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.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Iron-Inducible Nuclear Translocation of a Myb3 Transcription Factor in the Protozoan Parasite Trichomonas vaginalis

2012 ◽  
Vol 11 (12) ◽  
pp. 1441-1450 ◽  
Author(s):  
Hong-Ming Hsu ◽  
Yu Lee ◽  
Dharmu Indra ◽  
Shu-Yi Wei ◽  
Hsing-Wei Liu ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Trichomonas Vaginalis ◽  
Structural Integrity ◽  
Nuclear Translocation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Nuclear Localization Signals ◽  
Localization Signal

ABSTRACTInTrichomonas vaginalis, a novel nuclear localization signal spanning the folded R2R3 DNA-binding domain of a Myb2 protein was previously identified. To study whether a similar signal is used for nuclear translocation by other Myb proteins, nuclear translocation of Myb3 was examined in this report. When overexpressed, hemagglutinin-tagged Myb3 was localized to nuclei of transfected cells, with a cellular distribution similar to that of endogenous Myb3. Fusion to a bacterial tetracycline repressor, R2R3, of Myb3 that spans amino acids (aa) 48 to 156 was insufficient for nuclear translocation of the fusion protein, unless its C terminus was extended to aa 167. The conserved isoleucine in helix 2 of R2R3, which is important for Myb2's structural integrity in maintaining DNA-binding activity and nuclear translocation, was also vital for the former activity of Myb3, but less crucial for the latter. Sequential nuclear influx and efflux of Myb3, which require further extension of the nuclear localization signal to aa 180, were immediately induced after iron repletion. Sequence elements that regulate nuclear translocation with cytoplasmic retention, nuclear influx, and nuclear efflux were identified within the C-terminal tail. These results suggest that the R2R3 DNA-binding domain also serves as a common module for the nuclear translocation of both Myb2 and Myb3, but there are intrinsic differences between the two nuclear localization signals.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631 ◽  
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

Creation of a protein vector construct including an SSBTne DNA-binding domain and VirD2 nuclear localization signal

2008 ◽  
Vol 34 (5) ◽  
pp. 329-331
Author(s):  
D. V. Grishin ◽  
V. P. Gudov ◽  
O. V. Sergienko ◽  
V. G. Lunin ◽  
P. N. Kharchenko
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal

Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor

1993 ◽  
Vol 13 (11) ◽  
pp. 6858-6865
Author(s):  
M W Russo ◽  
C Matheny ◽  
J Milbrandt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Zinc Fingers ◽  
Fold Increase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domains ◽  
Cellular Factor ◽  
Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

scholarly journals Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

2007 ◽  
Vol 21 (6) ◽  
pp. 1297-1311 ◽  
Author(s):  
Kai Sun ◽  
Vedrana Montana ◽  
Karthikeyani Chellappa ◽  
Yann Brelivet ◽  
Dino Moras ◽  
...  
Keyword(s):  
Dna Binding ◽  
Nuclear Receptors ◽  
Nuclear Localization ◽  
Thyroid Hormone Receptor ◽  
Zinc Fingers ◽  
Binding Domain ◽  
Retinoid X Receptor ◽  
Common Mechanism ◽  
Dna Binding Domain ◽  
Cytoplasmic Localization

Abstract Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4α (HNF4α). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4α and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4α (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4α as well as decreased endogenous HNF4α protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor α, retinoid X receptor α, and thyroid hormone receptor β) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor α and peroxisome proliferator-activated receptor α. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

scholarly journals Transcriptional activity of the zinc finger protein NGFI-A is influenced by its interaction with a cellular factor.

1993 ◽  
Vol 13 (11) ◽  
pp. 6858-6865 ◽  
Author(s):  
M W Russo ◽  
C Matheny ◽  
J Milbrandt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Transcriptional Activity ◽  
Zinc Fingers ◽  
Fold Increase ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Activation Domains ◽  
Cellular Factor ◽  
Inhibitory Domain

NGFI-A is an immediate-early gene that encodes a transcription factor whose DNA-binding domain is composed of three zinc fingers. To define the domains responsible for its transcriptional activity, a mutational analysis was conducted with an NGFI-A molecule in which the zinc fingers were replaced by the GAL4 DNA-binding domain. In a cotransfection assay, four activation domains were found within NGFI-A. Three of the activation domains are similar to those characterized previously: one contains a large number of acidic residues, another is enriched in proline and glutamine residues, and another has some sequence homology to a domain found in Krox-20. The fourth bears no resemblance to previously described activation domains. NGFI-A also contains an inhibitory domain whose removal resulted in a 15-fold increase in NGFI-A activity. This increase in activity occurred in all mammalian cell types tested but not in Drosophila S2 cells. Competition experiments in which increasing amounts of the inhibitory domain were cotransfected along with NGFI-A demonstrated a dose-dependent increase in NGFI-A activity. A point mutation within the inhibitory domain of the competitor (I293F) abolished this property. When the analogous mutation was introduced into native NGFI-A, a 17-fold increase in activity was observed. The inhibitory effect therefore appears to be the result of an interaction between this domain and a titratable cellular factor which is weakened by this mutation. Downmodulation of transcription factor activity through interaction with a cellular factor has been observed in several other systems, including the regulation of transcription factor E2F by retinoblastoma protein, and in studies of c-Jun.

scholarly journals Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B.

1996 ◽  
Vol 16 (10) ◽  
pp. 5444-5449 ◽  
Author(s):  
H Suyang ◽  
R Phillips ◽  
I Douglas ◽  
S Ghosh
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Stable Complex ◽  
Dna Binding Domain ◽  
Nf Kappa B ◽  
Prolonged Stimulation ◽  
I Kappa B Alpha ◽  
Localization Signal

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.

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