BMAL1 Shuttling Controls Transactivation and Degradation of the CLOCK/BMAL1 Heterodimer

ABSTRACT CLOCK and BMAL1 are bHLH-PAS-containing transcription factors that bind to E-box elements and are indispensable for expression of core circadian clock components such as the Per and Cry genes. A key step in expression is the heterodimerization of CLOCK and BMAL1 and their accumulation in the nucleus with an approximately 24-h periodicity. We show here that nucleocytoplasmic shuttling of BMAL1 is essential for transactivation and for degradation of the CLOCK/BMAL1 heterodimer. Using serial deletions and point mutants, we identified a functional nuclear localization signal and Crm1-dependent nuclear export signals in BMAL1. Transient-transfection experiments revealed that heterodimerization of CLOCK and BMAL1 accelerates their turnover, as well as E-box-dependent clock gene transcription. Moreover, in embryonic mouse fibroblasts, robust transcription of Per2 is tightly associated with massive degradation of the CLOCK/BMAL1 heterodimer. CRY proteins suppressed this process during the transcription-negative phase and led to nuclear accumulation of the CLOCK/BMAL1 heterodimer. Thus, these findings suggest that the decrease of BMAL1 abundance during the circadian cycle reflects robust transcriptional activation of clock genes rather than inhibition of BMAL1 synthesis.

Download Full-text

Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.9041-9054.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 9041-9054 ◽

Cited By ~ 181

Author(s):

Christophe E. Pierreux ◽

Francisco J. Nicolás ◽

Caroline S. Hill

Keyword(s):

Growth Factor ◽

Transcriptional Activation ◽

Nuclear Export ◽

Transforming Growth Factor ◽

Nuclear Export Signal ◽

Transforming Growth Factor Β ◽

Nuclear Accumulation ◽

Leptomycin B ◽

Signaling Complex ◽

Localization Signal

ABSTRACT Smad4 plays a pivotal role in all transforming growth factor β (TGF-β) signaling pathways. Here we describe six widely expressed alternatively spliced variants of human Smad4 with deletions of different exons in the linker, the region of Smad4 that separates the two well-conserved MH1 and MH2 domains. All these Smad4 variants form complexes with activated Smad2 and Smad3 and are incorporated into DNA-binding complexes with the transcription factor Fast-1, regardless of the amount of linker they contain. However, sequences encoded by exons 5 to 7 in the linker are essential for transcriptional activation. Most importantly, our observation that different Smad4 isoforms have different subcellular localizations has led us to the identification of a functional CRM1-dependent nuclear export signal in the Smad4 linker and a constitutively active nuclear localization signal in the N-terminal MH1 domain. In the absence of TGF-β signaling, we conclude that Smad4 is rapidly and continuously shuttling between the nucleus and the cytoplasm, the distribution of Smad4 between the nucleus and the cytoplasm being dictated by the relative strengths of the nuclear import and export signals. We demonstrate that inhibition of CRM1-mediated nuclear export by treatment of cells with leptomycin B results in endogenous Smad4 accumulating very rapidly in the nucleus. Endogenous Smad2 and Smad3 are completely unaffected by leptomycin B treatment, indicating that the nucleocytoplasmic shuttling is specific for Smad4. We propose that, upon TGF-β signaling, complex formation between Smad4 and activated Smad2 or -3 leads to nuclear accumulation of Smad4 through inhibition of its nuclear export. We demonstrate that after prolonged TGF-β signaling Smad2 becomes dephosphorylated and Smad2 and Smad4 accumulate back in the cytoplasm.

Download Full-text

An NF-κB–driven lncRNA orchestrates colitis and circadian clock

Science Advances ◽

10.1126/sciadv.abb5202 ◽

2020 ◽

Vol 6 (42) ◽

pp. eabb5202

Author(s):

Shuai Wang ◽

Yanke Lin ◽

Feng Li ◽

Zifei Qin ◽

Ziyue Zhou ◽

...

Keyword(s):

Circadian Clock ◽

Clock Gene ◽

Clock Genes ◽

Experimental Colitis ◽

Circadian Clock Gene ◽

Its Gene ◽

E Box ◽

Direct Binding ◽

Central Clock ◽

Clock Protein

We uncover a cycling and NF-κB–driven lncRNA (named Lnc-UC) that epigenetically modifies transcription of circadian clock gene Rev-erbα, thereby linking circadian clock to colitis. Cycling expression of Lnc-UC is generated by the central clock protein Bmal1 via an E-box element. NF-κB activation in experimental colitis transcriptionally drives Lnc-UC through direct binding to two κB sites. Lnc-UC ablation disrupts colonic expressions of clock genes in mice; particularly, Rev-erbα is down-regulated and its diurnal rhythm is blunted. Consistently, Lnc-UC promotes expression of Rev-erbα (a known dual NF-κB/Nlrp3 repressor) to inactivate NF-κB signaling and Nlrp3 inflammasome in macrophages. Furthermore, Lnc-UC ablation sensitizes mice to experimental colitis and abolishes the diurnal rhythmicity in disease severity. Mechanistically, Lnc-UC physically interacts with Cbx1 protein to reduce its gene silencing activity via H3K9me3, thereby enhancing Rev-erbα transcription and expression. In addition, we identify a human Lnc-UC that has potential to promote Rev-erbα expression and restrain inflammations.

Download Full-text

Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1999.0504 ◽

1999 ◽

Vol 354 (1389) ◽

pp. 1601-1609 ◽

Cited By ~ 58

Author(s):

R. T. Hay ◽

L. Vuillard ◽

J. M. P. Desterro ◽

M. S. Rodriguez

Keyword(s):

Nuclear Localization ◽

Transcriptional Activation ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Cytoplasmic Process ◽

Leptomycin B ◽

Rapid Degradation ◽

Localization Signal ◽

Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8845-8854.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8845-8854 ◽

Cited By ~ 77

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.

Download Full-text

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

Journal of Cell Science ◽

10.1242/jcs.115.6.1137 ◽

2002 ◽

Vol 115 (6) ◽

pp. 1137-1148 ◽

Cited By ~ 1

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.

Download Full-text

Nuclear Entry Mechanism of Rat PER2 (rPER2): Role of rPER2 in Nuclear Localization of CRY Protein

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6651-6659.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6651-6659 ◽

Cited By ~ 64

Author(s):

Koyomi Miyazaki ◽

Miho Mesaki ◽

Norio Ishida

Keyword(s):

Nuclear Localization ◽

Clock Gene ◽

Clock Genes ◽

Nuclear Entry ◽

Cry Protein ◽

Terminal Domain ◽

Carboxyl Terminal Domain ◽

Localization Signal ◽

Carboxyl Terminal

ABSTRACT Mammalian PERIOD2 protein (PER2) is the product of a clock gene that controls circadian rhythms, because PER2-deficient mice have an arrhythmic phenotype. The nuclear entry regulation of clock gene products is a key step in proper circadian rhythm formation in bothDrosophila and mammals, because the periodic transcription of clock genes is controlled by an intracellular, oscillating, negative feedback loop. The present study used deletion mutants of rat PER2 (rPER2) to identify the functional nuclear localization signal (NLS) in rPER2. The elimination of putative NLS (residues 778 to 794) from the rPER2 fragment resulted in the loss of nuclear entry activity. Adding the NLS to the cytosolic protein (bacterial alkaline phosphatase) translocates the fusion protein to the nuclei. The data indicate the presence of a functional NLS in rPER2. Furthermore, intact rPER2 was preferentially translocated from the cytoplasm to the nucleus when coexpressed with human CRY1 (hCRY1). However, rPER2 mutants lacking a carboxyl-terminal domain could not enter the nucleus even in the presence of hCRY1. In addition, coexpression of the nuclear localization domain (residues 512 to 794) lacking rPER2 and CRY1 changed the subcellular localization of CRY1 from the nucleus to the cytoplasm. In vitro protein interaction studies demonstrated that the carboxyl-terminal domain of rPER2 is essential for binding to CRY1. The data suggested that both the rPER2 NLS and carboxyl-terminal CRY binding domain are essential for nuclear entry of the rPER2-CRY1 complex.

Download Full-text

Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity

The Journal of Cell Biology ◽

10.1083/jcb.200709091 ◽

2008 ◽

Vol 181 (7) ◽

pp. 1141-1154 ◽

Cited By ~ 85

Author(s):

Feng-Qian Li ◽

Adaobi Mofunanya ◽

Kimberley Harris ◽

Ken-Ichi Takemaru

Keyword(s):

Transcriptional Activation ◽

Nuclear Export ◽

Molecular Mechanisms ◽

Affinity Purification ◽

Nuclear Accumulation ◽

Binding Motif ◽

Transcriptional Coactivator ◽

Evolutionarily Conserved ◽

Canonical Wnt ◽

Affinity Purification Mass Spectrometry

β-Catenin functions in both cell–cell adhesion and as a transcriptional coactivator in the canonical Wnt pathway. Nuclear accumulation of β-catenin is the hallmark of active Wnt signaling and is frequently observed in human cancers. Although β-catenin shuttles in and out of the nucleus, the molecular mechanisms underlying its translocation remain poorly understood. Chibby (Cby) is an evolutionarily conserved molecule that inhibits β-catenin–mediated transcriptional activation. Here, we identified 14-3-3ε and 14-3-3ζ as Cby-binding partners using affinity purification/mass spectrometry. 14-3-3 proteins specifically recognize serine 20 within the 14-3-3–binding motif of Cby when phosphorylated by Akt kinase. Notably, 14-3-3 binding results in sequestration of Cby into the cytoplasm. Moreover, Cby and 14-3-3 form a stable tripartite complex with β-catenin, causing β-catenin to partition into the cytoplasm. Our results therefore suggest a novel paradigm through which Cby acts in concert with 14-3-3 proteins to facilitate nuclear export of β-catenin, thereby antagonizing β-catenin signaling.

Download Full-text

CDK-dependent phosphorylation of Alp7–Alp14 (TACC–TOG) promotes its nuclear accumulation and spindle microtubule assembly

Molecular Biology of the Cell ◽

10.1091/mbc.e13-11-0679 ◽

2014 ◽

Vol 25 (13) ◽

pp. 1969-1982 ◽

Cited By ~ 19

Author(s):

Naoyuki Okada ◽

Takashi Toda ◽

Masayuki Yamamoto ◽

Masamitsu Sato

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Coiled Coil ◽

Microtubule Assembly ◽

Nuclear Accumulation ◽

Cyclin Dependent Kinase ◽

Mitotic Entry ◽

Localization Signal ◽

Export Signal ◽

Overexpressed Gene

As cells transition from interphase to mitosis, the microtubule cytoskeleton is reorganized to form the mitotic spindle. In the closed mitosis of fission yeast, a microtubule-associated protein complex, Alp7–Alp14 (transforming acidic coiled-coil–tumor overexpressed gene), enters the nucleus upon mitotic entry and promotes spindle formation. However, how the complex is controlled to accumulate in the nucleus only during mitosis remains elusive. Here we demonstrate that Alp7–Alp14 is excluded from the nucleus during interphase using the nuclear export signal in Alp14 but is accumulated in the nucleus during mitosis through phosphorylation of Alp7 by the cyclin-dependent kinase (CDK). Five phosphorylation sites reside around the nuclear localization signal of Alp7, and the phosphodeficient alp7-5A mutant fails to accumulate in the nucleus during mitosis and exhibits partial spindle defects. Thus our results reveal one way that CDK regulates spindle assembly at mitotic entry: CDK phosphorylates the Alp7–Alp14 complex to localize it to the nucleus.

Download Full-text

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

eLife ◽

10.7554/elife.15460 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 30

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.

Download Full-text

A Putative NES Mediates Cytoplasmic Localization of Apoptin in Normal Cells

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/36.12.817 ◽

2004 ◽

Vol 36 (12) ◽

pp. 817-823 ◽

Cited By ~ 16

Author(s):

Qing-Ming Wang ◽

Guo-Cai Fan ◽

Ji-Zhong Chen ◽

Hui-Peng Chen ◽

Fu-Chu He

Keyword(s):

Tumor Cells ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Chicken Anemia Virus ◽

Nuclear Accumulation ◽

Cytoplasmic Localization ◽

Normal Cells ◽

Localization Signal ◽

Anemia Virus ◽

Export Signal

Abstract Apoptin, a protein expressed by chicken anemia virus, is found predominantly in the cytoplasm in normal cells, whereas it localizes in the nucleus in transformed and malignant cells. However, the mechanisms that regulate the different subcellular localization of Apoptin in normal and tumor cells have not been fully clarified. In this work, a putative nuclear export signal (NES) in Apoptin was predicted. It was testified that the putative NES (pNES) of Apoptin was not a functional NES, but actually acted as a cytoplasmic retention signal. Deletion of the pNES led to the nuclear accumulation of Apoptin in normal cells. In addition, when a strong nuclear localization signal was introduced into Apoptin, it exclusively translocated to the nucleus in normal cells. These observations indicated that the cytoplasmic localization of Apoptin in normal cells results from the balance between cytoplasmic retention and nuclear import. On the other hand, the pNES was also proved to be necessary for Apoptin multimerization. Mutants lacking the pNES did not form obviously visible globular aggregates in normal or tumor cells.

Download Full-text