuclear Accumulation ◽  
Fusion Protein Expression ◽  
Kinetics Of

Proteins of the NF-κB transcription factor family normally reside in the cytoplasm of cells in a complex with IκB inhibitor proteins. Stimulation with TNFα leads to proteosomal degradation of the IκB proteins and nuclear translocation of the NF-κB proteins. Expression of p65 and IκBα fused to fluorescent proteins was used to measure the dynamics of these processes in transfected HeLa cells. Simultaneous visualisation of p65-dsRed translocation and IκBα-EGFP degradation indicated that in the presence of dual fluorescent fusion protein expression,the half-time of IκBα-EGFP degradation was reduced and that of p65 translocation was significantly increased when compared with cells expressing the single fluorescent fusion proteins. These results suggest that the ratio of IκBα and p65 determine the kinetics of transcription factor translocation into the nucleus and indicate that the complex of p65 and IκBα is the true substrate for TNFα stimulation in mammalian cells. When cells were treated with the CRM-1-dependent nuclear export inhibitor,leptomycin B (LMB), there was nuclear accumulation of IκBα-EGFP and p65-dsRed, with IκBα-EGFP accumulating more rapidly. No NF-κB-dependent transcriptional activation was seen in response to LMB treatment. Following 1 hour treatment with LMB, significant IκBα-EGFP nuclear accumulation, but low levels of p65-dsRed nuclear accumulation, was observed. When these cells were stimulated with TNFα, degradation of IκBα-EGFP was observed in both the cytoplasm and nucleus. A normal transient transcription response was observed in the same cells using luminescence imaging of NF-κB-dependent transcription. These observations suggest that both normal activation and post-induction repression of NF-κB-dependent transcription occur even when nuclear export of NF-κB is inhibited. The results provide functional evidence that other factors, such as modification of p65 by phosphorylation, or interaction with other proteins such as transcriptional co-activators/co-repressors, may critically modulate the kinetics of transcription through this signalling pathway.

scholarly journals MondoA, a Novel Basic Helix-Loop-Helix–Leucine Zipper Transcriptional Activator That Constitutes a Positive Branch of a Max-Like Network

2000 ◽  
Vol 20 (23) ◽  
pp. 8845-8854 ◽  
Author(s):  
Andrew N. Billin ◽  
Alanna L. Eilers ◽  
Kathryn L. Coulter ◽  
Jennifer S. Logan ◽  
Donald E. Ayer
Keyword(s):  
Transcriptional Activation ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Leucine Zipper ◽  
Functional Characterization ◽  
Nuclear Accumulation ◽  
Cytoplasmic Localization ◽  
Basic Helix Loop Helix ◽  
Helix Loop Helix

ABSTRACT Max is a common dimerization partner for a family of transcription factors (Myc, Mad [or Mxi]), and Mnt [or Rox] proteins) that regulate cell growth, proliferation, and apoptosis. We recently characterized a novel Max-like protein, Mlx, which interacts with Mad1 and Mad4. Here we describe the cloning and functional characterization of a new family of basic helix-loop-helix–leucine zipper heterodimeric partners for Mlx termed the Mondo family. MondoA forms homodimers weakly and does not interact with Max or members of the Myc or Mad families. MondoA and Mlx associate in vivo, and surprisingly, they are localized primarily to the cytoplasm of cultured mammalian cells. Treatment of cells with the nuclear export inhibitor leptomycin B results in the nuclear accumulation of MondoA and Mlx, demonstrating that they shuttle between the cytoplasmic and nuclear compartments rather than having exclusively cytoplasmic localization. MondoA preferentially forms heterodimers with Mlx, and this heterocomplex can bind to, and activate transcription from, CACGTG E-boxes when targeted to the nucleus via a heterologous nuclear localization signal. The amino termini of the Mondo proteins are highly conserved among family members and contain separable and autonomous cytoplasmic localization and transcription activation domains. Therefore, Mlx can mediate transcriptional repression in conjunction with the Mad family and can mediate transcriptional activation via the Mondo family. We propose that Mlx, like Max, functions as the center of a transcription factor network.

scholarly journals Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Richard Panayiotou ◽  
Francesc Miralles ◽  
Rafal Pawlowski ◽  
Jessica Diring ◽  
Helen R Flynn ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Subcellular Localisation ◽  
Nuclear Actin ◽  
Inhibitory Function ◽  
Actin Concentration

The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A.

scholarly journals Nuclear Export of the Transcription Factor NirA Is a Regulatory Checkpoint for Nitrate Induction in Aspergillus nidulans

2006 ◽  
Vol 27 (3) ◽  
pp. 791-802 ◽  
Author(s):  
Andreas Bernreiter ◽  
Ana Ramon ◽  
Javier Fernández-Martínez ◽  
Harald Berger ◽  
Lidia Araújo-Bazan ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Constitutive Expression ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Nitrate Induction

ABSTRACT NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA c 1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA c 1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirAc1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

scholarly journals mTOR-dependent phosphorylation controls TFEB nuclear export

2018 ◽  
Author(s):  
Gennaro Napolitano ◽  
Alessandra Esposito ◽  
Heejun Choi ◽  
Valerio Benedetti ◽  
Maria Matarese ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Multisite Phosphorylation ◽  
Limiting Step ◽  
Activation Of Transcription ◽  
Transcription Factor Eb ◽  
Export Signal

AbstractThe transcriptional activation of catabolic processes during starvation is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient re-feeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data reveal that modulation of TFEB nuclear export via phosphorylation plays a major role in the modulation of TFEB localization and activity.

scholarly journals Functional domains of interferon regulatory factor I (IRF-1)

1998 ◽  
Vol 335 (1) ◽  
pp. 147-157 ◽  
Author(s):  
Fred SCHAPER ◽  
Sabine KIRCHHOFF ◽  
Guido POSERN ◽  
Mario KÖSTER ◽  
André OUMARD ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Consensus Sequence ◽  
Binding Domain ◽  
Functional Domains ◽  
Dna Binding Domain ◽  
Transcriptional Activation Domain

Interferon (IFN) regulatory factors (IRFs) are a family of transcription factors among which are IRF-1, IRF-2, and IFN consensus sequence binding protein (ICSBP). These factors share sequence homology in the N-terminal DNA-binding domain. IRF-1 and IRF-2 are further related and have additional homologous sequences within their C-termini. Whereas IRF-2 and ICSBP are identified as transcriptional repressors, IRF-1 is an activator. In the present work, the identification of functional domains in murine IRF-1 with regard to DNA-binding, nuclear translocation, heterodimerization with ICSBP and transcriptional activation are demonstrated. The minimal DNA-binding domain requires the N-terminal 124 amino acids plus an arbitrary C-terminal extension. By using mutants of IRF-1 fusion proteins with green fluorescent protein and monitoring their distribution in living cells, a nuclear location signal (NLS) was identified and found to be sufficient for nuclear translocation. Heterodimerization was confirmed by a two-hybrid system adapted to mammalian cells. The heterodimerization domain in IRF-1 was defined by studies in vitroand was shown to be homologous with a sequence in IRF-2, suggesting that IRF-2 also heterodimerizes with ICSBP through this sequence. An acidic domain in IRF-1 was found to be required and to be sufficient for transactivation. Epitope mapping of IRF-1 showed that regions within the NLS, the heterodimerization domain and the transcriptional activation domain are exposed for possible contacts with interacting proteins.

scholarly journals The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

2003 ◽  
Vol 77 (14) ◽  
pp. 7945-7956 ◽  
Author(s):  
Christopher F. Basler ◽  
Andrea Mikulasova ◽  
Luis Martinez-Sobrido ◽  
Jason Paragas ◽  
Elke Mühlberger ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Virus Infection ◽  
Transcriptional Activation ◽  
Ebola Virus ◽  
Nuclear Translocation ◽  
Sendai Virus ◽  
Vero Cells ◽  
Nuclear Accumulation ◽  
Regulatory Factor ◽  
Cellular Transcription Factor

ABSTRACT The Ebola virus VP35 protein was previously found to act as an interferon (IFN) antagonist which could complement growth of influenza delNS1 virus, a mutant influenza virus lacking the influenza virus IFN antagonist protein, NS1. The Ebola virus VP35 could also prevent the virus- or double-stranded RNA-mediated transcriptional activation of both the beta IFN (IFN-β) promoter and the IFN-stimulated ISG54 promoter (C. Basler et al., Proc. Natl. Acad. Sci. USA 97:12289-12294, 2000). We now show that VP35 inhibits virus infection-induced transcriptional activation of IFN regulatory factor 3 (IRF-3)-responsive mammalian promoters and that VP35 does not block signaling from the IFN-α/β receptor. The ability of VP35 to inhibit this virus-induced transcription correlates with its ability to block activation of IRF-3, a cellular transcription factor of central importance in initiating the host cell IFN response. We demonstrate that VP35 blocks the Sendai virus-induced activation of two promoters which can be directly activated by IRF-3, namely, the ISG54 promoter and the ISG56 promoter. Further, expression of VP35 prevents the IRF-3-dependent activation of the IFN-α4 promoter in response to viral infection. The inhibition of IRF-3 appears to occur through an inhibition of IRF-3 phosphorylation. VP35 blocks virus-induced IRF-3 phosphorylation and subsequent IRF-3 dimerization and nuclear translocation. Consistent with these observations, Ebola virus infection of Vero cells activated neither transcription from the ISG54 promoter nor nuclear accumulation of IRF-3. These data suggest that in Ebola virus-infected cells, VP35 inhibits the induction of antiviral genes, including the IFN-β gene, by blocking IRF-3 activation.

scholarly journals Akt-Dependent Cytokine Production in Mast Cells

2000 ◽  
Vol 192 (5) ◽  
pp. 729-740 ◽  
Author(s):  
Jiro Kitaura ◽  
Koichi Asai ◽  
Mari Maeda-Yamamoto ◽  
Yuko Kawakami ◽  
Ushio Kikkawa ◽  
...  
Keyword(s):  
Mast Cells ◽  
Transcriptional Activation ◽  
Tyrosine Kinases ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Glycogen Synthase ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Proinflammatory Mediators ◽  
Tnf Α

Cross-linking of FcεRI induces the activation of three protein tyrosine kinases, Lyn, Syk, and Bruton's tyrosine kinase (Btk), leading to the secretion of a panel of proinflammatory mediators from mast cells. This study showed phosphorylation at Ser-473 and enzymatic activation of Akt/protein kinase B, the crucial survival kinase, upon FcεRI stimulation in mouse mast cells. Phosphorylation of Akt is regulated positively by Btk and Syk and negatively by Lyn. Akt in turn can regulate positively the transcriptional activity of interleukin (IL)-2 and tumor necrosis factor (TNF)-α promoters. Transcription from the nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein 1 (AP-1) sites within these promoters is under the control of Akt activity. Accordingly, the signaling pathway involving IκB-α, a cytoplasmic protein that binds NF-κB and inhibits its nuclear translocation, appears to be regulated by Akt in mast cells. Catalytic activity of glycogen synthase kinase (GSK)-3β, a serine/threonine kinase that phosphorylates NF-AT and promotes its nuclear export, seems to be inhibited by Akt. Importantly, Akt regulates the production and secretion of IL-2 and TNF-α in FcεRI-stimulated mast cells. Altogether, these results revealed a novel function of Akt in transcriptional activation of cytokine genes via NF-κB, NF-AT, and AP-1 that contributes to the production of cytokines.

scholarly journals Mitogen-Activated Protein Kinase Regulates Nuclear Association of Human Progesterone Receptors

2003 ◽  
Vol 17 (4) ◽  
pp. 628-642 ◽  
Author(s):  
Ming Qiu ◽  
Abby Olsen ◽  
Emily Faivre ◽  
Kathryn B. Horwitz ◽  
Carol A. Lange
Keyword(s):  
Nuclear Export ◽  
Nuclear Translocation ◽  
Steroid Hormone ◽  
Progesterone Receptors ◽  
Steroid Hormone Receptors ◽  
Nuclear Accumulation ◽  
Prolonged Treatment ◽  
Leptomycin B ◽  
Down Regulation ◽  
Progestin Treatment

Abstract Breast cancers often have increased MAPK activity; this pathway may drive breast cancer cell growth by targeting steroid hormone receptors. MAPK phosphorylates human progesterone receptors (PRs) on Ser294, thus regulating several aspects of PR activity. To study the role of PR Ser294 phosphorylation on subcellular distribution, we stably expressed wild-type (wt) or S294A (Ser294 to Ala) PR-B in several cell types. PRs phosphorylated on Ser294 were nuclear. Activation of MAPK induced Ser294 phosphorylation and rapid nuclear translocation of wt, but not S294A, PR-B; both receptors concentrated in the nucleus after progestin treatment. The MAPK kinase inhibitor, U0126, blocked epidermal growth factor but not progestin-induced Ser294 phosphorylation and translocation of wt PR, indicating a novel mechanism for nuclear localization. After progestin treatment, wt PR-B underwent ligand-dependent down-regulation, while S294A PR-B persisted in nuclei. Prolonged treatment with U0126 or the nuclear export inhibitor, leptomycin B, promoted nuclear accumulation of wt PR-B and blocked ligand-dependent PR down-regulation, suggesting that PR degradation occurs in the cytoplasm and requires MAPK-dependent nuclear export. Stabilization of PRs by leptomycin B also blocked PR transcriptional activity, indicating a link between nucleocytoplasmic shuttling, receptor stability, and function. These results support a regulatory role for MAPK in nuclear steroid hormone receptor subcellular localization and coupling to multiple PR functions.

scholarly journals Rotavirus inhibits IFN-induced STAT nuclear translocation by a mechanism that acts after STAT binding to importin-α

2014 ◽  
Vol 95 (8) ◽  
pp. 1723-1733 ◽  
Author(s):  
Gavan Holloway ◽  
Vi T. Dang ◽  
David A. Jans ◽  
Barbara S. Coulson
Keyword(s):  
Nuclear Export ◽  
Nuclear Translocation ◽  
Strain Analysis ◽  
Nuclear Accumulation ◽  
Type I ◽  
Group A Rotaviruses ◽  
Importin Α ◽  
Group A ◽  
Infected Cells ◽  
Novel Strategy

The importance of innate immunity to rotaviruses is exemplified by the range of strategies evolved by rotaviruses to interfere with the IFN response. We showed previously that rotaviruses block gene expression induced by type I and II IFNs, through a mechanism allowing activation of signal transducer and activator of transcription (STAT) 1 and STAT2 but preventing their nuclear accumulation. This normally occurs through activated STAT1/2 dimerization, enabling an interaction with importin α5 that mediates transport into the nucleus. In rotavirus-infected cells, STAT1/2 inhibition may limit the antiviral actions of IFN produced early in infection. Here we further analysed the block to STAT1/2 nuclear accumulation, showing that activated STAT1 accumulates in the cytoplasm in rotavirus-infected cells. STAT1/2 nuclear accumulation was inhibited by rotavirus even in the presence of the nuclear export inhibitor Leptomycin B, demonstrating that enhanced nuclear export is not involved in STAT1/2 cytoplasmic retention. The ability to inhibit STAT nuclear translocation was completely conserved amongst the group A rotaviruses tested, including a divergent avian strain. Analysis of mutant rotaviruses indicated that residues after amino acid 47 of NSP1 are dispensable for STAT inhibition. Furthermore, expression of any of the 12 Rhesus monkey rotavirus proteins did not inhibit IFN-stimulated STAT1 nuclear translocation. Finally, co-immunoprecipitation experiments from transfected epithelial cells showed that STAT1/2 binds importin α5 normally following rotavirus infection. These findings demonstrate that rotavirus probably employs a novel strategy to inhibit IFN-induced STAT signalling, which acts after STAT activation and binding to the nuclear import machinery.

scholarly journals A yeast model system for functional analysis of β-catenin signaling

2002 ◽  
Vol 158 (6) ◽  
pp. 1067-1078 ◽  
Author(s):  
Margaret S. Lee ◽  
Karen A. D'Amour ◽  
Jackie Papkoff
Keyword(s):  
Reporter Gene ◽  
Gene Transcription ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Signal Transduction Pathway ◽  
Model System ◽  
Nuclear Accumulation ◽  
Molecular Events

We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of β-catenin (β-cat) signaling. Coexpression of mammalian β-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both β-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with β-cat, reduces β-cat binding to TCF4, and leads to increased cytoplasmic localization of β-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to β-cat and restores reporter gene transcription. Additional β-cat signaling components, Axin and glycogen synthase kinase 3β, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein β-transducin repeat-containing protein reduces the stability of β-cat and decreases reporter activation. Thus, we have reconstituted a functional β-cat signal transduction pathway in yeast and show that β-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.

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