Nuclear accumulation of the E2F heterodimer regulated by subunit composition and alternative splicing of a nuclear localization signal

1996 ◽  
Vol 109 (10) ◽  
pp. 2443-2452 ◽  
Author(s):  
S. de la Luna ◽  
M.J. Burden ◽  
C.W. Lee ◽  
N.B. La Thangue
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Alternative Splicing ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Nuclear Accumulation ◽  
Dna Binding Activity ◽  
Localization Signal

The cellular transcription factor E2F plays a critical role in integrating cell cycle progression with the transcription apparatus by virtue of a physical interaction and control by key regulators of the cell cycle, such as pRb, cyclins and cyclin-dependent kinases. Generic E2F DNA binding activity arises when a member of two families of proteins, E2F and DP, form heterodimeric complexes, an interaction which results in co-operative transcriptional and DNA binding activity. Here, we characterise a new and hitherto unexpected mechanism of control influencing the activity of E2F which is mediated at the level of intracellular location through a dependence on heterodimer formation for nuclear translocation. Nuclear accumulation is dramatically influenced by two distinct processes: alternative splicing of a nuclear localization signal and subunit composition of the E2F heterodimer. These data define a new level of control in the E2F transcription factor whereby interplay between subunits dictates the levels of nuclear DNA binding activity.

scholarly journals I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.

1992 ◽  
Vol 3 (12) ◽  
pp. 1339-1352 ◽  
Author(s):  
P A Ganchi ◽  
S C Sun ◽  
W C Greene ◽  
D W Ballard
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Localization Signal

The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65.

Xenopus nuclear factor 7 (xnf7) possesses an NLS that functions efficiently in both oocytes and embryos

1993 ◽  
Vol 105 (2) ◽  
pp. 389-395
Author(s):  
X. Li ◽  
L.D. Etkin
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Accumulation ◽  
Nuclear Factor ◽  
Zinc Finger Gene ◽  
Early Embryos ◽  
Localization Signal ◽  
Nuclear Phosphoprotein

Xenopus nuclear factor 7 (xnf7) is a nuclear phosphoprotein that is encoded by a member of a novel zinc finger gene family and likely functions as a transcription factor. It possesses a nuclear localization signal (NLS) similar to the bipartite basic NLS of nucleoplasmin, but unlike nucleoplasmin, which re-enters nuclei immediately after fertilization, xnf7 remains cytoplasmic until the mid-blastula transition (MBT). We have measured the accumulation of injected labeled xnf7 protein or protein produced from synthetic xnf7 transcripts in the oocyte nuclei (GV). The data show that the NLS of xnf7 functions efficiently in oocytes. Mutations in either of the bipartite basic domains of the xnf7 NLS inhibit nuclear accumulation, while mutations in the spacer sequences have no effect. The xnf7 NLS linked to pyruvate kinase directs the efficient accumulation of this protein into nuclei of early embryos prior to the MBT. These data suggest that retention of the xnf7 protein during development is the result of a mechanism that interferes with the xnf7 NLS function.

scholarly journals A Highly Organized Structure Mediating Nuclear Localization of a Myb2 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

2011 ◽  
Vol 10 (12) ◽  
pp. 1607-1617 ◽  
Author(s):  
Chien-Hsin Chu ◽  
Lung-Chun Chang ◽  
Hong-Ming Hsu ◽  
Shu-Yi Wei ◽  
Hsing-Wei Liu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Trichomonas Vaginalis ◽  
Simian Virus 40 ◽  
Binding Activity ◽  
T Antigen ◽  
Structural Domain ◽  
Dna Binding Activity

ABSTRACT Nuclear proteins usually contain specific peptide sequences, referred to as nuclear localization signals (NLSs), for nuclear import. These signals remain unexplored in the protozoan pathogen, Trichomonas vaginalis . The nuclear import of a Myb2 transcription factor was studied here using immunodetection of a hemagglutinin-tagged Myb2 overexpressed in the parasite. The tagged Myb2 was localized to the nucleus as punctate signals. With mutations of its polybasic sequences, 48KKQK51 and 61KR62, Myb2 was localized to the nucleus, but the signal was diffusive. When fused to a C-terminal non-nuclear protein, the Myb2 sequence spanning amino acid (aa) residues 48 to 143, which is embedded within the R2R3 DNA-binding domain (aa 40 to 156), was essential and sufficient for efficient nuclear import of a bacterial tetracycline repressor (TetR), and yet the transport efficiency was reduced with an additional fusion of a firefly luciferase to TetR, while classical NLSs from the simian virus 40 T-antigen had no function in this assay system. Myb2 nuclear import and DNA-binding activity were substantially perturbed with mutation of a conserved isoleucine (I74) in helix 2 to proline that altered secondary structure and ternary folding of the R2R3 domain. Disruption of DNA-binding activity alone by point mutation of a lysine residue, K51, preceding the structural domain had little effect on Myb2 nuclear localization, suggesting that nuclear translocation of Myb2, which requires an ordered structural domain, is independent of its DNA binding activity. These findings provide useful information for testing whether myriad Mybs in the parasite use a common module to regulate nuclear import.

scholarly journals M-phase-specific phosphorylation of the POU transcription factor GHF-1 by a cell cycle-regulated protein kinase inhibits DNA binding.

1995 ◽  
Vol 15 (12) ◽  
pp. 6694-6701 ◽  
Author(s):  
C Caelles ◽  
H Hennemann ◽  
M Karin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Binding Activity ◽  
Dependent Manner ◽  
Mitotic Kinase ◽  
Dna Binding Activity ◽  
M Phase ◽  
A Cell

GHF-1 is a member of the POU family of homeodomain proteins. It is a cell-type-specific transcription factor responsible for determination and expansion of growth hormone (GH)- and prolactin-expressing cells in the anterior pituitary. It was previously suggested that cyclic AMP (cAMP)-responsive protein kinase A (PKA) phosphorylates GHF-1 at a site within the N-terminal arm of its homeodomain, thereby inhibiting its binding to the GH promoter. These results, however, are inconsistent with the physiological stimulation of GH production by the cAMP pathway. As reported here, cAMP agonists and PKA do not inhibit GHF-1 activity in living cells and although they stimulate the phosphorylation of GHF-1, the inhibitory phosphoacceptor site within the homeodomain is not affected. Instead, this site, Thr-220, is subject to M-phase-specific phosphorylation. As a result, GHF-1 DNA binding activity is transiently inhibited during the M phase. This activity is regained once cells enter G1, a phase during which GHF-1 phosphorylation is minimal. Thr-220 of GHF-1 is the homolog of the mitotic phosphoacceptor site responsible for the M-phase-specific inhibition of Oct-1 DNA binding Ser-382. As this site is conserved in all POU proteins, it appears that all members of this group are similarly regulated. A specific kinase activity distinct in its substrate specificity and susceptibility to inhibitors from the Cdc2 mitotic kinase or PKA was identified in extracts of mitotic cells. This novel activity could be involved in regulating the DNA binding activity of all POU proteins in a cell cycle-dependent manner.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

scholarly journals The evaluation of anoxia responsive E2F DNA binding activity in the red eared slider turtle, Trachemys scripta elegans

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4755 ◽  
Author(s):  
Kyle K. Biggar ◽  
Kenneth B. Storey
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Complex Dynamics ◽  
Binding Activity ◽  
Trachemys Scripta ◽  
Model Organisms ◽  
Trachemys Scripta Elegans ◽  
Dna Binding Activity

In many cases, the DNA-binding activity of a transcription factor does not change, while its transcriptional activity is greatly influenced by the make-up of bound proteins. In this study, we assessed the protein composition and DNA-binding ability of the E2F transcription factor complex to provide insight into cell cycle control in an anoxia tolerant turtle through the use of a modified ELISA protocol. This modification also permits the use of custom DNA probes that are tailored to a specific DNA binding region, introducing the ability to design capture probes for non-model organisms. Through the use of EMSA and ELISA DNA binding assays, we have successfully determined the in vitro DNA binding activity and complex dynamics of the Rb/E2F cell cycle regulatory mechanisms in an anoxic turtle, Trachemys scripta elegans. Repressive cell cycle proteins (E2F4, Rb, HDAC4 and Suv39H1) were found to significantly increase at E2F DNA-binding sites upon anoxic exposure in anoxic turtle liver. The lack of p130 involvement in the E2F DNA-bound complex indicates that anoxic turtle liver may maintain G1 arrest for the duration of stress survival.

scholarly journals Serum induction of MEF2/RSRF expression in vascular myocytes is mediated at the level of translation.

1995 ◽  
Vol 15 (6) ◽  
pp. 3415-3423 ◽  
Author(s):  
E Suzuki ◽  
K Guo ◽  
M Kolman ◽  
Y T Yu ◽  
K Walsh
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Dna Binding ◽  
Cell Cycle Progression ◽  
Vascular Tissue ◽  
Binding Activity ◽  
Cycle Progression ◽  
Dna Binding Activity ◽  
Vascular Myocytes

Vascular smooth muscle cells (VSMCs) reversibly coordinate the expression of VSMC-specific genes and the genes required for cell cycle progression. Here we demonstrate that isoforms of the MEF2/RSRF transcription factor are expressed in VSMCs and in vascular tissue. The MEF2A DNA-binding activity was upregulated when quiescent VSMCs were stimulated to proliferate with serum mitogens. The serum-induction of MEF2A DNA-binding activity occurred approximately 4 h following serum activation, and this correlated with an increase in the level of MEF2A protein without changes in the level of MEF2A mRNA or protein stability. These results indicate that MEF2A induction by serum is regulated at the level of translation.

scholarly journals Metal-Responsive Transcription Factor 1 (MTF-1) Activity Is Regulated by a Nonconventional Nuclear Localization Signal and a Metal-Responsive Transactivation Domain

2009 ◽  
Vol 29 (23) ◽  
pp. 6283-6293 ◽  
Author(s):  
Uschi Lindert ◽  
Mirjam Cramer ◽  
Michael Meuli ◽  
Oleg Georgiev ◽  
Walter Schaffner
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domain ◽  
Localization Signal ◽  
Transcription Factor 1

ABSTRACT Metal-responsive transcription factor 1 (MTF-1) mediates both basal and heavy metal-induced transcription of metallothionein genes and also regulates other genes involved in the cell stress response and in metal homeostasis. In resting cells, MTF-1 localizes to both the cytoplasm and the nucleus but quantitatively accumulates in the nucleus upon metal load and under other stress conditions. Here we show that within the DNA-binding domain, a region spanning zinc fingers 1 to 3 (amino acids [aa] 137 to 228 in human MTF-1) harbors a nonconventional nuclear localization signal. This protein segment confers constitutive nuclear localization to a cytoplasmic marker protein. The deletion of the three zinc fingers impairs nuclear localization. The export of MTF-1 to the cytoplasm is controlled by a classical nuclear export signal (NES) embedded in the acidic activation domain. We show that this activation domain confers metal inducibility in distinct cell types when fused to a heterologous DNA-binding domain. Furthermore, the cause of a previously described stronger inducibility of human versus mouse MTF-1 could be narrowed down to a 3-aa difference in the NES; “humanizing” mouse MTF-1 at these three positions enhanced its metal inducibility to the level of human MTF-1.

The Re-Activation of FoxO3 Transcription Factor in Acute Myeloid Leukaemia Is Responsible for the Induction of a Quiescent Status of Leukaemic Progenitor Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 929-929
Author(s):  
Daniela Cilloni ◽  
Cristina Panuzzo ◽  
Francesca Arruga ◽  
Francesca Messa ◽  
Monica Pradotto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Stem Cell ◽  
Dna Binding ◽  
Prognostic Significance ◽  
Binding Activity ◽  
Cell Compartment ◽  
Transfected Cells ◽  
Dna Binding Activity ◽  
Acute Myeloid

Abstract The FoxO transcription factor promotes apoptosis and triggers cell cycle inhibition through multiple mechanisms. It is inactivated by PI3K/Akt induced phosphorylation thus resulting in nuclear exclusion and degradation. Moreover, several data demonstrated an abnormal activation of PI3K/Akt pathway in acute myeloid leukemia (AML) with contrasting prognostic significance. The aim of this study was to clarify the role of FoxO3 in AML and to investigate alternative pathways eventually responsible for FoxO3 inactivation. Importantly, we explore the effects of FoxO3 re-activation in CD34+ AML cells. Cell cycle was analyzed by FACS in CD34+ cell population as well as the levels of CD47, which has been demonstrated to increased during progression through the cell cycle and stem cell mobilization. Protein amount and localization were analyzed by Western blot and immunofluorescence, the DNA binding activity was measured by EMSA. 35 BM samples from AML patients at diagnosis and in 20 healthy donors were analyzed. Furthermore Spred1, known to be a FoxO3 target gene was quantified by RQ-PCR. We previously described the absence of Spred1 in AML patients and demonstrated that it promotes growth arrest and apoptosis in haematopoietic cells. Finally BM cells were incubated with a PI3K inhibitor LY294002 and the IKK inhibitor PS1145, alone and in combination. Moreover, the t(8;21) positive Kasumi cell line was transfected with pECE-FoxO3 to evaluate FoxO3 effects on cell growth and apoptosis. We found that, while FoxO3 in control cells is localized in both nucleus (mean value of intensity of 21,4 ±2) and cytoplasm (14,6±1,7), it is completely cytoplasmatic in AML cells (18,1±4,6 in cytoplasm vs 8,2±4 in the nucleus) and enters the nucleus after chemotherapy or in vitro incubation with LY29400. Moreover, FoxO3 DNA binding activity in AML patients is completely absent at diagnosis and restored after therapy. Also the mRNA of Spred1 is rather undetectable at diagnosis (2−ΔΔCt= 0,009±0,3) and shows normal levels during remission (2−ΔΔCt = 2±1,5) or after LY29400 incubation (2−ΔΔCt =0,8±0,3). In addition LY294002 and PS1145 treatment results in FoxO3 partial nuclear re-localization while their association induces a complete nuclear shuttle suggesting that both pathways could be implicated in FoxO3 inactivation. The restoring of FoxO3 function by LY29400 in CD34+ cells induces quiescence of this progenitor cell compartment as demonstrated by the comparison of cell cycle kinetics and the decreased expression of CD47 (p=0,01). Finally, FoxO3 overexpression in transfected cells results in a block of proliferation rate (66% of inhibition compared to empty vector transfected cells) and apoptosis induction (35% vs 12%). Taken together these data suggest that FoxO3 inactivation may be crucial for the leukemic progression and demonstrate that also IKK pathway contributes to this effect, providing the rationale for a therapeutic strategy. On the other hand, the re-activation of FoxO3 induces quiescence of the stem cell compartment so providing a mechanism of escape from chemotherapy induced apoptosis.

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